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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.
Iron-sulfur (Fe-S) clusters are essential protein cofactors. In enzymes, they are present either in the rhombic [2Fe-2S] or the cubic [4Fe-4S] form, where they are involved in catalysis and electron transfer and in the biosynthesis of metal-containing prosthetic groups like the molybdenum cofactor (Moco). Here, we give an overview of the assembly of Fe-S clusters in bacteria and humans and present their connection to the Moco biosynthesis pathway. In all organisms, Fe-S cluster assembly starts with the abstraction of sulfur froml-cysteine and its transfer to a scaffold protein. After formation, Fe-S clusters are transferred to carrier proteins that insert them into recipient apo-proteins. In eukaryotes like humans and plants, Fe-S cluster assembly takes place both in mitochondria and in the cytosol. Both Moco biosynthesis and Fe-S cluster assembly are highly conserved among all kingdoms of life. Moco is a tricyclic pterin compound with molybdenum coordinated through its unique dithiolene group. Moco biosynthesis begins in the mitochondria in a Fe-S cluster dependent step involving radical/S-adenosylmethionine (SAM) chemistry. An intermediate is transferred to the cytosol where the dithiolene group is formed, to which molybdenum is finally added. Further connections between Fe-S cluster assembly and Moco biosynthesis are discussed in detail.
ecoAO
(2017)
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.
Mammalian aldehyde oxidases (AOXs) are molybdo-flavoenzymes which are present in many tissues in various mammalian species, including humans and rodents. Different species contain a different number of AOX isoforms. In particular, the reasons why mammals other than humans express a multiplicity of tissue-specific AOX enzymes is unknown. In mouse, the isoforms mAOX1, mAOX3, mAOX4 and mAOX2 are present. We previously established a codon-optimized heterologous expression systems for the mAOX1-4 isoforms in Escherichia coli that gives yield to sufficient amounts of active protein for kinetic characterizations and sets the basis in this study for site-directed mutagenesis and structure-function studies. A direct and simultaneous comparison of the enzymatic properties and characteristics of the four enzymes on a larger number of substrates has never been performed. Here, thirty different structurally related aromatic, aliphatic and N-heterocyclic compounds were used as substrates, and the kinetic parameters of all four mAOX enzymes were directly compared. The results show that especially mAOX4 displays a higher substrate selectivity, while no major differences between mAOX1, mAOX2 and mAOX3 were identified. Generally, mAOX1 was the enzyme with the highest catalytic turnover for most substrates. To understand the factors that contribute to the substrate specificity of mAOX4, site-directed mutagenesis was applied to substitute amino acids in the substrate-binding funnel by the ones present in mAOX1, mAOX3, and mAOX2. An increase in activity was obtained by the amino acid exchange M1088V in the active site identified to be specific for mAOX4, to the amino acid identified in mAOX3.
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.
Same but different
(2018)
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.
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.