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The deficiency of the molybdenum cofactor (Moco) is an autosomal recessive disease, which leads to the loss of activity of all molybdoenzymes in humans with sulfite oxidase being the essential protein. Moco deficiency generally results in death in early childhood. Moco is a sulfur-containing cofactor synthesized in the cytosol with the sulfur being provided by a sulfur relay system composed of the L-cysteine desulfurase NFS1, MOCS3, and MOCS2A. Human MOCS3 is a dual-function protein that was shown to play an important role in Moco biosynthesis and in the mcm(5)s(2) U thio modifications of nucleosides in cytosolic tRNAs for Lys, Gln, and Glu. In this study, we constructed a homozygous MOCS3 knockout in HEK293T cells using the CRISPR/Cas9 system. The effects caused by the absence of MOCS3 were analyzed in detail. We show that sulfite oxidase activity was almost completely abolished, on the basis of the absence of Moco in these cells. In addition, mcm(5)s(2)U thio-modified tRNAs were not detectable. Because the L-cysteine desulfurase NFS1 was shown to act as a sulfur donor for MOCS3 in the cytosol, we additionally investigated the impact of a MOCS3 knockout on the cellular localization of NFS1. By different methods, we identified a MOCS3-independent novel localization of NFS1 at the centrosome.
Identification of YdhV as the First Molybdoenzyme Binding a Bis-Mo-MPT Cofactor in Escherichia coli
(2019)
The oxidoreductase YdhV in Escherichia coli has been predicted to belong to the family of molybdenum/tungsten cofactor (Moco/Wco)-containing enzymes. In this study, we characterized the YdhV protein in detail, which shares amino acid sequence homology with a tungsten-containing benzoyl-CoA reductase binding the bis-W-MPT (for metal-binding pterin) cofactor. The cofactor was identified to be of a bis-Mo-MPT type with no guanine nucleotides present, which represents a form of Moco that has not been found previously in any molybdoenzyme. Our studies showed that YdhV has a preference for bis-Mo-MPT over bis-W-MPT to be inserted into the enzyme. In-depth characterization of YdhV by X-ray absorption and electron paramagnetic resonance spectroscopies revealed that the bis-Mo-MPT cofactor in YdhV is redox active. The bis-Mo-MPT and bis-W-MPT cofactors include metal centers that bind the four sulfurs from the two dithiolene groups in addition to a cysteine and likely a sulfido ligand. The unexpected presence of a bis-Mo-MPT cofactor opens an additional route for cofactor biosynthesis in E. coli and expands the canon of the structurally highly versatile molybdenum and tungsten cofactors.
Human aldehyde oxidase (hAOX1) is a molybdenum enzyme with high toxicological importance, but its physiological role is still unknown. hAOX1 metabolizes different classes of xenobiotics and is one of the main drug-metabolizing enzymes in the liver, along with cytochrome P450. hAOX1 oxidizes and inactivates a large number of drug molecules and has been responsible for the failure of several phase I clinical trials. The interindividual variability of drug-metabolizing enzymes caused by single nucleotide polymorphisms (SNPs) is highly relevant in pharmaceutical treatments. In this study, we present the crystal structure of the inactive variant G1269R, revealing the first structure of a molybdenum cofactor (Moco)-free form of hAOX1. These data allowed to model, for the first time, the flexible Gate 1 that controls access to the active site. Furthermore, we inspected the thermostability of wild-type hAOX1 and hAOX1 with various SNPs (L438V, R1231H, G1269R or S1271L) by CD spectroscopy and ThermoFAD, revealing that amino acid exchanges close to the Moco site can impact protein stability up to 10 degrees C. These results correlated with biochemical and structural data and enhance our understanding of hAOX1 and the effect of SNPs in the gene encoding this enzyme in the human population. EnzymesAldehyde oxidase (); xanthine dehydrogenase (); xanthine oxidase (). DatabasesStructural data are available in the Protein Data Bank under the accession number .
Biosensors for the detection of benzaldehyde and g-aminobutyric acid (GABA) are reported using aldehyde oxidoreductase PaoABC from Escherichia coli immobilized in a polymer containing bound low potential osmium redox complexes. The electrically connected enzyme already electrooxidizes benzaldehyde at potentials below −0.15 V (vs. Ag|AgCl, 1 M KCl). The pH-dependence of benzaldehyde oxidation can be strongly influenced by the ionic strength. The effect is similar with the soluble osmium redox complex and therefore indicates a clear electrostatic effect on the bioelectrocatalytic efficiency of PaoABC in the osmium containing redox polymer. At lower ionic strength, the pH-optimum is high and can be switched to low pH-values at high ionic strength. This offers biosensing at high and low pH-values. A “reagentless” biosensor has been formed with enzyme wired onto a screen-printed electrode in a flow cell device. The response time to addition of benzaldehyde is 30 s, and the measuring range is between 10–150 µM and the detection limit of 5 µM (signal to noise ratio 3:1) of benzaldehyde. The relative standard deviation in a series (n = 13) for 200 µM benzaldehyde is 1.9%. For the biosensor, a response to succinic semialdehyde was also identified. Based on this response and the ability to work at high pH a biosensor for GABA is proposed by coimmobilizing GABA-aminotransferase (GABA-T) and PaoABC in the osmium containing redox polymer.
We have developed a three-dimensional (3D) graphene electrode suitable for the immobilization of human sulfite oxidase (hSO), which catalyzes the electrochemical oxidation of sulfite via direct electron transfer (DET). The electrode is fabricated by drop-casting graphene-polyethylenimine (G-P) composites on carbon papers (CPs) precoated with graphene oxide (GO). The negatively charged hSO can be adsorbed electrostatically on the positively charged matrix (G-P) on CP electrodes coated with GO (CPG), with a proper orientation for accelerated DET. Notably, further electrochemical reduction of G-P on CPG electrodes leads to a 9-fold increase of the saturation catalytic current density (j(m)) for sulfite oxidation reaching 24.4 +/- 0.3 mu A to cm(-2), the highest value among reported DET-based hSO bioelectrodes. The increased electron transfer rate plays a dominating role in the enhancement of direct enzymatic current because of the improved electric contact of hSO with the electrode, The optimized hSO bioelectrode shows a significant catalytic rate (k(cat): 25.6 +/- 0.3 s(-1)) and efficiency (k(cat)/K-m: 0.231 +/- 0.003 s(-1) mu M-1) compared to the reported hSO bioelectrodes. The assembly of the hSO bioanode and a commercial platinum biocathode allows the construction of sulfite/O-2 enzymatic biofuel cells (EBFCs) with flowing fuels. The optimized EBFC displays an open-circuit voltage (OCV) of 0.64 +/- 0.01 V and a maximum power density of 61 +/- 6 mu W cm(-2) (122 +/- 12 mW m(-3)) at 30 degrees C, which exceeds the best reported value by more than 6 times.
Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In recent years it has become obvious that the availability of iron plays an important role in the biosynthesis of Moco. First, the MoaA protein binds two (4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional NFe-4S) cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the L-cysteine desulfurase IscS, which is a shared protein with a main role in the assembly of Fe-S clusters. In this report, we investigated the transcriptional regulation of the moaABCDE operon by focusing on its dependence on cellular iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, our data show that the regulation of the moaABCDE operon at the level of transcription is only marginally influenced by the availability of iron. Nevertheless, intracellular levels of Moco were decreased under iron-limiting conditions, likely based on an inactive MoaA protein in addition to lower levels of the L-cysteine desulfurase IscS, which simultaneously reduces the sulfur availability for Moco production. IMPORTANCE FNR is a very important transcriptional factor that represents the master switch for the expression of target genes in response to anaerobiosis. Among the FNR-regulated operons in Escherichia coli is the moaABCDE operon, involved in Moco biosynthesis. Molybdoenzymes have essential roles in eukaryotic and prokaryotic organisms. In bacteria, molybdoenzymes are crucial for anaerobic respiration using alternative electron acceptors. This work investigates the connection of iron availability to the biosynthesis of Moco and the production of active molybdoenzymes.
The genus Shewanella is well known for its genetic diversity, its outstanding respiratory capacity, and its high potential for bioremediation. Here, a novel strain isolated from sediments of the Indian Ocean was characterized. A 16S rRNA analysis indicated that it belongs to the species Shewanella decolorationis. It was named Shewanella decolorationis LDS1. This strain presented an unusual ability to grow efficiently at temperatures from 24 degrees C to 40 degrees C without apparent modifications of its metabolism, as shown by testing respiratory activities or carbon assimilation, and in a wide range of salt concentrations. Moreover, S. decolorationis LDS1 tolerates high chromate concentrations. Indeed, it was able to grow in the presence of 4 mM chromate at 28 degrees C and 3 mM chromate at 40 degrees C. Interestingly, whatever the temperature, when the culture reached the stationary phase, the strain reduced the chromate present in the growth medium. In addition, S. decolorationis LDS1 degrades different toxic dyes, including anthraquinone, triarylmethane, and azo dyes. Thus, compared to Shewanella oneidensis, this strain presented better capacity to cope with various abiotic stresses, particularly at high temperatures. The analysis of genome sequence preliminary data indicated that, in contrast to S. oneidensis and S. decolorationis S12, S. decolorationis LDS1 possesses the phosphorothioate modification machinery that has been described as participating in survival against various abiotic stresses by protecting DNA. We demonstrate that its heterologous production in S. oneidensis allows it to resist higher concentrations of chromate. IMPORTANCE Shewanella species have long been described as interesting microorganisms in regard to their ability to reduce many organic and inorganic compounds, including metals. However, members of the Shewanella genus are often depicted as cold-water microorganisms, although their optimal growth temperature usually ranges from 25 to 28 degrees C under laboratory growth conditions. Shewanella decolorationis LDS1 is highly attractive, since its metabolism allows it to develop efficiently at temperatures from 24 to 40 degrees C, conserving its ability to respire alternative substrates and to reduce toxic compounds such as chromate or toxic dyes. Our results clearly indicate that this novel strain has the potential to be a powerful tool for bioremediation and unveil one of the mechanisms involved in its chromate resistance.
The small and large subunits of molybdopterin (MPT) synthase (MOCS2A and MOCS2B), are both encoded by the MOCS2 gene in overlapping and shifted open reading frames (ORFs), which is a highly unusual structure for eukaryotes. Theoretical analysis of genomic sequences suggested that the expression of these overlapping ORFs is facilitated by the use of alternate first exons leading to alternative transcripts. Here, we confirm the existence of these overlapping transcripts experimentally. Further, we identified a deletion in a molybdenum cofactor deficient patient, which removes the start codon for the small subunit (MOCS2A). We observed undisturbed production of both transcripts, while Western blot analysis demonstrated that MOCS2B, the large subunit, is unstable in the absence of MOCS2A. This reveals new insights into the expression of this evolutionary ancient anabolic system.
The pyranopterin dithiolene (PDT) ligand is an integral component of the molybdenum cofactor (Moco) found in all molybdoenzymes with the sole exception of nitrogenase. However, the roles of the PDT in catalysis are still unknown. The PDT is believed to be bound to the proteins by an extensive hydrogen bonding network, and it has been suggested that these interactions may function to fine-tune Moco for electron- and atom-transfer reactivity in catalysis. Here, we use resonance Raman (rR) spectroscopy to probe Moco-protein interactions using heavy-atom congeners of lumazine, molecules that bind tightly to both wild-type xanthine dehydrogenase (wt-XDH) and its Q102G and Q197A variants following enzymatic hydroxylation to the corresponding violapterin product molecules. The resulting enzyme-product complexes possess intense near-IR absorption, allowing high-quality rR spectra to be collected on wt-XDH and the Q102G and Q197A variants. Small negative frequency shifts relative to wt-XDH are observed for the low-frequency Moco vibrations. These results are interpreted in the context of weak hydrogen-bonding and/or electrostatic interactions between Q102 and the -NH2 terminus of the PDT, and between Q197 and the terminal oxo of the Mo equivalent to O group. The Q102A, Q102G, Q197A, and Q197E variants do not appreciably affect the kinetic parameters k(red) and k(red)/K-D, indicating that a primary role for these glutamine residues is to stabilize and coordinate Moco in the active site of XO family enzymes but to not directly affect the catalytic throughput. Raman frequency shifts between wt-XDH and its Q102G variant suggest that the changes in the electron density at the Mo ion that accompany Mo oxidation during electron-transfer regeneration of the catalytically competent active site are manifest in distortions at the distant PDT amino terminus. This implies a primary role for the PDT as a conduit for facilitating enzymatic electron-transfer reactivity in xanthine oxidase family enzymes.
Aldehyde oxidases (AOXs) are molybdo-flavoenzymes characterized by broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into the corresponding carboxylic acids and hydroxylating various heteroaromatic rings. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. The physiological function of mammalian AOX isoenzymes is still unclear, however, human AOX (hAOX1) is an emerging enzyme in phase-I drug metabolism. Indeed, the number of xenobiotics acting as hAOX1 substrates is increasing. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified within the hAOX1 gene. SNPs are a major source of inter-individual variability in the human population, and SNP-based amino acid exchanges in hAOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. In this report we selected ten novel SNPs resulting in amino acid exchanges in proximity to the FAD site of hAOX1 and characterized the purified enzymes after heterologous expression in Escherichia coli. The hAOX1 variants were characterized carefully by quantitative differences in their ability to produce superoxide radical. ROS represent prominent key molecules in physiological and pathological conditions in the cell. Our data reveal significant alterations in superoxide anion production among the variants. In particular the SNP-based amino acid exchange L438V in proximity to the isoalloxanzine ring of the FAD cofactor resulted in increased rate of superoxide radical production of 75%. Considering the high toxicity of the superoxide in the cell, the hAOX1-L438V SNP variant is an eventual candidate for critical or pathological roles of this natural variant within the human population.
Aldehyde oxidases (AOXs) are molybdoflavoenzymes with an important role in the metabolism and detoxification of heterocyclic compounds and aliphatic as well as aromatic aldehydes. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. Four different enzymes, mAOX1, mAOX3, mAOX4, and mAOX2, which are the products of distinct genes, are present in the mouse. A direct and simultaneous comparison of the enzymatic properties and characteristics of the four enzymes has never been performed. In this report, the four catalytically active mAOX enzymes were purified after heterologous expression in Escherichia coli. The kinetic parameters of the four mouse AOX enzymes were determined and compared with the use of six predicted substrates of physiologic and toxicological interest, i.e., retinaldehyde, N1-methylnicotinamide, pyridoxal, vanillin, 4-(dimethylamino) cinnamaldehyde (p-DMAC), and salicylaldehyde. While retinaldehyde, vanillin, p-DMAC, and salycilaldehyde are efficient substrates for the four mouse AOX enzymes, N1-methylnicotinamide is not a substrate of mAOX1 or mAOX4, and pyridoxal is notmetabolized by any of the purified enzymes. Overall, mAOX1, mAOX2, mAOX3, and mAOX4 are characterized by significantly different KM and kcat values for the active substrates. The four mouse AOXs are also characterized by quantitative differences in their ability to produce superoxide radicals. With respect to this last point, mAOX2 is the enzyme generating the largest rate of superoxide radicals of around 40% in relation to moles of substrate converted, and mAOX1, the homolog to the human enzyme, produces a rate of approximately 30% of superoxide radicals with the same substrate.
Transient Catalytic Voltammetry of Sulfite Oxidase Reveals Rate Limiting Conformational Changes
(2017)
Sulfite oxidases are metalloenzymes that oxidize sulfite to sulfate at a molybdenum active site. In vertebrate sulfite oxidases, the electrons generated at the Mo center are transferred to an external electron acceptor via a heme domain, which can adopt two conformations: a “closed” conformation, suitable for internal electron transfer, and an “open” conformation suitable for intermolecular electron transfer. This conformational change is an integral part of the catalytic cycle. Sulfite oxidases have been wired to electrode surfaces, but their immobilization leads to a significant decrease in their catalytic activity, raising the question of the occurrence of the conformational change when the enzyme is on an electrode. We recorded and quantitatively modeled for the first time the transient response of the catalytic cycle of human sulfite oxidase immobilized on an electrode. We show that conformational changes still occur on the electrode, but at a lower rate than in solution, which is the reason for the decrease in activity of sulfite oxidases upon immobilization.
The trafficking and delivery of sulfur to cofactors and nucleosides is a highly regulated and conserved process among all organisms. All sulfur transfer pathways generally have an L-cysteine desulfurase as an initial sulfur mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of sulfur-containing biomolecules like iron sulfur (Fe-S) clusters, thiamine, biotin, lipoic acid, the molybdenum cofactor (Moco), and thiolated nucleosides in tRNA. The human L-cysteine desulfurase NFS1 and the Escherichia coli homologue IscS share a level of amino acid sequence identity of similar to 60%. While E. coli IscS has a versatile role in the cell and was shown to have numerous interaction partners, NFS1 is mainly localized in mitochondria with a crucial role in the biosynthesis of Fe-S clusters. Additionally, NFS1 is also located in smaller amounts in the cytosol with a role in Moco biosynthesis and mcm(5)s(2)U34 thio modifications of nucleosides in tRNA. NFS1 and IscS were conclusively shown to have different interaction partners in their respective organisms. Here, we used functional complementation studies of an E. coli iscS deletion strain with human NFS1 to dissect their conserved roles in the transfer of sulfur to a specific target protein. Our results show that human NFS1 and E. coli IscS share conserved binding sites for proteins involved in Fe-S cluster assembly like IscU, but not with proteins for tRNA thio modifications or Moco biosynthesis. In addition, we show that human NFS1 was almost fully able to complement the role of IscS in Moco biosynthesis when its specific interaction partner protein MOCS3 from humans was also present.
The homodinuclear ruthenium(II) complex [{Ru(l-N4Me2)}(2)(-tape)](PF6)(4) {[1](PF6)(4)} (l-N4Me2=N,N-dimethyl-2,11-diaza[3.3](2,6)-pyridinophane, tape=1,6,7,12-tetraazaperylene) can store one or two electrons in the energetically low-lying * orbital of the bridging ligand tape. The corresponding singly and doubly reduced complexes [{Ru(l-N4Me2)}(2)(-tape(.-))](PF6)(3) {[2](PF6)(3)} and [{Ru(l-N4Me2)}(2)(-tape(2-))](PF6)(2) {[3](PF6)(2)}, respectively, were electrochemically generated, successfully isolated and fully characterized by single-crystal X-ray crystallography, spectroscopic methods and magnetic susceptibility measurements. The singly reduced complex [2](PF6)(3) contains the -radical tape(.-) and the doubly reduced [3](PF6)(2) the diamagnetic dianion tape(2-) as bridging ligand, respectively. Nucleophilic aromatic substitution at the bridging tape in [1](4+) by two sulfite units gave the complex [{Ru(l-N4Me2)}(2){-tape-(SO3)(2)}](2+) ([4](2+)). Complex dication [4](2+) was exploited as a redox mediator between an anaerobic homogenous reaction solution of an enzyme system (sulfite/sulfite oxidase) and the electrode via participation of the low-energy *-orbital of the disulfonato-substituted bridging ligand tape-(SO3)(2)(2-) (E-red1=-0.1V versus Ag/AgCl/1m KCl in water).
Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pK(a) of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase.
Phenothiazine-modified redox hydrogels were synthesized and used for the wiring of the aldehyde oxidoreductase PaoABC to electrode surfaces. The effects of the pH value and electrode surface modification on the biocatalytic activity of the layers were studied in the presence of vanillin as the substrate. The enzyme electrodes were successfully employed as bioanodes in vanillin/O-2 biofuel cells in combination with a high potential bilirubin oxidase biocathode. Open circuit voltages of around 700 mV could be obtained in a two compartment biofuel cell setup. Moreover, the use of a rather hydrophobic polymer with a high degree of crosslinking sites ensures the formation of stable polymer/enzyme films which were successfully used as bioanode in membrane-less biofuel cells. (C) 2015 Elsevier B.V. All rights reserved.
The Eukaryotic-Specific ISD11 Is a Complex-Orphan Protein with Ability to Bind the Prokaryotic IscS
(2016)
The eukaryotic protein Isd11 is a chaperone that binds and stabilizes the central component of the essential metabolic pathway responsible for formation of iron-sulfur clusters in mitochondria, the desulfurase Nfs1. Little is known about the exact role of Isd11. Here, we show that human Isd11 (ISD11) is a helical protein which exists in solution as an equilibrium between monomer, dimeric and tetrameric species when in the absence of human Nfs1 (NFS1). We also show that, surprisingly, recombinant ISD11 expressed in E. coli co-purifies with the bacterial orthologue of NFS1, IscS. Binding is weak but specific suggesting that, despite the absence of Isd11 sequences in bacteria, there is enough conservation between the two desulfurases to retain a similar mode of interaction. This knowledge may inform us on the conservation of the mode of binding of Isd11 to the desulfurase. We used evolutionary evidence to suggest Isd11 residues involved in the interaction.
Aldehyde oxidase (AOX1) is an enzyme with broad substrate specificity, catalyzing the oxidation of a wide range of endogenous and exogenous aldehydes as well as N-heterocyclic aromatic compounds. In humans, the enzyme’s role in phase I drug metabolism has been established and its importance is now emerging. However, the true physiologic function of AOX1 in mammals is still unknown. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified in human AOX1. SNPs are a major source of interindividual variability in the human population, and SNP-based amino acid exchanges in AOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. For the reliable analysis of the effect of amino acid exchanges in human proteins, the existence of reproducible expression systems for the production of active protein in ample amounts for kinetic, spectroscopic, and crystallographic studies is required. In our study we report an optimized expression system for hAOX1 in Escherichia coli using a codon-optimized construct. The codon-optimization resulted in an up to 15-fold increase of protein production and a simplified purification procedure. The optimized expression system was used to study three SNPs that result in amino acid changes C44W, G1269R, and S1271L. In addition, the crystal structure of the S1271L SNP was solved. We demonstrate that the recombinant enzyme can be used for future studies to exploit the role of AOX in drug metabolism, and for the identification and synthesis of new drugs targeting AOX when combined with crystallographic and modeling studies.
We report on the pH-dependent bioelectrocatalytic activity of the redox enzyme xanthine dehydrogenase (XDH) in the presence of sulfonated polyaniline PMSA1 (poly(2-methoxyaniline-5-sulfonic acid)-co-aniline). Ultraviolet-visible (UV-vis) spectroscopic measurements with both components in solution reveal electron transfer from the hypoxanthine (HX)-reduced enzyme to the polymer. The enzyme shows bioelectrocatalytic activity on indium tin oxide (ITO) electrodes, when the polymer is present. Depending on solution pH, different processes can be identified. It can be demonstrated that not only product-based communication with the electrode but also efficient polymer-supported bioelectrocatalysis occur. Interestingly, substrate dependent catalytic currents can be obtained in acidic and neutral solutions, although the highest activity of XDH with natural reaction partners is in the alkaline region. Furthermore, operation of the enzyme electrode without addition of the natural cofactor of XDH is feasible. Finally, macroporous ITO electrodes have been used as an immobilization platform for the fabrication of HX-sensitive electrodes. The study shows that the efficient polymer/enzyme interaction can be advantageously combined with the open structure of an electrode material of controlled pore size, resulting in good processability, stability, and defined signal transfer in the presence of a substrate.
The xanthine oxidase (XO) family comprises molybdenum-dependent enzymes that usually form homodimers (or dimers of heterodimers/trimers) organized in three domains that harbor two [2Fe-2S] clusters, one FAD, and a Mo cofactor. In this work, we crystallized an unusual member of the family, the periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli. This is the first example of an E. coli protein containing a molybdopterin-cytosine-dinucleotide cofactor and is the only heterotrimer of the XO family so far structurally characterized. The crystal structure revealed the presence of an unexpected [4Fe-4S] cluster, anchored to an additional 40 residues subdomain. According to phylogenetic analysis, proteins containing this cluster are widely spread in many bacteria phyla, putatively through repeated gene transfer events. The active site of PaoABC is highly exposed to the surface with no aromatic residues and an arginine (PaoC-R440) making a direct interaction with PaoC-E692, which acts as a base catalyst. In order to understand the importance of R440, kinetic assays were carried out, and the crystal structure of the PaoC-R440H variant was also determined.
Practical use of many bioelectronic and bioanalytical devices is limited by the need of expensive materials and time consuming fabrication. Here we demonstrate the use of nickel electrodes as a simple and cheap solid support material for bioelectronic applications. The naturally nanostructured electrodes showed a surprisingly high electromagnetic surface enhancement upon light illumination such that immobilization and electron transfer reactions of the model redox proteins cytochrome b(5) (Cyt b(5)) and cytochrome c (Cyt c) could be followed via surface enhanced resonance Raman spectroscopy. It could be shown that the nickel surface, when used as received, promotes a very efficient binding of the proteins upon preservation of their native structure. The immobilized redox proteins could efficiently exchange electrons with the electrode and could even act as an electron relay between the electrode and solubilized myoglobin. Our results open up new possibility for nickel electrodes as an exceptional good support for bioelectronic devices and biosensors on the one hand and for surface enhanced spectroscopic investigations on the other hand. (C) 2016 Elsevier B.V. All rights reserved.
The mitochondrial ATP-binding cassette (ABC) transporters ABCB7 in humans, Atm1 in yeast and ATM3 in plants, are highly conserved in their overall architecture and particularly in their glutathione binding pocket located within the transmembrane spanning domains. These transporters have attracted interest in the last two decades based on their proposed role in connecting the mitochondrial iron sulfur (Fe–S) cluster assembly with its cytosolic Fe–S cluster assembly (CIA) counterpart. So far, the specific compound that is transported across the membrane remains unknown. In this report we characterized the ABCB7-like transporter Rcc02305 in Rhodobacter capsulatus, which shares 47% amino acid sequence identity with its mitochondrial counterpart. The constructed interposon mutant strain in R. capsulatus displayed increased levels of intracellular reactive oxygen species without a simultaneous accumulation of the cellular iron levels. The inhibition of endogenous glutathione biosynthesis resulted in an increase of total glutathione levels in the mutant strain. Bioinformatic analysis of the amino acid sequence motifs revealed a potential aminotransferase class-V pyridoxal-50-phosphate (PLP) binding site that overlaps with the Walker A motif within the nucleotide binding domains of the transporter. PLP is a well characterized cofactor of L-cysteine desulfurases like IscS and NFS1 which has a role in the formation of a protein-bound persulfide group within these proteins. We therefore suggest renaming the ABCB7-like transporter Rcc02305 in R. capsulatus to PexA for PLP binding exporter. We further suggest that this ABC-transporter in R. capsulatus is involved in the formation and export of polysulfide species to the periplasm.
The control of bioelectrocatalytic processes by external stimuli for the indirect detection of non-redox active species was achieved using an esterase and a redox enzyme both integrated within a redox hydrogel. The poly( vinyl) imidazole Os(bpy)(2)Cl hydrogel displays pH-responsive properties. The esterase catalysed reaction leads to a local pH decrease causing protonation of imidazole moieties thus increasing hydrogel solvation and mobility of the tethered Os-complexes. This is the key step to enable improved electron transfer between an aldehyde oxidoreductase and the polymer-bound Os-complexes. The off-on switch is further integrated in a biofuel cell system for self-powered signal generation.
Formate dehydrogenase (FDH) enzymes are attractive catalysts for potential carbon dioxide conversion applications. The FDH from Rhodobacter capsulatus (RcFDH) binds a bis-molybdopterin-guanine-dinucleotide (bis-MGD) cofactor, facilitating reversible formate (HCOO-) to CO2 oxidation. We characterized the molecular structure of the active site of wildtype RcFDH and protein variants using X-ray absorption spectroscopy (XAS) at the Mo K-edge. This approach has revealed concomitant binding of a sulfido ligand (Mo=S) and a conserved cysteine residue (S(Cys386)) to Mo(VI) in the active oxidized molybdenum cofactor (Moco), retention of such a coordination motif at Mo(V) in a chemically reduced enzyme, and replacement of only the S(Cys386) ligand by an oxygen of formate upon Mo(IV) formation. The lack of a Mo=S bond in RcFDH expressed in the absence of FdsC implies specific metal sulfuration by this bis-MGD binding chaperone. This process still functioned in the Cys386Ser variant, showing no Mo-S(Cys386) ligand, but retaining a Mo=S bond. The C386S variant and the protein expressed without FdsC were inactive in formate oxidation, supporting that both Moligands are essential for catalysis. Low-pH inhibition of RcFDH was attributed to protonation at the conserved His387, supported by the enhanced activity of the His387Met variant at low pH, whereas inactive cofactor species showed sulfido-to-oxo group exchange at the Mo ion. Our results support that the sulfido and S(Cys386) ligands at Mo and a hydrogen-bonded network including His387 are crucial for positioning, deprotonation, and oxidation of formate during the reaction cycle of RcFDH.
A direct electron transfer (DET) based sulphite/oxygen biofuel cell is reported that utilises human sulphite oxidase (hSOx) and Myrothecium verrucaria bilirubin oxidase (MvBOx) and nanostructured gold electrodes. For bioanode construction, the nanostructured gold microelectrodes were further modified with 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester) to which polyethylene imine was covalently attached. hSOx was adsorbed onto this chemically modified nanostructured electrode with high surface loading of electroactive enzyme and in presence of sulphite high anodic bioelectrocatalytic currents were generated with an onset potential of 0.05 V vs. NHE. The biocathode contained MyBOx directly adsorbed to the deposited gold nanoparticles for cathodic oxygen reduction starting at 0.71 V vs. NHE. Both enzyme electrodes were integrated to a DET-type biofuel cell. Power densities of 8 and 1 mu W cm(-2) were achieved at 0.15 V and 0.45 V of cell voltages, respectively, with the membrane based biodevices under aerobic conditions. (C) 2014 Elsevier B.V. All rights reserved.
The Electrically Wired Molybdenum Domain of Human Sulfite Oxidase is Bioelectrocatalytically Active
(2015)
We report electron transfer between the catalytic molybdenum cofactor (Moco) domain of human sulfite oxidase (hSO) and electrodes through a poly(vinylpyridine)-bound [osmium(N,N'-methyl-2,2'-biimidazole)(3)](2+/3+) complex as the electron-transfer mediator. The biocatalyst was immobilized in this low-potential redox polymer on a carbon electrode. Upon the addition of sulfite to the immobilized separate Moco domain, the generation of a significant catalytic current demonstrated that the catalytic center is effectively wired and active. The bioelectrocatalytic current of the wired separate catalytic domain reached 25% of the signal of the wired full molybdoheme enzyme hSO, in which the heme b(5) is involved in the electron-transfer pathway. This is the first report on a catalytically active wired molybdenum cofactor domain. The formal potential of this electrochemical mediator is between the potentials of the two cofactors of hSO, and as hSO can occupy several conformations in the polymer matrix, it is imaginable that electron transfer from the catalytic site to the electrode through the osmium center occurs for the hSO molecules in which the Moco domain is sufficiently accessible. The observation of catalytic oxidation currents at low potentials is favorable for applications in bioelectronic devices.
The use of selected engineered galactose oxidase (GOase) variants for the oxidation of amino alcohols to aldehydes under mild conditions in aqueous systems is reported. GOase variant F-2 catalyses the regioselective oxidation of N-carbobenzyloxy (Cbz)-protected 3-amino-1,2-propanediol to the corresponding -hydroxyaldehyde which was then used in an aldolase reaction. Another variant, M3-5, was found to exhibit activity towards free and N-Cbz-protected aliphatic and aromatic amino alcohols allowing the synthesis of lactams such as 3,4-dihydronaphthalen-1(2H)-one, 2-pyrrolidone and valerolactam in one-pot tandem reactions with xanthine dehydrogenase (XDH) or aldehyde oxidase (PaoABC).
Mononuclear molybdoenzymes catalyze a broad range of redox reactions and are highly conserved in all kingdoms of life. This study addresses the question of how the Mo cofactor (Moco) is incorporated into the apo form of human sulfite oxidase (hSO) by using site-directed spin labeling to determine intramolecular distances in the nanometer range. Comparative measurements of the holo and apo forms of hSO enabled the localization of the corresponding structural changes, which are localized to a short loop (residues 263-273) of the Moco-containing domain. A flap-like movement of the loop provides access to the Moco binding-pocket in the apo form of the protein and explains the earlier studies on the in vitro reconstitution of apo-hSO with Moco. Remarkably, the loop motif can be found in a variety of structurally similar molybdoenzymes among various organisms, thus suggesting a common mechanism of Moco incorporation.
The bioelectrocatalytic sulfite oxidation by human sulfite oxidase (hSO) on indium tin oxide (ITO) is reported, which is facilitated by functionalizing of the electrode surface with polyethylenimine (PEI)-entrapped CdS nanoparticles and enzyme. hSO was assembled onto the electrode with a high surface loading of electroactive enzyme. In the presence of sulfite but without additional mediators, a high bioelectrocatalytic current was generated. Reference experiments with only PEI showed direct electron transfer and catalytic activity of hSO, but these were less pronounced. The application of the polyelectrolyte-entrapped quantum dots (QDs) on ITO electrodes provides a compatible surface for enzyme binding with promotion of electron transfer. Variations of the buffer solution conditions, e.g., ionic strength, pH, viscosity, and the effect of oxygen, were studied in order to understand intramolecular and heterogeneous electron transfer from hSO to the electrode. The results are consistent with a model derived for the enzyme by using flash photolysis in solution and spectroelectrochemistry and molecular dynamic simulations of hSO on monolayer-modified gold electrodes. Moreover, for the first time a photoelectrochemical electrode involving immobilized hSO is demonstrated where photoexcitation of the CdS/hSO-modified electrode lead to an enhanced generation of bioelectrocatalytic currents upon sulfite addition. Oxidation starts already at the redox potential of the electron transfer domain of hSO and is greatly increased by application of a small overpotential to the CdS/hSO-modified ITO.
Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-angstrom resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-angstrom resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.
Molybdenum and tungsten enzymes require specific chaperones for folding and cofactor insertion. PaoD is the chaperone of the periplasmic aldehyde oxidoreductase PaoABC. It is the last gene in the paoABCD operon in Escherichia coli and its presence is crucial for obtaining mature enzyme. PaoD is an unstable, 35 kDa, protein. Our biochemical studies showed that it is a dimer in solution with a tendency to form large aggregates, especially after freezing/thawing cycles. In order to improve stability, PaoD was thawed in the presence of two ionic liquids [C(4)mim]Cl and [C(2)OHmim]PF6 and no protein precipitation was observed. This allowed protein concentration and crystallization using polyethylene glycol or ammonium sulfate as precipitating agents. Saturation transfer difference - nuclear magnetic resonance (STD-NMR) experiments have also been performed in order to investigate the effect of the ionic liquids in the stabilization process, showing a clear interaction between the acidic ring protons of the cation and, most likely, negatively charged residues at the protein surface. DLS assays also show a reduction of the overall size of the protein aggregates in presence of ionic liquids. Furthermore, cofactor binding studies on PaoD showed that the protein is able to discriminate between molybdenum and tungsten bound to the molybdenum cofactor, since only a Mo-MPT form of the cofactor remained bound to PaoD.
The periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli is a molybdenum enzyme involved in detoxification of aldehydes in the cell. It is an example of an heterotrimeric enzyme of the xanthine oxidase family of enzymes which does not dimerize via its molybdenum cofactor binding domain. In order to structurally characterize PaoABC, X-ray crystallography and small angle X-ray scattering (SAXS) have been carried out. The protein crystallizes in the presence of 20% (w/v) polyethylene glycol 3350 using the hanging-drop vapour diffusion method. Although crystals were initially twinned, several experiments were done to overcome twinning and lowering the crystallization temperature (293 K to 277 K) was the solution to the problem. The non-twinned crystals used to solve the structure diffract X-rays to beyond 1.80 angstrom and belong to the C2 space group, with cell parameters a = 109.42 angstrom, b = 78.08 angstrom, c = 151.77 angstrom, = 99.77 degrees, and one molecule in the asymmetric unit. A molecular replacement solution was found for each subunit separately, using several proteins as search models. SAXS data of PaoABC were also collected showing that, in solution, the protein is also an heterotrimer.
Molybdoenzymes are complex enzymes in which the molybdenum cofactor (Moco) is deeply buried in the enzyme. Most molybdoenzymes contain a specific chaperone for the insertion of Moco. For the formate dehydrogenase FdsGBA from Rhodobacter capsulatus the two chaperones FdsC and FdsD were identified to be essential for enzyme activity, but are not a subunit of the mature enzyme. Here, we purified and characterized the FdsC protein after heterologous expression in Escherichia coli. We were able to copurify FdsC with the bound Moco derivate bis-molybdopterin guanine dinucleotide. This cofactor successfully was used as a source to reconstitute the activity of molybdoenzymes.
Structured summary of protein interactions:
FdsC and FdsC bind by molecular sieving (View interaction)
FdsD binds to RcMobA by surface plasmon resonance (View interaction)
FdsC binds to RcMobA by surface plasmon resonance (View interaction)
FdsC binds to FdsA by surface plasmon resonance (View interaction)
In the genome of Drosophila melanogaster, four genes coding for aldehyde oxidases (AOX1-4) were identified on chromosome 3. Phylogenetic analysis showed that the AOX gene cluster evolved via independent duplication events in the vertebrate and invertebrate lineages. The functional role and the substrate specificity of the distinct Drosophila AOX enzymes is unknown. Two loss-of-function mutant alleles in this gene region, low pyridoxal oxidase (Po-lpo) and aldehyde oxidase-1 (Aldox-1(n1)) are associated with a phenotype characterized by undetectable AOX enzymatic activity. However, the genes involved and the corresponding mutations have not yet been identified. In this study we characterized the activities, substrate specificities and expression profiles of the four AOX enzymes in D. melanogaster. We show that the Po-lpo-associated phenotype is the consequence of a structural alteration of the AOX1 gene. We identified an 11-bp deletion in the Po-lpo allele, resulting in a frame-shift event, which removes the molybdenum cofactor domain of the encoded enzyme. Furthermore, we show that AOX2 activity is detectable only during metamorphosis and characterize a Minos-AOX2 insertion in this developmental gene that disrupts its activity. We demonstrate that the Aldox-1(n1) phenotype maps to the AOX3 gene and AOX4 activity is not detectable in our assays.
Dendritic polyglycerol-poly(ethylene glycol)-based polymer networks for biosensing application
(2014)
This work describes the formation of a new dendritic polyglycerol-poly(ethylene glycol)-based 3D polymer network as a matrix for immobilization of the redox enzyme periplasmatic aldehyde oxidoreductase to create an electrochemical biosensor. The novel network is built directly on the gold surface, where it simultaneously stabilizes the enzyme for up to 4 days. The prepared biosensors can be used for amperometric detection of benzaldehyde in the range of 0.8-400 mu M.
Site-directed spin labeling of the unnatural amino acid p-acetylphenylalanine (p-AcPhe) using oxime based coupling chemistry is successfully applied to investigate human sulfite oxidase (hSO), a protein containing an essential cysteine residue, which impedes the use of thiol based coupling chemistry. The protein was found to be sensitive toward typical reaction conditions of oxime coupling, namely, acidic reaction conditions and elevated temperatures. Thus, coupling at neutral pH and room temperature is mandatory. Three catalysts described in the literature to accelerate the reaction rate have been tested. Best spin labeling efficiencies were observed for p-methoxyaniline, while the other catalysts described in the literature to have even better performance for oxime coupling at neutral pH were substantially less active or led to precipitation of the protein. A clear correlation of spin labeling efficiency with the local environment of the residue is found, shedding some light on the importance of the sterically demanding reaction complex between p-AcPhe, the aniline catalyst, and the spin label for the reaction rate. The analysis of the line shape has shown that its interpretation in terms of local environment is more challenging as compared to the well-established spin labels based on cysteine chemistry. To this end the results presented here indicate that the larger steric demand of the spin labeled p-AcPhe can induce structural effects instead of reporting on them.
Catalytic bio-chemo and bio-bio tandem oxidation reactions for amide and carboxylic acid synthesis
(2014)
A catalytic toolbox for three different water-based one-pot cascades to convert aryl alcohols to amides and acids and cyclic amines to lactams, involving combination of oxidative enzymes (monoamine oxidase, xanthine dehydrogenase, galactose oxidase and laccase) and chemical oxidants (TBHP or Cul(cat)/H2O2) at mild temperatures, is presented. Mutually compatible conditions were found to afford products in good to excellent yields.
Background: Kinetic characterization of wild-type xanthine dehydrogenase and variants. Results: Comparison of the pH dependence of both k(red) and k(red)/K-d, as well as k(cat) and k(cat)/K-m. Conclusion: Ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of xanthine. Significance: Examining the contributions of Glu(232) to catalysis is essential for understanding the mechanism of xanthine dehydrogenase.
The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu(232) in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that k(red), the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu(232) being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both k(red) and k(red)/K-d from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pK(a) of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme.
Background: Localization and identification of interaction partners of two splice variants of the human 3-mercaptopyruvate sulfurtransferase TUM1. Results: We show that TUM1 interacts with proteins involved in Moco and FeS cluster biosynthesis. Conclusion: Human TUM1 is a dual localized protein in the cytosol and mitochondria with distinct roles in sulfur transfer and interaction partners. Significance: The study contributes to the sulfur transfer pathway for the biosynthesis of sulfur-containing biofactors.
The human tRNA thiouridine modification protein (TUM1), also designated as 3-mercaptopyruvate sulfurtransferase (MPST), has been implicated in a wide range of physiological processes in the cell. The roles range from an involvement in thiolation of cytosolic tRNAs to the generation of H2S as signaling molecule both in mitochondria and the cytosol. TUM1 is a member of the sulfurtransferase family and catalyzes the conversion of 3-mercaptopyruvate to pyruvate and protein-bound persulfide. Here, we purified and characterized two novel TUM1 splice variants, designated as TUM1-Iso1 and TUM1-Iso2. The purified proteins showed similar kinetic behavior and comparable pH and temperature dependence. Cellular localization studies, however, showed a different localization pattern between the isoforms. TUM1-Iso1 is exclusively localized in the cytosol, whereas TUM1-Iso2 showed a dual localization both in the cytosol and mitochondria. Interaction studies were performed with the isoforms both in vitro using the purified proteins and in vivo by fluorescence analysis in human cells, using the split-EGFP system. The studies showed that TUM1 interacts with the l-cysteine desulfurase NFS1 and the rhodanese-like protein MOCS3, suggesting a dual function of TUM1 both in sulfur transfer for the biosynthesis of the molybdenum cofactor, and for the thiolation of tRNA. Our studies point to distinct roles of each TUM1 isoform in the sulfur transfer processes in the cell, with different compartmentalization of the two splice variants of TUM1.
The molybdenum cofactor is modified by the addition of GMP or CMP to the C4' phosphate of molybdopterin forming the molybdopterin guanine dinucleotide or molybdopterin cytosine dinucleotide cofactor, respectively. The two reactions are catalyzed by specific enzymes as follows: the GTP: molybdopterin guanylyltransferase MobA and the CTP: molybdopterin cytidylyltransferase MocA. Both enzymes show 22% amino acid sequence identity and are specific for their respective nucleotides. Crystal structure analysis of MobA revealed two conserved motifs in the N-terminal domain of the protein involved in binding of the guanine base. Based on these motifs, we performed site-directed mutagenesis studies to exchange the amino acids to the sequence found in the paralogue MocA. Using a fully defined in vitro system, we showed that the exchange of five amino acids was enough to obtain activity with both GTP and CTP in either MocA or MobA. Exchange of the complete N-terminal domain of each protein resulted in the total inversion of nucleotide specificity activity, showing that the N-terminal domain determines nucleotide recognition and binding. Analysis of protein-protein interactions showed that the C-terminal domain of either MocA or MobA determines the specific binding to the respective acceptor protein.
The enzyme xanthine dehydrogenase (XDH) from the purple photosynthetic bacterium Rhodobacter capsulatus catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid as part of purine metabolism. The native electron acceptor is NAD(+) but herein we show that uric acid in its 2-electron oxidized form is able to act as an artificial electron acceptor from XDH in an electrochemically driven catalytic system. Hypoxanthine oxidation is also observed with the novel production of uric acid in a series of two consecutive 2-electron oxidation reactions via xanthine. XDH exhibits native activity in terms of its pH optimum and inhibition by allopurinol.
Aldehyde oxidase (AOX) is characterized by a broad substrate specificity, oxidizing aromatic azaheterocycles, such as N(1)-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. In the past decade, AOX has been recognized increasingly to play an important role in the metabolism of drugs through its complex cofactor content, tissue distribution, and substrate recognition. In humans, only one AOX gene (AOX1) is present, but in mouse and other mammals different AOX homologs were identified. The multiple AOX isoforms are expressed tissue-specifically in different organisms, and it is believed that they recognize distinct substrates and carry out different physiological tasks. AOX is a dimer with a molecular mass of approximately 300 kDa, and each subunit of the homodimeric enzyme contains four different cofactors: the molybdenum cofactor, two distinct [2Fe-2S] clusters, and one FAD. We purified the AOX homolog from mouse liver (mAOX3) and established a system for the heterologous expression of mAOX3 in Escherichia coli. The purified enzymes were compared. Both proteins show the same characteristics and catalytic properties, with the difference that the recombinant protein was expressed and purified in a 30% active form, whereas the native protein is 100% active. Spectroscopic characterization showed that FeSII is not assembled completely in mAOX3. In addition, both proteins were crystallized. The best crystals were from native mAOX3 and diffracted beyond 2.9 angstrom. The crystals belong to space group P1, and two dimers are present in the unit cell.
Ten square-based pyramidal molybdenum complexes with different sulfur donor ligands, that is, a variety of dithiolenes and sulfides, were prepared, which mimic coordination motifs of the molybdenum cofactors of molybdenum-dependent oxidoreductases. The model compounds were investigated by Mo K-edge X-ray absorption spectroscopy (XAS) and (with one exception) their molecular structures were analyzed by X-ray diffraction to derive detailed information on bond lengths and geometries of the first coordination shell of molybdenum. Only small variations in Mo=O and Mo-S bond lengths and their respective coordination angles were observed for all complexes including those containing Mo(CO)(2) or Mo(mu-S)(2)Mo motifs. XAS analysis (edge energy) revealed higher relative oxidation levels in the molybdenum ion in compounds with innocent sulfur-based ligands relative to those in dithiolene complexes, which are known to exhibit noninnocence, that is, donation of substantial electron density from ligand to metal. In addition, longer average Mo-S and Mo=O bonds and consequently lower.(Mo=O) stretching frequencies in the IR spectra were observed for complexes with dithiolene-derived ligands. The results emphasize that the noninnocent character of the dithiolene ligand influences the electronic structure of the model compounds, but does not significantly affect their metal coordination geometry, which is largely determined by the Mo(IV) or (V) ion itself. The latter conclusion also holds for the molybdenum site geometries in the oxidized Mo-VI cofactor of DMSO reductase and the reduced Mo-IV cofactor of arsenite oxidase. The innocent behavior of the dithiolene molybdopterin ligands observed in the enzymes is likely to be related to cofactor-protein interactions.
Background: In Moco biosynthesis, sulfur is transferred from L-cysteine to MPT synthase, catalyzing the conversion of cPMP to MPT.
Results: The rhodanese-like protein YnjE is a novel protein involved in Moco biosynthesis.
Conclusion: YnjE enhances the rate of conversion of cPMP to MPT and interacts with MoeB and IscS. S
ignificance: To understand the mechanism of sulfur transfer and the role of rhodaneses in the cell.
The pathway of molybdenum cofactor biosynthesis has been studied in detail by using proteins from Mycobacterium species, which contain several homologs associated with the first steps of Moco biosynthesis. While all Mycobacteria species contain a MoeZR, only some strains have acquired an additional homolog, MoeBR, by horizontal gene transfer. The role of MoeBR and MoeZR was studied in detail for the interaction with the two MoaD-homologs involved in Moco biosynthesis, MoaD1 and MoaD2, in addition to the CysO protein involved in cysteine biosynthesis. We show that both proteins have a role in Moco biosynthesis, while only MoeZR, but not MoeBR, has an additional role in cysteine biosynthesis. MoeZR and MoeBR were able to complement an E. coli moeB mutant strain, but only in conjunction with the Mycobacterial MoaD1 or MoaD2 proteins. Both proteins were able to sulfurate MoaD1 and MoaD2 in vivo, while only MoeZR additionally transferred the sulfur to CysO. Our in vivo studies show that Mycobacteria have acquired several homologs to maintain Moco biosynthesis. MoeZR has a dual role in Moco- and cysteine biosynthesis and is involved in the sulfuration of MoaD and CysO, whereas MoeBR only has a role in Moco biosynthesis, which is not an essential function for Mycobacteria.
The TorD family of specific chaperones is divided into four subfamilies dedicated to molybdoenzyme biogenesis and a fifth one, exemplified by YcdY of Escherichia coli, for which no defined partner has been identified so far. We propose that YcdY is the chaperone of YcdX, a zinc protein involved in the swarming motility process of E. coli, since YcdY interacts with YcdX and increases its activity in vitro.
We use substituted polyanilines for the construction of new polymer electrodes for interaction studies with the redox protein cytochrome c (cyt c) and the enzyme sulfite oxidase (SO). For these purposes four different polyaniline copolymers are chemically synthesized. Three of them are copolymers, containing 2-methoxyaniline-5-sulfonic acid with variable ratios of aniline; the fourth copolymer consists of 3-amino-benzoic acid and aniline. The results show that all polymers are suitable for being immobilized as thin stable films on gold wire and indium tin oxide (ITO) electrode surfaces from DMSO solution. This can be demonstrated by cyclic voltammetry and UV-Vis spectroscopy measurements. Moreover, cyt c can be electrochemically detected not only in solution, but also immobilized on top of the polymer films. Furthermore, the appearance of a significant catalytic current has been demonstrated for the sulfonated polyanilines, when the polymer-coated protein electrode is being measured upon addition of sulfite oxidase, confirming the establishment of a bioanalytical signal chain. Best results have been obtained for the polymer with highest sulfonation grade. The redox switching of the polymer by the enzymatic reaction can also be analyzed by following the spectral properties of the polymer electrode.
Aldehyde oxidase (AO) is a complex molybdo-flavoprotein that belongs to the xanthine oxidase family. AO is active as a homodimer, and each 150-kDa monomer binds two distinct [2Fe2S] clusters, FAD, and the molybdenum cofactor. AO has an important role in the metabolism of drugs based on its broad substrate specificity oxidizing aromatic aza-heterocycles, for example, N-1-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. Sequencing the 35 coding exons of the human AOX1 gene in a sample of 180 Italian individuals led to the identification of relatively frequent, synonymous, missense and nonsense single-nucleotide polymorphisms (SNPs). Human aldehyde oxidase (hAOX1) was purified after heterologous expression in Escherichia coli. The recombinant protein was obtained with a purity of 95% and a yield of 50 mu g/l E. coli culture. Site-directed mutagenesis of the hAOX1 cDNA allowed the purification of protein variants bearing the amino acid changes R802C, R921H, N1135S, and H1297R, which correspond to some of the identified SNPs. The hAOX1 variants were purified and compared with the wild-type protein relative to activity, oligomerization state, and metal content. Our data show that the mutation of each amino acid residue has a variable impact on the ability of hAOX1 to metabolize selected substrates. Thus, the human population is characterized by the presence of functionally inactive hAOX1 allelic variants as well as variants encoding enzymes with different catalytic activities. Our results indicate that the presence of these allelic variants should be considered for the design of future drugs.
We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are beta-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus.
Silver nanoparticles with identical plasmonic properties but different surface functionalities are synthesized and tested as chemically selective surface-enhanced resonance Raman (SERR) amplifiers in a two-component protein solution. The surface plasmon resonances of the particles are tuned to 413 nm to match the molecular resonance of protein heme cofactors. Biocompatible functionalization of the nanoparticles with a thin film of chitosan yields selective SERR enhancement of the anionic protein cytochrome b(5), whereas functionalization with SiO2 amplifies only the spectra of the cationic protein cytochrome c. As a result, subsequent addition of the two differently functionalized particles yields complementary information on the same mixed protein sample solution. Finally, the applicability of chitosan-coated Ag nanoparticles for protein separation was tested by in situ resonance Raman spectroscopy.
We report the mediated electrocatalytic voltammetry of the molybdoenzyme xanthine dehydrogenase (XDH) from Rhodobacter capsulatus at a thiol-modified Au electrode. The 2-electron acceptor N-methylphenazinium methanesulfonate (phenazine methosulfate, PMS) is an effective artificial electron transfer partner for XDH instead of its native electron acceptor NAD(+). XDH catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and xanthine to uric acid. Cyclic voltammetry was used to generate the active (oxidized) form of the mediator. Simulation of the catalytic voltammetry across a broad range of substrate and PMS concentrations at different sweep rates was achieved with the program DigiSim to yield a set of consistent rate and equilibrium constants that describe the catalytic system. This provides the first example of the mediated electrochemistry of a xanthine dehydrogenase (or oxidase) that is uncomplicated by interference from product oxidation. A remarkable two-step, sequential oxidation of hypoxanthine to uric acid via xanthine by XDH is observed.
To identify copper homeostasis genes in Rhodobacter capsulatus, we performed random transposon Tn5 mutagenesis. Screening of more than 10,000 Tn5 mutants identified tellurite resistance gene trgB as a so far unrecognized major copper tolerance determinant. The trgB gene is flanked by tellurite resistance gene trgA and cysteine synthase gene cysK2. While growth of trgA mutants was only moderately restricted by tellurite, trgB and cysK2 mutants were severely affected by tellurite, which implies that viability under tellurite stress requires increased cysteine levels. Mutational analyses revealed that trgB was the only gene in this chromosomal region conferring cross-tolerance towards copper. Expression of the monocistronic trgB gene required promoter elements overlapping the trgA coding region as shown by nested deletions. Neither copper nor tellurite affected trgB transcription as demonstrated by reverse transcriptase PCR and trgB-lacZ fusions. Addition of tellurite or copper gave rise to increased cellular tellurium and copper concentrations, respectively, as determined by inductively coupled plasma-optical emission spectroscopy. By contrast, cellular iron concentrations remained fairly constant irrespective of tellurite or copper addition. This is the first study demonstrating a direct link between copper and tellurite response in bacteria.
The present study reports a facile approach for sulfite biosensing, based on enhanced direct electron transfer of a human sulfite oxidase (hSO) immobilized on a gold nanoparticles modified electrode. The spherical core shell AuNPs were prepared via a new method by reduction of HAuCl4 with branched poly(ethyleneimine) in an ionic liquids resulting particles with a diameter less than 10 nm. These nanoparticles were covalently attached to a mercaptoundecanoic acid modified Au-electrode where then hSO was adsorbed and an enhanced interfacial electron transfer and electrocatalysis was achieved. UV/Vis and resonance Raman spectroscopy, in combination with direct protein voltammetry, are employed for the characterization of the system and reveal no perturbation of the structural integrity of the redox protein. The proposed biosensor exhibited a quick steady-state current response, within 2 s, a linear detection range between 0.5 and 5.4 mu M with a high sensitivity (1.85 nA mu M-1). The investigated system provides remarkable advantages in the possibility to work at low applied potential and at very high ionic strength. Therefore these properties could make the proposed system useful in the development of bioelectronic devices and its application in real samples.
Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 angstrom. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity.
A Biosensor for aromatic aldehydes comprising the mediator dependent PaoABC-Aldehyde oxidoreductase
(2013)
A novel aldehyde oxidoreductase (PaoABC) from Escherichia coli was utilized for the development of an oxygen insensitive biosensor for benzaldehyde. The enzyme was immobilized in polyvinyl alcohol and currents were measured for aldehyde oxidation with different one and two electron mediators with the highest sensitivity for benzaldehyde in the presence of hexacyanoferrate(III). The benzaldehyde biosensor was optimized with respect to mediator concentration, enzyme loading and pH using potassium hexacyanoferrate(III). The linear measuring range is between 0.5200 mu M benzaldehyde. In correspondence with the substrate selectivity of the enzyme in solution the biosensor revealed a preference for aromatic aldehydes and less effective conversion of aliphatic aldehydes. The biosensor is oxygen independent, which is a particularly attractive feature for application. The biosensor can be applied to detect contaminations with benzaldehyde in solvents such as benzyl alcohol, where traces of benzaldehyde in benzyl alcohol down to 0.0042?% can be detected.
The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a Delta tusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the Delta tusA mutant. Characterization of the Delta tusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes.
In humans, the L-cysteine desulfurase NFS1 plays a crucial role in the mitochondrial iron-sulfur cluster biosynthesis and in the thiomodification of mitochondrial and cytosolic tRNAs. We have previously demonstrated that purified NFS1 is able to transfer sulfur to the C-terminal domain of MOCS3, a cytosolic protein involved in molybdenum cofactor biosynthesis and tRNA thiolation. However, no direct evidence existed so far for the interaction of NFS1 and MOCS3 in the cytosol of human cells. Here, we present direct data to show the interaction of NFS1 and MOCS3 in the cytosol of human cells using Forster resonance energy transfer and a split-EGFP system. The colocalization of NFS1 and MOCS3 in the cytosol was confirmed by immunodetection of fractionated cells and localization studies using confocal fluorescence microscopy. Purified NFS1 was used to reconstitute the lacking molybdoenzyme activity of the Neurospora crassa nit-1 mutant, giving additional evidence that NFS1 is the sulfur donor for Moco biosynthesis in eukaryotes in general.
Ag-TiO2 and Au-TiO2 hybrid electrodes were designed by covalent attachment of TiO2 nanoparticles to Ag or Au electrodes via an organic linker. The optical and electronic properties of these systems were investigated using the cytochrome b(5) (Cyt b(5)) domain of sulfite oxidase, exclusively attached to the TiO2 surface, as a Raman marker and model redox enzyme. Very strong SERR signals of Cyt b(5) were obtained for Ag-supported systems due to plasmonic field enhancement of Ag. Time-resolved surface-enhanced resonance Raman spectroscopic measurements yielded a remarkably fast electron transfer kinetic (k = 60 s(-1)) of Cyt b(5) to Ag. A much lower Raman intensity was observed for Au-supported systems with undefined and slow redox behavior. We explain this phenomenon on the basis of the different potential of zero charge of the two metals that largely influence the electronic properties of the TiO2 island film.
The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo-S and Mo-O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme.
Ironsulfur clusters (ISCs) are important prosthetic groups that define the functions of many proteins. Proteins with ISCs (called ironsulfur or FeS proteins) are present in mitochondria, the cytosol, the endoplasmic reticulum and the nucleus. They participate in various biological pathways including oxidative phosphorylation (OXPHOS), the citric acid cycle, iron homeostasis, heme biosynthesis and DNA repair. Here, we report a homozygous mutation in LYRM4 in two patients with combined OXPHOS deficiency. LYRM4 encodes the ISD11 protein, which forms a complex with, and stabilizes, the sulfur donor NFS1. The homozygous mutation (c.203GT, p.R68L) was identified via massively parallel sequencing of 1000 mitochondrial genes (MitoExome sequencing) in a patient with deficiency of complexes I, II and III in muscle and liver. These three complexes contain ISCs. Sanger sequencing identified the same mutation in his similarly affected cousin, who had a more severe phenotype and died while a neonate. Complex IV was also deficient in her skeletal muscle. Several other FeS proteins were also affected in both patients, including the aconitases and ferrochelatase. Mutant ISD11 only partially complemented for an ISD11 deletion in yeast. Our in vitro studies showed that the l-cysteine desulfurase activity of NFS1 was barely present when co-expressed with mutant ISD11. Our findings are consistent with a defect in the early step of ISC assembly affecting a broad variety of FeS proteins. The differences in biochemical and clinical features between the two patients may relate to limited availability of cysteine in the newborn period and suggest a potential approach to therapy.
Sulfite oxidase (SO) is an essential molybdoenzyme for humans, catalyzing the final step in the degradation of sulfur-containing amino acids and lipids, which is the oxidation of sulfite to sulfate. The catalytic site of SO consists of a molybdenum ion bound to the dithiolene sulfurs of one molybdopterin (MPT) molecule, carrying two oxygen ligands, and is further coordinated by the thiol sulfur of a conserved cysteine residue. We have exchanged four non-active site cysteines in the molybdenum cofactor (Moco) binding domain of human SO (SOMD) with serine using site-directed mutagenesis. This facilitated the specific replacement of the active site Cys207 with selenocysteine during protein expression in Escherichia coli. The sulfite oxidizing activity (k(cat)/K-M) of SeSOMD4Ser was increased at least 1.5-fold, and the pH optimum was shifted to a more acidic value compared to those of SOMD4Ser and SOMD4Cys(wt) X-ray absorption spectroscopy revealed a Mow Se bond length of 2.51 A, likely caused by the specific binding of Sec207 to the molybdenum, and otherwise rather similar square-pyramidal S/Se(Cys)(O2MoS2)-S-VI(MPT) site structures in the three constructs. The low-pH form of the Mo(V) electron paramagnetic resonance (EPR) signal of SeSOM4Ser was altered compared to those of SOMD4Ser and SOMD4cy,(,), with g, in particular shifted to a lower magnetic field, due to the Se ligation at the molybdenum. In contrast, the Mo(V) EPR signal of the high-pH form was unchanged. The substantially stronger effect of substituting selenocysteine for cysteine at low pH as compared to high pH is most likely due to the decreased covalency of the Mo Se bond.
The formate dehydrogenase from Rhodobactercapsulatus (RcFDH) is an oxygen-tolerant protein with an ()(2) subunit composition that is localized in the cytoplasm. It belongs to the group of metal and NAD(+)-dependent FDHs with the coordination of a molybdenum cofactor, four [Fe4S4] clusters and one [Fe2S2] cluster associated with the -subunit, one [Fe4S4] cluster and one FMN bound to the -subunit, and one [Fe2S2] cluster bound to the -subunit. RcFDH was heterologously expressed in Escherichiacoli and characterized. Cofactor analysis showed that the bis-molybdopterin guanine dinucleotide cofactor is bound to the FdsA subunit containing a cysteine ligand at the active site. A turnover rate of 2189min(-1) with formate as substrate was determined. The back reaction for the reduction of CO2 was catalyzed with a k(cat) of 89min(-1). The preference for formate oxidation shows an energy barrier for CO2 reduction of the enzyme. Furthermore, the FMN-containing and [Fe4S4]-containing -subunit together with the [Fe2S2]-containing -subunit forms a diaphorase unit with activities for both NAD(+) reduction and NADH oxidation. In addition to the structural genes fdsG, fdsB, and fdsA, the fds operon in R.capsulatus contains the fdsC and fdsD genes. Expression studies showed that RcFDH is only active when both FdsC and FdsD are present. Both proteins are proposed to be involved in bis-molybdopterin guanine dinucleotide modification and insertion into RcFDH.
An unusual behavior of the periplasmic aldehyde oxidoreductase (PaoABC) from Escherichia coil has been observed from electrochemical investigations of the enzyme catalyzed oxidation of aromatic aldehydes with different mediators under different conditions of ionic strength. The enzyme has similarity to other molybdoenzymes of the xanthine oxidase family, but the catalytic behavior turned out to be very different. Under steady state conditions the turnover of PaoABC is maximal at pH 4 for the negatively charged ferricyanide and at pH 9 for a positively charged osmium complex. Stopped-flow kinetic measurements of the catalytic half reaction showed that oxidation of benzaldehyde proceeds also above pH 7. Thus, benzaldehyde oxidation can proceed under acidic and basic conditions using this enzyme, a property which has not been described before for molybdenum hydroxylases. It is also suggested that the electron transfer with artificial electron acceptors and PaoABC can proceed at different protein sites and depends on the nature of the electron acceptor in addition to the ionic strength. (C) 2013 Elsevier B.V. All rights reserved.
In order to elucidate factors that determine substrate specificity and activity of mammalian molybdo-flavoproteins we performed site directed mutagenesis of mouse aldehyde oxidase 3 (mAOX3). The sequence alignment of different aldehyde oxidase (AOX) isoforms identified variations in the active site of mAOX3 in comparison to other AOX proteins and xanthine oxidoreductases (XOR). Based on the structural alignment of mAOX3 and bovine XOR, differences in amino acid residues involved in substrate binding in XORs in comparison to AOXs were identified. We exchanged several residues in the active site to the ones found in other AOX homologues in mouse or to residues present in bovine XOR in order to examine their influence on substrate selectivity and catalytic activity. Additionally we analyzed the influence of the [2Fe-2S] domains of mAOX3 on its kinetic properties and cofactor saturation. We applied UV-VIS and EPR monitored redox-titrations to determine the redox potentials of wild type mAOX3 and mAOX3 variants containing the iron-sulfur centers of mAOX1. In addition, a combination of molecular docking and molecular dynamic simulations (MD) was used to investigate factors that modulate the substrate specificity and activity of wild type and AOX variants. The successful conversion of an AOX enzyme to an XOR enzyme was achieved exchanging eight residues in the active site of mAOX3. It was observed that the absence of the K889H exchange substantially decreased the activity of the enzyme towards all substrates analyzed, revealing that this residue has an important role in catalysis.
A Rhodobacter capsulatus member of a universal permease family imports molybdate and other oxyanions
(2010)
Molybdenum (Mo) is an important trace element that is toxic at high concentrations. To resolve the mechanisms underlying Mo toxicity, Rhodobacter capsulatus mutants tolerant to high Mo concentrations were isolated by random transposon Tn5 mutagenesis. The insertion sites of six independent isolates mapped within the same gene predicted to code for a permease of unknown function located in the cytoplasmic membrane. During growth under Mo-replete conditions, the wild-type strain accumulated considerably more Mo than the permease mutant. For mutants defective for the permease, the high-affinity molybdate importer ModABC, or both transporters, in vivo Mo-dependent nitrogenase (Mo-nitrogenase) activities at different Mo concentrations suggested that ModABC and the permease import molybdate in nanomolar and micromolar ranges, respectively. Like the permease mutants, a mutant defective for ATP sulfurylase tolerated high Mo concentrations, suggesting that ATP sulfurylase is the main target of Mo inhibition in R. capsulatus. Sulfate-dependent growth of a double mutant defective for the permease and the high-affinity sulfate importer CysTWA was reduced compared to those of the single mutants, implying that the permease plays an important role in sulfate uptake. In addition, permease mutants tolerated higher tungstate and vanadate concentrations than the wild type, suggesting that the permease acts as a general oxyanion importer. We propose to call this permease PerO (for oxyanion permease). It is the first reported bacterial molybdate transporter outside the ABC transporter family.
The phototrophic purple bacterium Rhodobacter capsulatus encodes two transcriptional regulators, MopA and MopB, with partially overlapping and specific functions in molybdate-dependent gene regulation. Both MopA and MopB consist of an N-terminal DNA-binding helix-turn-helix domain and a C-terminal molybdate-binding di-MOP domain. They formed homodimers as apo-proteins and in the molybdate-bound state as shown by yeast two-hybrid (Y2H) studies, glutaraldehyde cross-linking, gel filtration chromatography, and copurification experiments. Y2H studies suggested that both the DNA- binding and the molybdate-binding domains contribute to dimer formation. Analysis of molybdate binding to MopA and MopB revealed a binding stoichiometry of four molybdate oxyanions per homodimer. Specific interaction partners of MopA and MopB were the molybdate transporter ATPase ModC and the molbindin-like Mop protein, respectively. Like other molbindins, the R. capsulatus Mop protein formed hexamers, which were stabilized by binding of six molybdate oxyanions per hexamer. Heteromer formation of MopA and MopB was shown by Y2H studies and copurification experiments. Reporter gene activity of a strictly MopA-dependent mop-lacZ fusion in mutant strains defective for either mopA, mopB, or both suggested that MopB negatively modulates expression of the mop promoter. We propose that depletion of the active MopA homodimer pool by formation of MopA-MopB heteromers might represent a fine-tuning mechanism controlling mop gene expression.
An efficient electrocatalytic biosensor for sulfite detection was developed by co-immobilizing sulfite oxidase and cytochrome c with polyaniline sulfonic acid in a layer-by-layer assembly. QCM, UV-Vis spectroscopy and cyclic voltammetry revealed increasing loading of electrochemically active protein with the formation of multilayers. The sensor operates reagentless at low working potential. A catalytic oxidation current was detected in the presence of sulfite at the modified gold electrode, polarized at +0.1 V ( vs. Ag/AgCl 1 M KCl). The stability of the biosensor performance was characterized and optimized. A 17-bilayer electrode has a linear range between 1 and 60 mu M sulfite with a sensitivity of 2.19 mA M-1 sulfite and a response time of 2 min. The electrode retained a stable response for 3 days with a serial reproducibility of 3.8% and lost 20% of sensitivity after 5 days of operation. It is possible to store the sensor in a dry state for more than 2 months. The multilayer electrode was used for determination of sulfite in unspiked and spiked samples of red and white wine. The recovery and the specificity of the signals were evaluated for each sample.
We have purified and characterized a specific CTP: molybdopterin cytidylyltransferase for the biosynthesis of the molybdopterin (MPT) cytosine dinucleotide (MCD) cofactor in Escherichia coli. The protein, named MocA, shows 22% amino acid sequence identity to E. coli MobA, the specific GTP: molybdopterin guanylyltransferase for molybdopterin guanine dinucleotide biosynthesis. MocA is essential for the activity of the MCD-containing enzymes aldehyde oxidoreductase Yag-TSR and the xanthine dehydrogenases XdhABC and XdhD. Using a fully defined in vitro assay, we showed that MocA, Mo-MPT, CTP, and MgCl2 are required and sufficient for MCD biosynthesis in vitro. The activity of MocA is specific for CTP; other nucleotides such as ATP and GTP were not utilized. In the defined in vitro system a turnover number of 0.37 +/- 0.01 min(-1) was obtained. A1:1 binding ratio of MocA to Mo-MPT and CTP was determined to monomeric MocA with dissociation constants of 0.23 +/- 0.02 mu M for CTP and 1.17 +/- 0.18 mu M for Mo-MPT. We showed that MocA was also able to convert MPT to MCD in the absence of molybdate, however, with only one catalytic turnover. The addition of molybdate after one turnover gave rise to a higher MCD production, revealing that MCD remains bound to MocA in the absence of molybdate. This work presents the first characterization of a specific enzyme involved in MCD biosynthesis in bacteria.
Three DNA regions carrying genes encoding putative homologs of xanthine dehydrogenases were identified in Escherichia coli, named xdhABC, xdhD, and yagTSRQ. Here, we describe the purification and characterization of gene products of the yagTSRQ operon, a molybdenum-containing iron-sulfur flavoprotein from E. coli, which is located in the periplasm. The 135 kDa enzyme comprised a noncovalent (alpha beta gamma) heterotrimer with a large (78.1 kDa) molybdenum cofactor (Moco)-containing YagR subunit, a medium (33.9 kDa) FAD-containing YagS subunit, and a small (21.0 kDa) 2 x [2Fe2S]-containing YagT subunit. YagQ is not a subunit of the mature enzyme, and the protein is expected to be involved in Moco modification and insertion into YagTSR. Analysis of the form of Moco present in YagTSR revealed the presence of the molybdopterin cytosine dinucleotide cofactor. Two different [2Fe2S] clusters, typical for this class of enzyme, were identified by EPR. YagTSR represents the first example of a molybdopterin cytosine dinucleotide-containing protein in E. coli. Kinetic characterization of the enzyme revealed that YagTSR converts a broad spectrum of aldehydes, with a preference for aromatic aldehydes. Ferredoxin instead of NAD(+) or molecular oxygen was used as terminal electron acceptor. Complete growth inhibition of E. coli cells devoid of genes from the yagTSRQ operon was observed by the addition of cinnamaldehyde to a low-pH medium. This finding shows that YagTSR might have a role in the detoxification of aromatic aldehydes for E. coli under certain growth conditions.
Crystal structure of YnjE from Escherichia coli, a sulfurtransferase with three rhodanese domains
(2009)
Rhodaneses/sulfurtransferases are ubiquitous enzymes that catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor via an active site cysteine that is modified to a persulfide during the reaction. Here, we present the first crystal structure of a triple-domain rhodanese-like protein, namely YnjE from Escherichia coli, in two states where its active site cysteine is either unmodified or present as a persulfide. Compared to well- characterized tandem domain rhodaneses, which are composed of one inactive and one active domain, YnjE contains an extra N-terminal inactive rhodanese-like domain. Phylogenetic analysis reveals that YnjE triple-domain homologs can be found in a variety of other gamma-proteobacteria, in addition, some single-, tandem-, four and even six-domain variants exist. All YnjE rhodaneses are characterized by a highly conserved active site loop (CGTGWR) and evolved independently from other rhodaneses, thus forming their own subfamily. On the basis of structural comparisons with other rhodaneses and kinetic studies, YnjE, which is more similar to thiosulfate:cyanide sulfurtransferases than to 3- mercaptopyruvate:cyanide sulfurtransferases, has a different substrate specificity that depends not only on the composition of the active site loop with the catalytic cysteine at the first position but also on the surrounding residues. In vitro YnjE can be efficiently persulfurated by the cysteine desulfurase IscS. The catalytic site is located within an elongated cleft, formed by the central and C-terminal domain and is lined by bulky hydrophobic residues with the catalytic active cysteine largely shielded from the solvent.
Mechanism of substrate and inhibitor binding of Rhodobacter capsulatus xanthine dehydrogenase
(2009)
Rhodobacter capsulatus xanthine dehydrogenase (XDH) is an (alpha beta)(2) heterotetrameric cytoplasmic enzyme that resembles eukaryotic xanthine oxidoreductases in respect to both amino acid sequence and structural fold. To obtain a detailed understanding of the mechanism of substrate and inhibitor binding at the active site, we solved crystal structures of R. capsulatus XDH in the presence of its substrates hypoxanthine, xanthine, and the inhibitor pterin-6- aldehyde using either the inactive desulfo form of the enzyme or an active site mutant (E(B)232Q) to prevent substrate turnover. The hypoxanthine-and xanthine-bound structures reveal the orientation of both substrates at the active site and show the importance of residue GluB-232 for substrate positioning. The oxygen atom at the C-6 position of both substrates is oriented toward Arg(B)-310 in the active site. Thus the substrates bind in an orientation opposite to the one seen in the structure of the reduced enzyme with the inhibitor oxypurinol. The tightness of the substrates in the active site suggests that the intermediate products must exit the binding pocket to allow first the attack of the C-2, followed by oxidation of the C-8 atom to form the final product uric acid. Structural studies of pterin-6-aldehyde, a potent inhibitor of R. capsulatus XDH, contribute further to the understanding of the relative positioning of inhibitors and substrates in the binding pocket. Steady state kinetics reveal a competitive inhibition pattern with a K-i of 103.57 +/- 18.96 nM for pterin-6-aldehyde.
A biosensor, based on a redoxactive osmium polymer and sulfite oxidase on screen-printed electrodes, is presented here as a promising method for the detection of sulfite. A catalytic oxidative current was generated when a sample containing sulfite was pumped over the carbon screen-printed electrode modified with osmium redox polymer wired sulfite oxidase. A stationary value was reached after approximately 50 s and a complete measurement lasted no more than 3 min. The electrode polarized at -0.1 V (vs. Ag vertical bar AgCl 1M KCl) permits minimizing the influence of interfering substances, since these compounds can be unspecific oxidized at higher potentials. Because of the good stability of the protein film on the electrode surface, a well functioning biosensor-flow system was possible to construct. The working stability and reproducibility were further enhanced by the addition of bovine serum albumin generating a more long-term stable and biocompatible protein environment. The optimized biosensor showed a stable signal for more than a week of operation and a coefficient of variation of 4.8% for 12 successive measurements. The lower limit of detection of the sensor was 0.5 mu M sulfite and the response was linear until 100 mu M. The high sensitivity permitted a 1:500 dilution of wine samples. The immobilization procedure and the operational conditions granted minimized interferences. Additionally, repeating the immobilization procedure to form several layers of wired SO further increased the sensitivity of such a sensor. Finally. the applicability of the developed sulfite biosensor was tested on real samples, such as white and red wines.
The persulfide sulfur formed on an active site cysteine residue of pyridoxal 5'-phosphate-dependent cysteine desulfurases is subsequently incorporated into the biosynthetic pathways of a variety of sulfur-containing cofactors and thionucleosides. In molybdenum cofactor biosynthesis, MoeB activates the C terminus of the MoaD subunit of molybdopterin (MPT) synthase to form MoaD-adenylate, which is subsequently converted to a thiocarboxylate for the generation of the dithiolene group of MPT. It has been shown that three cysteine desulfurases (CsdA, SufS, and IscS) of Escherichia coli can transfer sulfur from L-cysteine to the thiocarboxylate of MoaD in vitro. Here, we demonstrate by surface plasmon resonance analyses that IscS, but not CsdA or SufS, interacts with MoeB and MoaD. MoeB and MoaD can stimulate the IscS activity up to 1.6-fold. Analysis of the sulfuration level of MoaD isolated from strains defective in cysteine desulfurases shows a largely decreased sulfuration level of the protein in an iscS deletion strain but not in a csdA/sufS deletion strain. We also show that another iscS deletion strain of E. coli accumulates compound Z, a direct oxidation product of the immediate precursor of MPT, to the same extent as an MPT synthase-deficient strain. In contrast, analysis of the content of compound Z in Delta csdA and Delta sufS strains revealed no such accumulation. These findings indicate that IscS is the primary physiological sulfur-donating enzyme for the generation of the thiocarboxylate of MPT synthase in MPT biosynthesis.
The human MOCS3 protein contains an N-terminal domain similar to the Escherichia coli MoeB protein and a C- terminal segment displaying similarities to the sulfurtransferase rhodanese. MOCS3 is proposed to catalyze both the adenylation and the subsequent generation of a thiocarboxylate group at the C-terminus of the smaller subunit of molybdopterin (MPT) synthase during Moco biosynthesis in humans. Recent studies have shown that the MOCS3 rhodanese-like domain (MOCS3-RLD) catalyzes the transfer of sulfur from thiosulfate to cyanide and is also able to provide the sulfur for the thiocarboxylation of MOCS2A in a defined in vitro system for the generation of MPT from precursor Z. MOCS3-RLD contains four cysteine residues of which only C412 in the six amino acid active loop is conserved in homologous proteins from other organisms. ESI-MS/MS studies gave direct evidence for the formation of a persulfide group that is exclusively formed on C412. Simultaneous mutagenesis of the remaining three cysteine residues showed that none of them is involved in the sulfur transfer reaction in vitro. A disulfide bridge was identified to be formed between C316 and C324, and possible roles of the three noncatalytic cysteine residues are discussed. By ESI-MS/MS a partially gluconoylated N- terminus of the His(6)-tagged MOCS3-RLD was identified (mass increment of 178 Da) which resulted in a heterogeneity of the protein but did not influence sulfurtransferase activity
Molybdenum cofactor deficiency (MIM#252150) is a severe autosomal- recessive disorder with a devastating outcome. The cofactor is the product of a complex biosynthetic pathway involving four different genes (MOCS1, MOCS2, MOCS3 and GEPH). This disorder is caused almost exclusively by mutations in the MOCS1 or MOCS2 genes. Mutations affecting this biosynthetic pathway result in a lethal phenotype manifested by progressive neurological damage via the inactivation of the molybdenum cofactor-dependent enzyme, sulphite oxidase. Here we describe a total of ten novel disease-causing mutations in the MOCS1 and MOCS2 genes. Nine out of these ten mutations were classified as pathogenic in nature, since they create a stop codon, affect constitutive splice site positions, or change strictly conserved motifs. The tenth mutation abolishes the stop codon of the MOCS2B gene, thus elongating the corresponding protein. The mutation was expressed in vitro and was found to abolish the binding affinities of the large subunit of molybdopterin synthase (MOCS2B) for both precursor Z and the small subunit of molybdopterin synthase (MOCS2A)
After heterologous expression in Escherichia coli, the Azotobacter vinelandii rhodanese RhdA is purified in a persulfurated form (RhdA-SSH). We identified L-cysteine as the most effective sulfur source in producing RhdA-SSH. An E. coli soluble extract was required for in vitro persulfuration of RhdA, and the addition of pyridoxal-5'-phosphate increased RhdA-SSH production, indicating a likely involvement of a cysteine desulfurase. We were able to show the formation of a covalent complex between IscS and RhdA. By combining a time-course fluorescence assay and mass spectrometry analysis, we demonstrated the transfer of sulfur from E. coli IscS to RhdA. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved
Human sulfite oxidase (hSO) was immobilised on SAM-coated silver electrodes under preservation of the native heme pocket structure of the cytochrome b5 (Cyt b5) domain and the functionality of the enzyme. The redox properties and catalytic activity of the entire enzyme were studied by surface enhanced resonance Raman (SERR) spectroscopy and cyclic voltammetry (CV) and compared to the isolated heme domain when possible. It is shown that heterogeneous electron transfer and catalytic activity of hSO sensitively depend on the local environment of the enzyme. Increasing the ionic strength of the buffer solution leads to an increase of the heterogeneous electron transfer rate from 17 s(-1) to 440 s(- 1) for hSO as determined by SERR spectroscopy. CV measurements demonstrate an increase of the apparent turnover rate for the immobilised hSO from 0.85 s(-1) in 100 mM buffer to 5.26 s(-1) in 750 mM buffer. We suggest that both effects originate from the increased mobility of the surface-bound enzyme with increasing ionic strength. In agreement with surface potential calculations we propose that at high ionic strength the enzyme is immobilised via the dimerisation domain to the SAM surface. The flexible loop region connecting the Moco and the Cyt b5 domain allows alternating contact with the Moco interaction site and the SAM surface, thereby promoting the sequential intramolecular and heterogeneous electron transfer from Moco via Cyt b5 to the electrode. At lower ionic strength, the contact time of the Cyt b5 domain with the SAM surface is longer, corresponding to a slower overall electron transfer process.
Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a cytoplasmic enzyme with an (alpha beta) 2 heterodimeric structure that is highly identical to homodimeric eukaryotic xanthine oxidoreductases. The crystal structure revealed that the molybdenum cofactor (Moco) is deeply buried within the protein. A protein involved in Moco insertion and XDH maturation has been identified, which was designated XdhC. XdhC was shown to be essential for the production of active XDH but is not a subunit of the purified enzyme. Here we describe the purification of XdhC and the detailed characterization of its role for XDH maturation. We could show that XdhC binds Moco in stoichiometric amounts, which subsequently can be inserted into Moco-free apo-XDH. A specific interaction between XdhC and XdhB was identified. We show that XdhC is required for the stabilization of the sulfurated form of Moco present in enzymes of the xanthine oxidase family. Our findings imply that enzyme-specific proteins exist for the biogenesis of molybdoenzymes, coordinating Moco binding and insertion into their respective target proteins. So far, the requirement of such proteins for molybdoenzyme maturation has been described only for prokaryotes