TY - JOUR A1 - Zupok, Arkadiusz A1 - Iobbi-Nivol, Chantal A1 - Mejean, Vincent A1 - Leimkühler, Silke T1 - The regulation of Moco biosynthesis and molybdoenzyme gene expression by molybdenum and iron in bacteria JF - Metallomics : integrated biometal science N2 - Bacterial molybdoenzymes are key enzymes involved in the global sulphur, nitrogen and carbon cycles. These enzymes require the insertion of the molybdenum cofactor (Moco) into their active sites and are able to catalyse a large range of redox-reactions. Escherichia coli harbours nineteen different molybdoenzymes that require a tight regulation of their synthesis according to substrate availability, oxygen availability and the cellular concentration of molybdenum and iron. The synthesis and assembly of active molybdoenzymes are regulated at the level of transcription of the structural genes and of translation in addition to the genes involved in Moco biosynthesis. The action of global transcriptional regulators like FNR, NarXL/QP, Fur and ArcA and their roles on the expression of these genes is described in detail. In this review we focus on what is known about the molybdenum- and iron-dependent regulation of molybdoenzyme and Moco biosynthesis genes in the model organism E. coli. The gene regulation in E. coli is compared to two other well studied model organisms Rhodobacter capsulatus and Shewanella oneidensis. Y1 - 2019 U6 - https://doi.org/10.1039/c9mt00186g SN - 1756-5901 SN - 1756-591X VL - 11 IS - 10 SP - 1602 EP - 1624 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Zupok, Arkadiusz A1 - Górka, Michał Jakub A1 - Siemiatkowska, Beata A1 - Skirycz, Aleksandra A1 - Leimkühler, Silke T1 - Iron-Dependent Regulation of Molybdenum Cofactor Biosynthesis Genes in Escherichia coli JF - Journal of bacteriology N2 - 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. KW - Escherichia coli KW - FNR KW - iron regulation KW - iron-sulfur cluster KW - anaerobic respiration KW - molybdenum cofactor Y1 - 2019 U6 - https://doi.org/10.1128/JB.00382-19 SN - 0021-9193 SN - 1098-5530 VL - 201 IS - 17 PB - American Society for Microbiology CY - Washington ER - TY - JOUR A1 - Zhang, Wanjiao A1 - Urban, Alexander A1 - Mihara, Hisaaki A1 - Leimkühler, Silke A1 - Kurihara, Tatsuo A1 - Esaki, Nobuyoshi T1 - IscS functions as a primary sulfur-donating enzyme by interacting specifically with MoeB and MoaD in the biosynthesis of molybdopterin in escherichia coli N2 - 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. Y1 - 2010 UR - http://www.jbc.org/ U6 - https://doi.org/10.1074/jbc.M109.082172 SN - 0021-9258 ER - TY - JOUR A1 - Zeng, Ting A1 - Pankratov, Dmitry A1 - Falk, Magnus A1 - Leimkühler, Silke A1 - Shleev, Sergey A1 - Wollenberger, Ursula T1 - Miniature direct electron transfer based sulphite/oxygen enzymatic fuel cells JF - Biosensors and bioelectronics : the principal international journal devoted to research, design development and application of biosensors and bioelectronics N2 - 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. KW - Enzymatic fuel cell KW - Microscale electrode KW - Direct electron transfer KW - Sulphite oxidase KW - Bilirubin oxidase Y1 - 2015 U6 - https://doi.org/10.1016/j.bios.2014.10.080 SN - 0956-5663 SN - 1873-4235 VL - 66 SP - 39 EP - 42 PB - Elsevier CY - Oxford ER - TY - JOUR A1 - Zeng, Ting A1 - Leimkühler, Silke A1 - Wollenberger, Ulla A1 - Fourmond, Vincent T1 - Transient Catalytic Voltammetry of Sulfite Oxidase Reveals Rate Limiting Conformational Changes JF - Journal of the American Chemical Society N2 - 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. Y1 - 2017 U6 - https://doi.org/10.1021/jacs.7b05480 SN - 0002-7863 VL - 139 SP - 11559 EP - 11567 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Zeng, Ting A1 - Leimkühler, Silke A1 - Koetz, Joachim A1 - Wollenberger, Ursula T1 - Effective Electrochemistry of Human Sulfite Oxidase Immobilized on Quantum-Dots-Modified Indium Tin Oxide Electrode JF - ACS applied materials & interfaces N2 - 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. KW - human sulfite oxidase KW - direct electrochemistry KW - bioelectrocatalysis KW - photocurrent KW - CdS quantum dots Y1 - 2015 U6 - https://doi.org/10.1021/acsami.5b06665 SN - 1944-8244 VL - 7 IS - 38 SP - 21487 EP - 21494 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Zeng, Ting A1 - Frasca, Stefano A1 - Rumschöttel, Jens A1 - Koetz, Joachim A1 - Leimkühler, Silke A1 - Wollenberger, Ursula T1 - Role of Conductive Nanoparticles in the Direct Unmediated Bioelectrocatalysis of Immobilized Sulfite Oxidase JF - Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis KW - Direct electron transfer KW - Protein voltammetry KW - Human sulfite oxidase KW - Bioelectrocatalysis KW - Nanoparticles Y1 - 2016 U6 - https://doi.org/10.1002/elan.201600246 SN - 1040-0397 SN - 1521-4109 VL - 28 SP - 2303 EP - 2310 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Yokoyama, Kenichi A1 - Leimkühler, Silke T1 - The role of FeS clusters for molybdenum cofactor biosynthesis and molybdoenzymes in bacteria JF - Biochimica et biophysica acta : Molecular cell research N2 - The biosynthesis of the molybdenum cofactor (Moco) has been intensively studied, in addition to its insertion into molybdoenzymes. In particular, a link between the assembly of molybdoenzymes and the biosynthesis of FeS clusters has been identified in the recent years: 1) the synthesis of the first intermediate in Moco biosynthesis requires an FeS-cluster containing protein, 2) the sulfurtransferase for the dithiolene group in Moco is also involved in the synthesis of FeS clusters, thiamin and thiolated tRNAs, 3) the addition of a sulfido-ligand to the molybdenum atom in the active site additionally involves a sulfurtransferase, and 4) most molybdoenzymes in bacteria require FeS clusters as redox active cofactors. In this review we will focus on the biosynthesis of the molybdenum cofactor in bacteria, its modification and insertion into molybdoenzymes, with an emphasis to its link to FeS cluster biosynthesis and sulfur transfer. (C) 2014 Elsevier B.V. All rights reserved. KW - Molybdenum-iron-iron-sulfur cluster KW - Molybdenum cofactor KW - tRNA KW - Sulfur transfer KW - L-Cysteine desulfurase Y1 - 2015 U6 - https://doi.org/10.1016/j.bbamcr.2014.09.021 SN - 0167-4889 SN - 0006-3002 VL - 1853 IS - 6 SP - 1335 EP - 1349 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Yildiz, Tugba A1 - Leimkühler, Silke T1 - TusA is a versatile protein that links translation efficiency to cell division in Escherichia coli JF - Journal of bacteriology N2 - To enable accurate and efficient translation, sulfur modifications are introduced posttranscriptionally into nucleosides in tRNAs. The biosynthesis of tRNA sulfur modifications involves unique sulfur trafficking systems for the incorporation of sulfur atoms in different nucleosides of tRNA. One of the proteins that is involved in inserting the sulfur for 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U34) modifications in tRNAs is the TusA protein. TusA, however, is a versatile protein that is also involved in numerous other cellular pathways. Despite its role as a sulfur transfer protein for the 2-thiouridine formation in tRNA, a fundamental role of TusA in the general physiology of Escherichia coli has also been discovered. Poor viability, a defect in cell division, and a filamentous cell morphology have been described previously for tusA-deficient cells. In this report, we aimed to dissect the role of TusA for cell viability. We were able to show that the lack of the thiolation status of wobble uridine (U-34) nucleotides present on Lys, Gln, or Glu in tRNAs has a major consequence on the translation efficiency of proteins; among the affected targets are the proteins RpoS and Fis. Both proteins are major regulatory factors, and the deregulation of their abundance consequently has a major effect on the cellular regulatory network, with one consequence being a defect in cell division by regulating the FtsZ ring formation.
IMPORTANCE More than 100 different modifications are found in RNAs. One of these modifications is the mnm(5)s(2)U modification at the wobble position 34 of tRNAs for Lys, Gln, and Glu. The functional significance of U34 modifications is substantial since it restricts the conformational flexibility of the anticodon, thus providing translational fidelity. We show that in an Escherichia coli TusA mutant strain, involved in sulfur transfer for the mnm(5)s(2)U34 thio modifications, the translation efficiency of RpoS and Fis, two major cellular regulatory proteins, is altered. Therefore, in addition to the transcriptional regulation and the factors that influence protein stability, tRNA modifications that ensure the translational efficiency provide an additional crucial regulatory factor for protein synthesis. KW - iron-sulfur clusters KW - tRNA thio modifications KW - FtsZ ring formation KW - cell KW - division KW - TusA KW - RpoS KW - Fis KW - FtsZ Y1 - 2021 U6 - https://doi.org/10.1128/JB.00659-20 SN - 1098-5530 VL - 203 IS - 7 PB - American Society for Microbiology CY - Washington ER - TY - JOUR A1 - Yan, Robert A1 - Friemel, Martin A1 - Aloisi, Claudia A1 - Huynen, Martijn A1 - Taylor, Ian A. A1 - Leimkühler, Silke A1 - Pastore, Annalisa T1 - The Eukaryotic-Specific ISD11 Is a Complex-Orphan Protein with Ability to Bind the Prokaryotic IscS JF - PLoS one N2 - 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. Y1 - 2016 U6 - https://doi.org/10.1371/journal.pone.0157895 SN - 1932-6203 VL - 11 SP - 383 EP - 395 PB - PLoS CY - San Fransisco ER - TY - JOUR A1 - Yan, Jiawei A1 - Frøkjær, Emil Egede A1 - Engelbrekt, Christian A1 - Leimkühler, Silke A1 - Ulstrup, Jens A1 - Wollenberger, Ulla A1 - Xiao, Xinxin A1 - Zhang, Jingdong T1 - Voltammetry and single-molecule in situ scanning tunnelling microscopy of the redox metalloenzyme human sulfite oxidase JF - ChemElectroChem N2 - Human sulfite oxidase (hSO) is a homodimeric two-domain enzyme central in the biological sulfur cycle. A pyranopterin molybdenum cofactor (Moco) is the catalytic site and a heme b(5) group located in the N-terminal domain. The two domains are connected by a flexible linker region. Electrons produced at the Moco in sulfite oxidation, are relayed via heme b(5) to electron acceptors or an electrode surface. Inter-domain conformational changes between an open and a closed enzyme conformation, allowing "gated" electron transfer has been suggested. We first recorded cyclic voltammetry (CV) of hSO on single-crystal Au(111)-electrode surfaces modified by self-assembled monolayers (SAMs) both of a short rigid thiol, cysteamine and of a longer structurally flexible thiol, omega-amino-octanethiol (AOT). hSO on cysteamine SAMs displays a well-defined pair of voltammetric peaks around -0.207 V vs. SCE in the absence of sulfite substrate, but no electrocatalysis. hSO on AOT SAMs displays well-defined electrocatalysis, but only "fair" quality voltammetry in the absence of sulfite. We recorded next in situ scanning tunnelling spectroscopy (STS) of hSO on AOT modified Au(111)-electrodes, disclosing, a 2-5 % surface coverage of strong molecular scale contrasts, assigned to single hSO molecules, notably with no contrast difference in the absence and presence of sulfite. In situ STS corroborated this observation with a sigmoidal tunnelling current/overpotential correlation. KW - cyclic voltammetry KW - human sulfite oxidase KW - in  situ scanning KW - tunnelling spectroscopy KW - self-assembled molecular monolayers KW - single-crystal gold electrodes Y1 - 2021 U6 - https://doi.org/10.1002/celc.202001258 SN - 2196-0216 VL - 8 IS - 1 SP - 164 EP - 171 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Wiethaus, Jessica A1 - Mueller, Alexandra A1 - Neumann, Meina A1 - Neumann, Sandra A1 - Leimkühler, Silke A1 - Narberhaus, Franz A1 - Masepohl, Bernd T1 - Specific interactions between four Molybdenum-binding proteins contribute to Mo-dependent gene regulation in Rhodobacter capsulatus N2 - 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. Y1 - 2009 UR - http://jb.asm.org/ U6 - https://doi.org/10.1128/Jb.00526-09 SN - 0021-9193 ER - TY - JOUR A1 - Voss, Martin A1 - Nimtz, Manfred A1 - Leimkühler, Silke T1 - Elucidation of the dual role of Mycobacterial MoeZR in Molybdenum Cofactor Biosynthesis and Cysteine Biosynthesis JF - PLoS one N2 - 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. Y1 - 2011 U6 - https://doi.org/10.1371/journal.pone.0028170 SN - 1932-6203 VL - 6 IS - 11 PB - PLoS CY - San Fransisco ER - TY - JOUR A1 - Tiedemann, Kim A1 - Iobbi-Nivol, Chantal A1 - Leimkühler, Silke T1 - The Role of the Nucleotides in the Insertion of the bis-Molybdopterin Guanine Dinucleotide Cofactor into apo-Molybdoenzymes JF - Molecules N2 - The role of the GMP nucleotides of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor of the DMSO reductase family has long been a subject of discussion. The recent characterization of the bis-molybdopterin (bis-Mo-MPT) cofactor present in the E. coli YdhV protein, which differs from bis-MGD solely by the absence of the nucleotides, now enables studying the role of the nucleotides of bis-MGD and bis-MPT cofactors in Moco insertion and the activity of molybdoenzymes in direct comparison. Using the well-known E. coli TMAO reductase TorA as a model enzyme for cofactor insertion, we were able to show that the GMP nucleotides of bis-MGD are crucial for the insertion of the bis-MGD cofactor into apo-TorA. KW - bis-MGD KW - chaperone KW - molybdenum cofactor KW - TMAO reductase Y1 - 2022 U6 - https://doi.org/10.3390/molecules27092993 SN - 1420-3049 VL - 27 SP - 1 EP - 15 PB - MDPI CY - Basel, Schweiz ET - 9 ER - TY - JOUR A1 - Terao, Mineko A1 - Romao, Maria Joao A1 - Leimkühler, Silke A1 - Bolis, Marco A1 - Fratelli, Maddalena A1 - Coelho, Catarina A1 - Santos-Silva, Teresa A1 - Garattini, Enrico T1 - Structure and function of mammalian aldehyde oxidases JF - Archives of toxicology : official journal of EUROTOX N2 - Mammalian aldehyde oxidases (AOXs; EC1.2.3.1) are a group of conserved proteins belonging to the family of molybdo-flavoenzymes along with the structurally related xanthine dehydrogenase enzyme. AOXs are characterized by broad substrate specificity, oxidizing not only aromatic and aliphatic aldehydes into the corresponding carboxylic acids, but also hydroxylating a series of heteroaromatic rings. The number of AOX isoenzymes expressed in different vertebrate species is variable. The two extremes are represented by humans, which express a single enzyme (AOX1) in many organs and mice or rats which are characterized by tissue-specific expression of four isoforms (AOX1, AOX2, AOX3, and AOX4). In vertebrates each AOX isoenzyme is the product of a distinct gene consisting of 35 highly conserved exons. The extant species-specific complement of AOX isoenzymes is the result of a complex evolutionary process consisting of a first phase characterized by a series of asynchronous gene duplications and a second phase where the pseudogenization and gene deletion events prevail. In the last few years remarkable advances in the elucidation of the structural characteristics and the catalytic mechanisms of mammalian AOXs have been made thanks to the successful crystallization of human AOX1 and mouse AOX3. Much less is known about the physiological function and physiological substrates of human AOX1 and other mammalian AOX isoenzymes, although the importance of these proteins in xenobiotic metabolism is fairly well established and their relevance in drug development is increasing. This review article provides an overview and a discussion of the current knowledge on mammalian AOX. KW - Aldehyde oxidase KW - Molybdo-flavoenzymes KW - Xanthine oxidoreductase KW - Drug metabolism Y1 - 2016 U6 - https://doi.org/10.1007/s00204-016-1683-1 SN - 0340-5761 SN - 1432-0738 VL - 90 SP - 753 EP - 780 PB - Springer CY - Heidelberg ER - TY - JOUR A1 - Terao, Mineko A1 - Garattini, Enrico A1 - Romão, Maria João A1 - Leimkühler, Silke T1 - Evolution, expression, and substrate specificities of aldehyde oxidase enzymes in eukaryotes JF - The journal of biological chemistry N2 - Aldehyde oxidases (AOXs) are a small group of enzymes belonging to the larger family of molybdo-flavoenzymes, along with the well-characterized xanthine oxidoreductase. The two major types of reactions that are catalyzed by AOXs are the hydroxylation of heterocycles and the oxidation of aldehydes to their corresponding carboxylic acids. Different animal species have different complements of AOX genes. The two extremes are represented in humans and rodents; whereas the human genome contains a single active gene (AOX1), those of rodents, such as mice, are endowed with four genes (Aox1-4), clustering on the same chromosome, each encoding a functionally distinct AOX enzyme. It still remains enigmatic why some species have numerous AOX enzymes, whereas others harbor only one functional enzyme. At present, little is known about the physiological relevance of AOX enzymes in humans and their additional forms in other mammals. These enzymes are expressed in the liver and play an important role in the metabolisms of drugs and other xenobiotics. In this review, we discuss the expression, tissue-specific roles, and substrate specificities of the different mammalian AOX enzymes and highlight insights into their physiological roles. KW - metalloenzyme KW - molybdenum KW - mouse KW - drug metabolism KW - flavoprotein KW - xenobiotic KW - oxidase KW - oxygen radicals KW - iron-sulfur protein KW - aldehyde oxidase (AOX) KW - enzyme evolution KW - metal-containing enzyme KW - molybdenum cofactor (Moco) KW - molybdo-flavoenzyme KW - 2Fe-2S cluster KW - flavin adenine dinucleotide (FAD) Y1 - 2020 U6 - https://doi.org/10.1074/jbc.REV119.007741 SN - 0021-9258 SN - 1083-351X VL - 295 IS - 16 SP - 5377 EP - 5389 PB - American Society for Biochemistry and Molecular Biology CY - Rockville ER - TY - JOUR A1 - Tang, Jing A1 - Werchmeister, Rebecka Maria Larsen A1 - Preda, Loredana A1 - Huang, Wei A1 - Zheng, Zhiyong A1 - Leimkühler, Silke A1 - Wollenberger, Ulla A1 - Xiao, Xinxin A1 - Engelbrekt, Christian A1 - Ulstrup, Jens A1 - Zhang, Jingdong T1 - Three-dimensional sulfite oxidase bioanodes based on graphene functionalized carbon paper for sulfite/O-2 biofuel cells JF - ACS catalysis N2 - 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. KW - enzymatic biofuel cell KW - reduced graphene oxide KW - sulfite oxidase KW - carbon paper KW - direct electron transfer Y1 - 2019 U6 - https://doi.org/10.1021/acscatal.9b01715 SN - 2155-5435 VL - 9 IS - 7 SP - 6543 EP - 6554 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Tanabe, Tomohisa Sebastian A1 - Leimkühler, Silke A1 - Dahl, Christiane ED - Poole, RK T1 - The functional diversity of the prokaryotic sulfur carrier protein TusA JF - Advances in microbial physiology N2 - Persulfide groups participate in a wide array of biochemical pathways and are chemically very versatile. The TusA protein has been identified as a central element supplying and transferring sulfur as persulfide to a number of important biosynthetic pathways, like molybdenum cofactor biosynthesis or thiomodifications in nucleosides of tRNAs. In recent years, it has furthermore become obvious that this protein is indispensable for the oxidation of sulfur compounds in the cytoplasm. Phylogenetic analyses revealed that different TusA protein variants exists in certain organisms, that have evolved to pursue specific roles in cellular pathways. The specific TusA-like proteins thereby cannot replace each other in their specific roles and are rather specific to one sulfur transfer pathway or shared between two pathways. While certain bacteria like Escherichia coli contain several copies of TusA-like proteins, in other bacteria like Allochromatium vinosum a single copy of TusA is present with an essential role for this organism. Here, we give an overview on the multiple roles of the various TusA-like proteins in sulfur transfer pathways in different organisms to shed light on the remaining mysteries of this versatile protein. Y1 - 2019 SN - 978-0-12-817715-0 SN - 978-0-12-817714-3 U6 - https://doi.org/10.1016/bs.ampbs.2019.07.004 SN - 0065-2911 VL - 75 SP - 233 EP - 277 PB - Elsevier Acad. Press CY - Amsterdam ER - TY - JOUR A1 - Tadjoung Waffo, Armel Franklin A1 - Mitrova, Biljana A1 - Tiedemann, Kim A1 - Iobbi-Nivol, Chantal A1 - Leimkühler, Silke A1 - Wollenberger, Ulla T1 - Electrochemical trimethylamine n-oxide biosensor with enzyme-based oxygen-scavenging membrane for long-term operation under ambient air JF - Biosensors : open access journal N2 - An amperometric trimethylamine N-oxide (TMAO) biosensor is reported, where TMAO reductase (TorA) and glucose oxidase (GOD) and catalase (Cat) were immobilized on the electrode surface, enabling measurements of mediated enzymatic TMAO reduction at low potential under ambient air conditions. The oxygen anti-interference membrane composed of GOD, Cat and polyvinyl alcohol (PVA) hydrogel, together with glucose concentration, was optimized until the O-2 reduction current of a Clark-type electrode was completely suppressed for at least 3 h. For the preparation of the TMAO biosensor, Escherichia coli TorA was purified under anaerobic conditions and immobilized on the surface of a carbon electrode and covered by the optimized O-2 scavenging membrane. The TMAO sensor operates at a potential of -0.8 V vs. Ag/AgCl (1 M KCl), where the reduction of methylviologen (MV) is recorded. The sensor signal depends linearly on TMAO concentrations between 2 mu M and 15 mM, with a sensitivity of 2.75 +/- 1.7 mu A/mM. The developed biosensor is characterized by a response time of about 33 s and an operational stability over 3 weeks. Furthermore, measurements of TMAO concentration were performed in 10% human serum, where the lowest detectable concentration is of 10 mu M TMAO. KW - trimethylamine N-oxide KW - biosensor KW - TMAO-reductase KW - oxygen scavenger KW - immobilized enzyme KW - multienzyme electrode KW - viologen Y1 - 2021 U6 - https://doi.org/10.3390/bios11040098 SN - 2079-6374 VL - 11 IS - 4 PB - MDPI CY - Basel ER - TY - JOUR A1 - Stripp, Sven T. A1 - Duffus, Benjamin R. A1 - Fourmond, Vincent A1 - Leger, Christophe A1 - Leimkühler, Silke A1 - Hirota, Shun A1 - Hu, Yilin A1 - Jasniewski, Andrew A1 - Ogata, Hideaki A1 - Ribbe, Markus W. T1 - Second and outer coordination sphere effects in nitrogenase, hydrogenase, formate dehydrogenase, and CO dehydrogenase JF - Chemical reviews : CR N2 - Gases like H-2, N-2, CO2, and CO are increasingly recognized as critical feedstock in "green" energy conversion and as sources of nitrogen and carbon for the agricultural and chemical sectors. However, the industrial transformation of N-2, CO2, and CO and the production of H-2 require significant energy input, which renders processes like steam reforming and the Haber-Bosch reaction economically and environmentally unviable. Nature, on the other hand, performs similar tasks efficiently at ambient temperature and pressure, exploiting gas-processing metalloenzymes (GPMs) that bind low-valent metal cofactors based on iron, nickel, molybdenum, tungsten, and sulfur. Such systems are studied to understand the biocatalytic principles of gas conversion including N-2 fixation by nitrogenase and H-2 production by hydrogenase as well as CO2 and CO conversion by formate dehydrogenase, carbon monoxide dehydrogenase, and nitrogenase. In this review, we emphasize the importance of the cofactor/protein interface, discussing how second and outer coordination sphere effects determine, modulate, and optimize the catalytic activity of GPMs. These may comprise ionic interactions in the second coordination sphere that shape the electron density distribution across the cofactor, hydrogen bonding changes, and allosteric effects. In the outer coordination sphere, proton transfer and electron transfer are discussed, alongside the role of hydrophobic substrate channels and protein structural changes. Combining the information gained from structural biology, enzyme kinetics, and various spectroscopic techniques, we aim toward a comprehensive understanding of catalysis beyond the first coordination sphere. Y1 - 2022 U6 - https://doi.org/10.1021/acs.chemrev.1c00914 SN - 0009-2665 SN - 1520-6890 VL - 122 IS - 14 SP - 11900 EP - 11973 PB - American Chemical Society CY - Washington, DC ER - TY - JOUR A1 - Spricigo, Roberto A1 - Richter, Claudia A1 - Leimkühler, Silke A1 - Gorton, Lo A1 - Scheller, Frieder W. A1 - Wollenberger, Ursula T1 - Sulfite biosensor based on osmium redox polymer wired sulfite oxidase N2 - 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. Y1 - 2010 UR - http://www.sciencedirect.com/science/journal/09277757 U6 - https://doi.org/10.1016/j.colsurfa.2009.09.001 SN - 0927-7757 ER - TY - JOUR A1 - Spricigo, Roberto A1 - Leimkühler, Silke A1 - Gorton, Lo A1 - Scheller, Frieder W. A1 - Wollenberger, Ursula T1 - The Electrically Wired Molybdenum Domain of Human Sulfite Oxidase is Bioelectrocatalytically Active JF - European journal of inorganic chemistry : a journal of ChemPubSoc Europe N2 - 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. KW - Metalloenzymes KW - Enzyme catalysis KW - Immobilization KW - Osmium Y1 - 2015 U6 - https://doi.org/10.1002/ejic.201500034 SN - 1434-1948 SN - 1099-0682 IS - 21 SP - 3526 EP - 3531 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Spricigo, Roberto A1 - Dronov, Roman A1 - Lisdat, Fred A1 - Leimkühler, Silke A1 - Scheller, Frieder W. A1 - Wollenberger, Ursula T1 - Electrocatalytic sulfite biosensor with human sulfite oxidase co-immobilized with cytochrome c in a polyelectrolyte-containing multilayer N2 - 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. Y1 - 2009 UR - http://www.springerlink.com/content/100417 U6 - https://doi.org/10.1007/s00216-008-2432-y SN - 1618-2642 ER - TY - JOUR A1 - Sivanesan, Arumugam A1 - Ly, Khoa H. A1 - Adamkiewicz, Witold A1 - Stiba, Konstanze A1 - Leimkühler, Silke A1 - Weidinger, Inez M. T1 - Tunable electric field enhancement and redox chemistry on TiO2 Island films via covalent attachment to Ag or Au nanostructures JF - The journal of physical chemistry : C, Nanomaterials and interfaces N2 - 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. Y1 - 2013 U6 - https://doi.org/10.1021/jp4032578 SN - 1932-7447 VL - 117 IS - 22 SP - 11866 EP - 11872 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Sivanesan, Arumugam A1 - Kalaivani, Govindasamy A1 - Fischer, Anna A1 - Stiba, Konstanze A1 - Leimkühler, Silke A1 - Weidinger, Inez M. T1 - Complementary surface-enhanced resonance raman Spectroscopic Biodetection of mixed protein solutions by Chitosan- and Silica-Coated Plasmon-Tuned Silver Nanoparticles JF - Analytical chemistry N2 - 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. Y1 - 2012 U6 - https://doi.org/10.1021/ac301001a SN - 0003-2700 VL - 84 IS - 13 SP - 5759 EP - 5764 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Sezer, Murat A1 - Spricigo, Roberto A1 - Utesch, Tillmann A1 - Millo, Diego A1 - Leimkühler, Silke A1 - Mroginski, Maria A. A1 - Wollenberger, Ursula A1 - Hildebrandt, Peter A1 - Weidinger, Inez M. T1 - Redox properties and catalytic activity of surface-bound human sulfite oxidase studied by a combined surface enhanced resonance Raman spectroscopic and electrochemical approach N2 - 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. Y1 - 2010 UR - http://www.rsc.org/Publishing/Journals/CP/index.asp U6 - https://doi.org/10.1039/B927226g SN - 1463-9076 ER - TY - JOUR A1 - Schwanhold, Nadine A1 - Iobbi-Nivol, Chantal A1 - Lehmann, Angelika A1 - Leimkühler, Silke T1 - Same but different BT - Comparison of two system-specific molecular chaperones for the maturation of formate dehydrogenases JF - PLoS one N2 - The maturation of bacterial molybdoenzymes is a complex process leading to the insertion of the bulky bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor into the apoenzyme. Most molybdoenzymes were shown to contain a specific chaperone for the insertion of the bis-MGD cofactor. Formate dehydrogenases (FDH) together with their molecular chaperone partner seem to display an exception to this specificity rule, since the chaperone FdhD has been proven to be involved in the maturation of all three FDH enzymes present in Escherichia colt. Multiple roles have been suggested for FdhD-like chaperones in the past, including the involvement in a sulfur transfer reaction from the L-cysteine desulfurase IscS to bis-MGD by the action of two cysteine residues present in a conserved CXXC motif of the chaperones. However, in this study we show by phylogenetic analyses that the CXXC motif is not conserved among FdhD-like chaperones. We compared in detail the FdhD-like homologues from Rhodobacter capsulatus and E. colt and show that their roles in the maturation of FDH enzymes from different subgroups can be exchanged. We reveal that bis-MGDbinding is a common characteristic of FdhD-like proteins and that the cofactor is bound with a sulfido-ligand at the molybdenum atom to the chaperone. Generally, we reveal that the cysteine residues in the motif CXXC of the chaperone are not essential for the production of active FDH enzymes. Y1 - 2018 U6 - https://doi.org/10.1371/journal.pone.0201935 SN - 1932-6203 VL - 13 IS - 11 PB - PLoS CY - San Fransisco ER - TY - JOUR A1 - Schrapers, Peer A1 - Hartmann, Tobias A1 - Kositzki, Ramona A1 - Dau, Holger A1 - Reschke, Stefan A1 - Schulzke, Carola A1 - Leimkühler, Silke A1 - Haumann, Michael T1 - 'Sulfido and Cysteine Ligation Changes at the Molybdenum Cofactor during Substrate Conversion by Formate Dehydrogenase (FDH) from Rhodobacter capsulatus JF - Inorganic chemistry N2 - 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. Y1 - 2015 U6 - https://doi.org/10.1021/ic502880y SN - 0020-1669 SN - 1520-510X VL - 54 IS - 7 SP - 3260 EP - 3271 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Sarauli, David A1 - Xu, Chenggang A1 - Dietzel, Birgit A1 - Stiba, Konstanze A1 - Leimkühler, Silke A1 - Schulz, Burkhard A1 - Lisdat, Fred T1 - Thin films of substituted polyanilines interactions with biomolecular systems JF - Soft matter N2 - 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. Y1 - 2012 U6 - https://doi.org/10.1039/c2sm07261k SN - 1744-683X VL - 8 IS - 14 SP - 3848 EP - 3855 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Sarauli, David A1 - Riedel, Marc A1 - Wettstein, Christoph A1 - Hahn, Robert A1 - Stiba, Konstanze A1 - Wollenberger, Ursula A1 - Leimkühler, Silke A1 - Schmuki, Patrik A1 - Lisdat, Fred T1 - Semimetallic TiO2 nanotubes new interfaces for bioelectrochemical enzymatic catalysis JF - Journal of materials chemistry N2 - Different self-organized TiO2 nanotube structures are shown to represent new interfaces for the achievement of bioelectrochemical enzymatic catalysis involving redox proteins and enzymes without further surface modification or the presence of mediators. Y1 - 2012 U6 - https://doi.org/10.1039/c2jm16427b SN - 0959-9428 VL - 22 IS - 11 SP - 4615 EP - 4618 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Sarauli, David A1 - Borowski, Anja A1 - Peters, Kristina A1 - Schulz, Burkhard A1 - Fattakhova-Rohlfing, Dina A1 - Leimkühler, Silke A1 - Lisdat, Fred T1 - Investigation of the pH-Dependent Impact of Sulfonated Polyaniline on Bioelectrocatalytic Activity of Xanthine Dehydrogenase JF - ACS catalysis N2 - 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. KW - enzyme bioelectrocatalysis KW - sulfonated polyanilines KW - xanthine dehydrogenase KW - pH-dependent electrochemistry KW - macroporous ITO electrodes Y1 - 2016 U6 - https://doi.org/10.1021/acscatal.6b02011 SN - 2155-5435 VL - 6 SP - 7152 EP - 7159 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Samuel, Prinson P. A1 - Horn, Sebastian A1 - Döring, Alexander A1 - Havelius, Kajsa G. V. A1 - Reschke, Stefan A1 - Leimkühler, Silke A1 - Haumann, Michael A1 - Schulzke, Carola T1 - A Crystallographic and Mo K-Edge XAS Study of Molybdenum Oxo Bis-,Mono-, and Non-Dithiolene Complexes - First-Sphere Coordination Geometry and Noninnocence of Ligands JF - European journal of inorganic chemistry : a journal of ChemPubSoc Europe N2 - 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. KW - Molybdenum KW - Enzyme models KW - X-ray absorption spectroscopy KW - Noninnocence KW - Bioinorganic chemistry Y1 - 2011 U6 - https://doi.org/10.1002/ejic.201100331 SN - 1434-1948 IS - 28 SP - 4387 EP - 4399 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Romao, Maria Joao A1 - Coelho, Catarina A1 - Santos-Silva, Teresa A1 - Foti, Alessandro A1 - Terao, Mineko A1 - Garattini, Enrico A1 - Leimkühler, Silke T1 - Structural basis for the role of mammalian aldehyde oxidases in the metabolism of drugs and xenobiotics JF - Current Opinion in Chemical Biology N2 - 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. Mammals are characterized by a complement of species specific AOX isoenzymes, that varies from one in humans (AOX1) to four in rodents (AOX1, AOX2, AOX3 and AOX4). The physiological function of mammalian AOX isoenzymes is unknown, although human AOX1 is an emerging enzyme in phase-I drug metabolism. Indeed, the number of therapeutic molecules under development which act as AOX substrates is increasing. The recent crystallization and structure determination of human AOX1 as well as mouse AOX3 has brought new insights into the mechanisms underlying substrate/inhibitor binding as well as the catalytic activity of this class of enzymes. Y1 - 2017 U6 - https://doi.org/10.1016/j.cbpa.2017.01.005 SN - 1367-5931 SN - 1879-0402 VL - 37 SP - 39 EP - 47 PB - Elsevier CY - Oxford ER - TY - JOUR A1 - Riedel, Simona A1 - Siemiatkowska, Beata A1 - Watanabe, Mutsumi A1 - Müller, Christina S. A1 - Schünemann, Volker A1 - Hoefgen, Rainer A1 - Leimkühler, Silke T1 - The ABCB7-Like Transporter PexA in Rhodobacter capsulatus Is Involved in the Translocation of Reactive Sulfur Species JF - Frontiers in Microbiology N2 - 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. KW - ABCB7 KW - persulfide KW - polysulfide KW - glutathione KW - ABC transporter KW - Walker A motif KW - pyridoxal-50-phosphate Y1 - 2019 U6 - https://doi.org/10.3389/fmicb.2019.00406 SN - 1664-302X VL - 10 PB - Frontiers Media CY - Lausanne ER - TY - JOUR A1 - Reschke, Stefan A1 - Sigfridsson, Kajsa G. V. A1 - Kaufmann, Paul A1 - Leidel, Nils A1 - Horn, Sebastian A1 - Gast, Klaus A1 - Schulzke, Carola A1 - Haumann, Michael A1 - Leimkühler, Silke T1 - Identification of a bis-molybdopterin intermediate in molybdenum cofactor biosynthesis in escherichia coli JF - The journal of biological chemistry N2 - 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. Y1 - 2013 U6 - https://doi.org/10.1074/jbc.M113.497453 SN - 0021-9258 SN - 1083-351X VL - 288 IS - 41 SP - 29736 EP - 29745 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - JOUR A1 - Reschke, Stefan A1 - Niks, Dimitri A1 - Wilson, Heather A1 - Sigfridsson, Kajsa G. V. A1 - Haumann, Michael A1 - Rajagopalan, K. V. A1 - Hine, Russ A1 - Leimkühler, Silke T1 - Effect of exchange of the cysteine molybdenum ligand with selenocysteine on the structure and function of the active site in human sulfite oxidase JF - Biochemistry N2 - 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. Y1 - 2013 U6 - https://doi.org/10.1021/bi4008512 SN - 0006-2960 VL - 52 IS - 46 SP - 8295 EP - 8303 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Reschke, Stefan A1 - Duffus, Benjamin R. A1 - Schrapers, Peer A1 - Mebs, Stefan A1 - Teutloff, Christian A1 - Dau, Holger A1 - Haumann, Michael A1 - Leimkühler, Silke T1 - Identification of YdhV as the First Molybdoenzyme Binding a Bis-Mo-MPT Cofactor in Escherichia coli JF - Biochemistry N2 - 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. Y1 - 2019 U6 - https://doi.org/10.1021/acs.biochem.9b00078 SN - 0006-2960 VL - 58 IS - 17 SP - 2228 EP - 2242 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Reeve, Holly A. A1 - Nicholson, Jake A1 - Altaf, Farieha A1 - Lonsdale, Thomas H. A1 - Preissler, Janina A1 - Lauterbach, Lars A1 - Lenz, Oliver A1 - Leimkühler, Silke A1 - Hollmann, Frank A1 - Paul, Caroline E. A1 - Vincent, Kylie A. T1 - A hydrogen-driven biocatalytic approach to recycling synthetic analogues of NAD(P)H JF - Chemical communications : ChemComm N2 - We demonstrate a recycling system for synthetic nicotinamide cofactor analogues using a soluble hydrogenase with turnover number of >1000 for reduction of the cofactor analogues by H-2. Coupling this system to an ene reductase, we show quantitative conversion of N-ethylmaleimide to N-ethylsuccinimide. The biocatalyst system retained >50% activity after 7 h. Y1 - 2022 U6 - https://doi.org/10.1039/d2cc02411j SN - 1359-7345 SN - 1364-548X VL - 58 IS - 75 SP - 10540 EP - 10543 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Redelberger, David A1 - Seduk, Farida A1 - Genest, Olivier A1 - Mejean, Vincent A1 - Leimkühler, Silke A1 - Iobbi-Nivol, Chantal T1 - YcdY Protein of Escherichia coli, an Atypical Member of the TorD Chaperone Family JF - Journal of bacteriology N2 - 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. Y1 - 2011 U6 - https://doi.org/10.1128/JB.05927-11 SN - 0021-9193 VL - 193 IS - 23 SP - 6512 EP - 6516 PB - American Society for Microbiology CY - Washington ER - TY - JOUR A1 - Rademacher, Corinna A1 - Hoffmann, Marie-Christine A1 - Lackmann, Jan-Wilm A1 - Moser, Roman A1 - Pfänder, Yvonne A1 - Leimkühler, Silke A1 - Narberhaus, Franz A1 - Masepohl, Bernd T1 - Tellurite resistance gene trgB confers copper tolerance to Rhodobacter capsulatus JF - BioMetals : an international journal on the role of metal ions in biology, biochemistry and medicine N2 - 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. KW - Copper KW - Tellurite KW - Nudix hydrolase KW - Metal homeostasis KW - Rhodobacter Y1 - 2012 U6 - https://doi.org/10.1007/s10534-012-9566-2 SN - 0966-0844 VL - 25 IS - 5 SP - 995 EP - 1008 PB - Springer CY - Dordrecht ER - TY - JOUR A1 - Pinyou, Piyanut A1 - Ruff, Adrian A1 - Poeller, Sascha A1 - Alsaoub, Sabine A1 - Leimkühler, Silke A1 - Wollenberger, Ursula A1 - Schuhmann, Wolfgang T1 - Wiring of the aldehyde oxidoreductase PaoABC to electrode surfaces via entrapment in low potential phenothiazine-modified redox polymers JF - Bioelectrochemistry : an international journal devoted to electrochemical aspects of biology and biological aspects of electrochemistry ; official journal of the Bioelectrochemical Society N2 - 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. KW - Aldehyde oxidoreductase KW - Enzyme electrode KW - Redox polymer KW - Phenothiazine KW - Biosensor KW - Biofuel cell Y1 - 2016 U6 - https://doi.org/10.1016/j.bioelechem.2015.12.005 SN - 1567-5394 SN - 1878-562X VL - 109 SP - 24 EP - 30 PB - Elsevier CY - Lausanne ER - TY - JOUR A1 - Paragas, Erickson M. A1 - Humphreys, Sara C. A1 - Min, Joshua A1 - Joswig-Jones, Carolyn A. A1 - Leimkühler, Silke A1 - Jones, Jeffrey P. T1 - ecoAO BT - a simple system for the study of human aldehyde oxidases role in drug metabolism JF - ACS OMEGA N2 - Although aldehyde oxidase (AO) is an important hepatic drug-metabolizing enzyme, it remains understudied and is consequently often overlooked in preclinical studies, an oversight that has resulted in the failure of multiple clinical trials. AO’s preclusion to investigation stems from the following: (1) difficulties synthesizing metabolic standards due to the chemospecificity and regiospecificity of the enzyme and (2) significant inherent variability across existing in vitro systems including liver cytosol, S9 fractions, and primary hepatocytes, which lack specificity and generate discordant expression and activity profiles. Here, we describe a practical bacterial biotransformation system, ecoAO, addressing both issues simultaneously. ecoAO is a cell paste of MoCo-producing Escherichia coli strain TP1017 expressing human AO. It exhibits specific activity toward known substrates, zoniporide, 4-trans-(N,N-dimethylamino)cinnamaldehyde, O6-benzylguanine, and zaleplon; it also has utility as a biocatalyst, yielding milligram quantities of synthetically challenging metabolite standards such as 2-oxo-zoniporide. Moreover, ecoAO enables routine determination of kcat and V/K, which are essential parameters for accurate in vivo clearance predictions. Furthermore, ecoAO has potential as a preclinical in vitro screening tool for AO activity, as demonstrated by its metabolism of 3-aminoquinoline, a previously uncharacterized substrate. ecoAO promises to provide easy access to metabolites with the potential to improve pharmacokinetic clearance predictions and guide drug development. Y1 - 2017 U6 - https://doi.org/10.1021/acsomega.7b01054 SN - 2470-1343 VL - 2 SP - 4820 EP - 4827 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Otto, Nils A1 - Marelja, Zvonimir A1 - Schoofs, Andreas A1 - Kranenburg, Holger A1 - Bittern, Jonas A1 - Yildirim, Kerem A1 - Berh, Dimitri A1 - Bethke, Maria A1 - Thomas, Silke A1 - Rode, Sandra A1 - Risse, Benjamin A1 - Jiang, Xiaoyi A1 - Pankratz, Michael A1 - Leimkühler, Silke A1 - Klämbt, Christian T1 - The sulfite oxidase Shopper controls neuronal activity by regulating glutamate homeostasis in Drosophila ensheathing glia JF - Nature Communications N2 - Specialized glial subtypes provide support to developing and functioning neural networks. Astrocytes modulate information processing by neurotransmitter recycling and release of neuromodulatory substances, whereas ensheathing glial cells have not been associated with neuromodulatory functions yet. To decipher a possible role of ensheathing glia in neuronal information processing, we screened for glial genes required in the Drosophila central nervous system for normal locomotor behavior. Shopper encodes a mitochondrial sulfite oxidase that is specifically required in ensheathing glia to regulate head bending and peristalsis. shopper mutants show elevated sulfite levels affecting the glutamate homeostasis which then act on neuronal network function. Interestingly, human patients lacking the Shopper homolog SUOX develop neurological symptoms, including seizures. Given an enhanced expression of SUOX by oligodendrocytes, our findings might indicate that in both invertebrates and vertebrates more than one glial cell type may be involved in modulating neuronal activity. Y1 - 2018 U6 - https://doi.org/10.1038/s41467-018-05645-z SN - 2041-1723 VL - 9 PB - Nature Publ. Group CY - London ER - TY - JOUR A1 - Otrelo-Cardoso, Ana Rita A1 - Schwuchow, Viola A1 - Rodrigues, David A1 - Cabrita, Eurico J. A1 - Leimkühler, Silke A1 - Romao, Maria Joao A1 - Santos-Silva, Teresa T1 - Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes JF - PLoS one N2 - 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. Y1 - 2014 U6 - https://doi.org/10.1371/journal.pone.0087295 SN - 1932-6203 VL - 9 IS - 1 PB - PLoS CY - San Fransisco ER - TY - JOUR A1 - Otrelo-Cardoso, Ana Rita A1 - da Silva Correia, Marcia Alexandra A1 - Schwuchow, Viola A1 - Svergun, Dmitri I. A1 - Romao, Maria Joao A1 - Leimkühler, Silke A1 - Santos-Silva, Teresa T1 - Structural Data on the Periplasmic Aldehyde Oxidoreductase PaoABC from Escherichia coli: SAXS and Preliminary X-ray Crystallography Analysis JF - International journal of molecular sciences N2 - 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. KW - periplasmic aldehyde oxidoreductase KW - X-ray crystallography KW - small angle X-ray scattering KW - crystal twinning Y1 - 2014 U6 - https://doi.org/10.3390/ijms15022223 SN - 1422-0067 VL - 15 IS - 2 SP - 2223 EP - 2236 PB - MDPI CY - Basel ER - TY - JOUR A1 - Ogunkola, Moses Olalekan A1 - Guiraudie-Capraz, Gaelle A1 - Féron, François A1 - Leimkühler, Silke T1 - The Human Mercaptopyruvate Sulfurtransferase TUM1 Is Involved in Moco Biosynthesis, Cytosolic tRNA Thiolation and Cellular Bioenergetics in Human Embryonic Kidney Cells JF - Biomolecules N2 - Sulfur is an important element that is incorporated into many biomolecules in humans. The incorporation and transfer of sulfur into biomolecules is, however, facilitated by a series of different sulfurtransferases. Among these sulfurtransferases is the human mercaptopyruvate sulfurtransferase (MPST) also designated as tRNA thiouridine modification protein (TUM1). The role of the human TUM1 protein has been suggested in a wide range of physiological processes in the cell among which are but not limited to involvement in Molybdenum cofactor (Moco) biosynthesis, cytosolic tRNA thiolation and generation of H2S as signaling molecule both in mitochondria and the cytosol. Previous interaction studies showed that TUM1 interacts with the L-cysteine desulfurase NFS1 and the Molybdenum cofactor biosynthesis protein 3 (MOCS3). Here, we show the roles of TUM1 in human cells using CRISPR/Cas9 genetically modified Human Embryonic Kidney cells. Here, we show that TUM1 is involved in the sulfur transfer for Molybdenum cofactor synthesis and tRNA thiomodification by spectrophotometric measurement of the activity of sulfite oxidase and liquid chromatography quantification of the level of sulfur-modified tRNA. Further, we show that TUM1 has a role in hydrogen sulfide production and cellular bioenergetics. KW - Moco biosynthesis KW - sulfite oxidase KW - cytosolic tRNA thiolation KW - 5-methoxycarbonylmethyl-2-thiouridine KW - H2S biosynthesis KW - cellular bioenergetics Y1 - 2023 U6 - https://doi.org/10.3390/biom13010144 SN - 2218-273X VL - 13 SP - 1 EP - 23 PB - MDPI CY - Basel, Schweiz ET - 1 ER - TY - JOUR A1 - Nishino, Takeshi A1 - Okamoto, Ken A1 - Leimkühler, Silke T1 - Enzymes of the Xanthine Oxidase Family JF - Molybdenum and tungsten enzymes : biochemistry N2 - Enzymes from the xanthine oxidase (XO) family of molybdenum enzymes are generally, with some exceptions, molybdenum iron–sulfur flavin hydroxylases. Mammalian xanthine oxidoreductase and aldehyde oxidase were among the first enzymes to be studied in detail more than 100 years ago and, surprisingly, they continue to be thoroughly studied in molecular detail with many open and unresolved questions remaining. Enzymes of the XO family are characterized by a molybdenum cofactor (Moco) active site with a MoVIOS(OH) ligand sphere where substrate hydroxylation of either aromatic or aliphatic carbon centers is catalyzed. During the reaction, electrons are transferred to the oxidizing substrate, most commonly O2 or NAD+, which react at the FAD site. Y1 - 2016 SN - 978-1-78262-391-5 SN - 978-1-78262-089-1 SN - 978-1-78262-881-1 U6 - https://doi.org/10.1039/9781782623915-00192 VL - 5 SP - 192 EP - 239 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Neumann, Meina A1 - Seduk, Farida A1 - Iobbi-Nivol, Chantal A1 - Leimkühler, Silke T1 - Molybdopterin Dinucleotide Biosynthesis in Escherichia coli identification of amino acid residues of molybdopterin dinucleotide transferases that determine specificity for binding of guanine or cytosine nucleotides JF - The journal of biological chemistry N2 - 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. Y1 - 2011 U6 - https://doi.org/10.1074/jbc.M110.155671 SN - 0021-9258 VL - 286 IS - 2 SP - 1400 EP - 1408 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - JOUR A1 - Neumann, Meina A1 - Schulte, Marc A1 - Jünemann, Nora A1 - Stöcklein, Walter F. M. A1 - Leimkühler, Silke T1 - Rhodobacter capsulatus XdhC is involved in molybdenum cofactor binding and insertion into xanthine dehydrogenase N2 - 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 Y1 - 2006 UR - http://www.jbc.org/ U6 - https://doi.org/10.1074/jbc.M601617200 ER - TY - JOUR A1 - Neumann, Meina A1 - Mittelstaedt, Gerd A1 - Seduk, Farida A1 - Iobbi-Nivol, Chantal A1 - Leimkühler, Silke T1 - MocA is a specific cytidylyltransferase involved in molybdopterin cytosine dinucleotide biosynthesis in Escherichia coli N2 - 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. Y1 - 2009 UR - http://www.jbc.org/ U6 - https://doi.org/10.1074/jbc.M109.008565 SN - 0021-9258 ER -