Molybdopterin Biosynthesis Research Articles

Effect of Palmitoylethanolamide Compared to a Placebo on the Gut Microbiome and Biochemistry in an Overweight Adult Population: A Randomised, Placebo Controlled, Double-Blind Study.

This study investigates the effects of palmitoylethanolamide (PEA) on the gut microbiome of overweight adults. Fifty-eight participants (twenty males, thirty-eight females) aged 18-65 years with a BMI range of 30-40 kg/m2 were recruited. Participants were randomised to receive PEA (n = 36) or a placebo (n = 22) for 12 weeks. Microbiota composition, richness, diversity, and metabolic functions, faecal short chain fatty acids and calprotectin, pathology markers, and health-related questionnaires were analysed throughout the 12 weeks of supplementation. PEA supplementation significantly reduced triglyceride levels and IL-2 concentrations. No significant differences were found in the overall microbiota composition between the groups, and microbiota richness and diversity remained consistent for both groups. Functional analysis demonstrated no differences in functional richness and diversity, but specific pathways were modified. PEA supplementation resulted in a decrease in the abundance of pathways related to aromatic compound degradation, NAD interconversion, and L-glutamate degradation, while pathways associated with molybdopterin biosynthesis and O-antigen building blocks exhibited increased abundance. Increased production of O-antigen results in smooth LPS associated with reduced pathogenic stealth and persistence. PEA supplementation may influence specific microbial species, metabolic pathways, and reduce serum triglyceride and IL-2 concentration, shedding light on the intricate relationship between PEA, the microbiome, and host health.

Molybdopterin biosynthesis pathway contributes to the regulation of SaeRS two-component system by ClpP in Staphylococcus aureus

ABSTRACT In Staphylococcus aureus, the SaeRS two-component system is essential for the bacterium’s hemolytic activity and virulence. The Newman strain of S. aureus contains a variant of SaeS sensor kinase, SaeS L18P. Previously, we showed that, in the strain Newman, SaeS L18P is degraded by the membrane-bound protease FtsH. Intriguingly, the knockout mutation of clpP, encoding the cytoplasmic protease ClpP, greatly reduces the expression of SaeS L18P. Here, we report that, in the strain Newman, the positive regulatory role of ClpP on the SaeS L18P expression is due to its destabilizing effect on FtsH and degradation of MoeA, a molybdopterin biosynthesis protein. Although the transcription of ftsH was not affected by ClpP, the expression level of FtsH was increased in the clpP mutant. The destabilizing effect appears to be indirect because ClpXP did not directly degrade FtsH in an in vitro assay. Through transposon mutagenesis, we found out that the moeA gene, encoding the molybdopterin biosynthesis protein A, suppresses the hemolytic activity of S. aureus along with the transcription and expression of SaeS L18P. In a proteolysis assay, ClpXP directly degraded MoeA, demonstrating that MoeA is a substrate of the protease. In a murine bloodstream infection model, the moeA mutant displayed reduced virulence and lower survival compared with the WT strain. Based on these results, we concluded that ClpP positively controls the expression of SaeS L18P in an FtsH and MoeA-dependent manner, and the physiological role of MoeA outweighs its suppressive effect on the SaeRS TCS during infection.

Genome-Wide Association Study Reveals Genomic Regions Associated With Molybdenum Accumulation in Wheat Grains.

Molybdenum (Mo) is an essential micronutrient for almost all organisms. Wheat, a major staple crop worldwide, is one of the main dietary sources of Mo. However, the genetic basis for the variation of Mo content in wheat grains remains largely unknown. Here, a genome-wide association study (GWAS) was performed on the Mo concentration in the grains of 207 wheat accessions to dissect the genetic basis of Mo accumulation in wheat grains. As a result, 77 SNPs were found to be significantly associated with Mo concentration in wheat grains, among which 52 were detected in at least two sets of data and distributed on chromosome 2A, 7B, and 7D. Moreover, 48 out of the 52 common SNPs were distributed in the 726,761,412–728,132,521 bp genomic region of chromosome 2A. Three putative candidate genes, including molybdate transporter 1;2 (TraesCS2A02G496200), molybdate transporter 1;1 (TraesCS2A02G496700), and molybdopterin biosynthesis protein CNX1 (TraesCS2A02G497200), were identified in this region. These findings provide new insights into the genetic basis for Mo accumulation in wheat grains and important information for further functional characterization and breeding to improve wheat grain quality.

Major aldehyde dehydrogenase AldFGH of Gluconacetobacter diazotrophicus is independent of pyrroloquinoline quinone but dependent on molybdopterin for acetic acid fermentation.

Acetic acid fermentation involves the oxidation of ethanol to acetic acid via acetaldehyde as the intermediate and is catalyzed by the membrane-bound alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) of acetic acid bacteria. Although ADH depends on pyrroloquinoline quinone (PQQ), the prosthetic group associated with ALDH remains a matter of debate. This study aimed to address the dependency of ALDH of Gluconacetobacter diazotrophicus strain PAL5 on PQQ and the physiological role of ALDH in acetic acid fermentation. We constructed deletion mutant strains for both the ALDH gene clusters of PAL5, aldFGH and aldSLC. In addition, the adhAB operon for ADH was eliminated, since it shows ALDH activity. The triple-deletion derivative ΔaldFGH ΔaldSLC ΔadhAB failed to show ALDH activity, which suggested that ALDH activity in PAL5 is derived from these three enzyme complexes. Since the single-gene cluster deletion derivative ΔaldFGH lost most ALDH activity, and accumulated much higher acetaldehyde than wild type under acetic acid fermentation conditions, we concluded that AldFGH functions as the major ALDH in PAL5. Furthermore, deletion of the PQQ biosynthesis gene cluster (pqqABCDE) abolished ADH activity completely, but did not affect ALDH activity. Instead, the molybdopterin biosynthesis gene deletion derivatives lost ALDH activity. Thus, we concluded that the AldFGH and AldSLC complexes of Ga. diazotrophicus PAL5 require a form of molybdopterin but not PQQ for ALDH activity. KEY POINTS: • AldFGH is the major aldehyde dehydrogenase in Gluconacetobacter diazotrophicus PAL5. • Acetaldehyde accumulated from ethanol in the absence of AldFGH. • Molybdopterin, rather than pyrroloquinoline quinone, is required for AldFGH.

Model Substrate/Inactivation Reactions for MoaA and Ribonucleotide Reductases: Loss of Bromo, Chloro, or Tosylate Groups from C2 of 1,5-Dideoxyhomoribofuranoses upon Generation of an α-Oxy Radical at C3.

We report studies on radical-initiated fragmentations of model 1,5-dideoxyhomoribofuranose derivatives with bromo, chloro, and tosyloxy substituents on C2. The effects of stereochemical inversion at C2 were probed with the corresponding arabino epimers. In all cases, the elimination of bromide, chloride, and tosylate anions occurred when the 3-hydroxyl group was unprotected. The isolation of deuterium-labeled furanone products established heterolytic cleavage followed by the transfer of deuterium from labeled tributylstannane. In contrast, 3-O-methyl derivatives underwent the elimination of bromine or chlorine radicals to give the 2,3-alkene with no incorporation of label in the methyl vinyl ether. More drastic fragmentation occurred with both of the 3-O-methyl-2-tosyloxy epimers to give an aromatized furan derivative with no deuterium label. Contrasting results observed with the present anhydroalditol models relative to our prior studies with analogously substituted nucleoside models have demonstrated that insights from biomimetic chemical reactions can provide illumination of mechanistic pathways employed by ribonucleotide reductases (RNRs) and the MoaA enzyme involved in the biosynthesis of molybdopterin.

Proteome Mapping of a Cyanobacterium Reveals Distinct Compartment Organization and Cell-Dispersed Metabolism.

Cyanobacteria are complex prokaryotes, incorporating a Gram-negative cell wall and internal thylakoid membranes (TMs). However, localization of proteins within cyanobacterial cells is poorly understood. Using subcellular fractionation and quantitative proteomics, we produced an extensive subcellular proteome map of an entire cyanobacterial cell, identifying ∼67% of proteins in Synechocystis sp. PCC 6803, ∼1000 more than previous studies. Assigned to six specific subcellular regions were 1,712 proteins. Proteins involved in energy conversion localized to TMs. The majority of transporters, with the exception of a TM-localized copper importer, resided in the plasma membrane (PM). Most metabolic enzymes were soluble, although numerous pathways terminated in the TM (notably those involved in peptidoglycan monomer, NADP+, heme, lipid, and carotenoid biosynthesis) or PM (specifically, those catalyzing lipopolysaccharide, molybdopterin, FAD, and phylloquinol biosynthesis). We also identified the proteins involved in the TM and PM electron transport chains. The majority of ribosomal proteins and enzymes synthesizing the storage compound polyhydroxybuyrate formed distinct clusters within the data, suggesting similar subcellular distributions to one another, as expected for proteins operating within multicomponent structures. Moreover, heterogeneity within membrane regions was observed, indicating further cellular complexity. Cyanobacterial TM protein localization was conserved in Arabidopsis (Arabidopsis thaliana) chloroplasts, suggesting similar proteome organization in more developed photosynthetic organisms. Successful application of this technique in Synechocystis suggests it could be applied to mapping the proteomes of other cyanobacteria and single-celled organisms. The organization of the cyanobacterial cell revealed here substantially aids our understanding of these environmentally and biotechnologically important organisms.

Proteomic analysis of the salt-adapted and directly salt-(NaCl and NaCl+Na2SO4 mixture) stressed cyanobacterium Anabaena fertilissima

Salinity is a serious threat to agriculture productivity. Beneficial microbes could be simple and low-cost biological methods to mitigate the salt toxicity. The present study describes proteome dynamics and salt stress tolerance of the salt-primed (pre-exposed to salt) and directly salt-stressed cyanobacterium Anabaena fertilissima. The difference in the proteome of differently salt-treated and the control cells was the abundance of proteins. Out of 130 proteins resolved in control, 51.8% remained constant, 25.5% were upregulated, and 22.7% protein spots were downregulated in salt-adapted cells (exposed to 500 mM NaCl). However, in cells exposed to 250 mM NaCl, percentage of constant, upregulated, and downregulated proteins was 56.8, 16, and 27.2, whereas in the cells exposed to equimolar NaCl+Na2SO4 mixture, these values were 41.8, 29.4, and 28.7, respectively. This indicated that an altered protein expression occurred in large number of protein species under different salt-stress regimes. Four proteins showing significant and reproducible changes under salt stress showed close homology with photosystem I reaction center subunit XII, epoxyqueuosine reductase, response regulator protein VraR, and molybdopterin biosynthesis protein. The kinetic analysis revealed that salt treatment increased the abundance of all the four identified proteins. Despite the exposure to higher NaCl concentration (500 mM), salt-adapted cells showed minimal hyper accumulation of these four proteins, followed by NaCl and NaCl+Na2SO4 mixture. Lesser accumulation of these identified proteins in salt mixture stressed cells than that of NaCl stressed cells revealed that the availability of sulfur during salt-stress relieved the salt-toxicity, while Cl− increased it.

Genetic signatures for Helicobacter pylori strains of West African origin.

Helicobacter pylori is a genetically diverse bacterial species that colonizes the stomach in about half of the human population. Most persons colonized by H. pylori remain asymptomatic, but the presence of this organism is a risk factor for gastric cancer. Multiple populations and subpopulations of H. pylori with distinct geographic distributions are recognized. Genetic differences among these populations might be a factor underlying geographic variation in gastric cancer incidence. Relatively little is known about the genomic features of African H. pylori strains compared to other populations of strains. In this study, we first analyzed the genomes of H. pylori strains from seven globally distributed populations or subpopulations and identified encoded proteins that exhibited the highest levels of sequence divergence. These included secreted proteins, an LPS glycosyltransferase, fucosyltransferases, proteins involved in molybdopterin biosynthesis, and Clp protease adaptor (ClpS). Among proteins encoded by the cag pathogenicity island, CagA and CagQ exhibited the highest levels of sequence diversity. We then identified proteins in strains of Western African origin (classified as hspWAfrica by MLST analysis) with sequences that were highly divergent compared to those in other populations of strains. These included ATP-dependent Clp protease, ClpS, and proteins of unknown function. Three of the divergent proteins sequences identified in West African strains were characterized by distinct insertions or deletions up to 8 amino acids in length. These polymorphisms in rapidly evolving proteins represent robust genetic signatures for H. pylori strains of West African origin.

Nitrogenase Cofactor Assembly: An Elemental Inventory.

Nitrogenase is known for its remarkable ability to catalyze the reduction of N2 to NH3, and C1 substrates to short-chain hydrocarbon products, under ambient conditions. The best-studied Mo-nitrogenase utilizes a complex metallocofactor as the site of substrate binding and reduction. Designated the M-cluster, this [MoFe7S9C(R-homocitrate)] cluster can be viewed as [MoFe3S3] and [Fe4S3] subclusters bridged by three μ2-sulfides and one μ6-interstitial carbide, with its Mo end further coordinated by an R-homocitrate moiety. The unique cofactor has attracted considerable attention ever since its discovery; however, the complexity of its structure has hindered mechanistic understanding and chemical synthesis of this cofactor. Motivated by the pressing questions related to the structure and function of the nitrogenase cofactor, one major thrust of our research has been to unravel the key biosynthetic steps of this metallocluster to cultivate a deeper understanding of these reactions and their effects on functionalizing the cofactor. In this Account, we will discuss our recent work that provides insights into how simple Fe and S atoms, along with a single C atom, a heterometallic Mo atom and an organic homocitrate entity, are assembled into one of the most complex metalloclusters known in Nature. Combined biochemical, spectroscopic and structural studies have led us to a working model of M-cluster assembly, which starts with the sequential synthesis of small [Fe2S2] and [Fe4S4] units by NifS/U, followed by the coupling and rearrangement of two [Fe4S4] clusters on NifB concomitant with the insertion of an interstitial carbide and a "9th sulfur" that give rise to a [Fe8S9C] core that is nearly indistinguishable in structure to the M-cluster except for the absence of Mo and homocitrate. This 8Fe core is then matured into an M-cluster on NifEN upon substitution of a Mo-homocitrate conjugate for one terminal Fe atom of the cluster prior to transfer of the M-cluster to its target binding site in the catalytic component of Mo-nitrogenase. Taking stock of the elemental inventory during the cofactor assembly process, the core Fe and S atoms are derived from modular fusion of FeS building blocks, going through 2Fe, 4Fe and 8Fe stages to generate an 8Fe core of the cofactor. However, such a flow of Fe/S along the biosynthetic pathway of the M-cluster is "intervened" by the insertion of C and Mo, which renders the cofactor unique in structure and reactivity. Insertion of C occurs through a novel, radical SAM-dependent mechanism, which involves SN2-type methyl transfer from SAM to a [Fe4S4] cluster pair, hydrogen abstraction of the transferred methyl group by a SAM-derived 5'-dA· radical, and further deprotonation of the resultant methylene radical concomitant with radical chemistry-based coupling and rearrangement of the [Fe4S4] cluster pair into an [Fe8S9C] core. Insertion of Mo, on the other hand, employs an ATPase-dependent mechanism that parallels metal trafficking in the biosynthesis of molybdopterin and CO dehydrogenase cofactors. These findings provide a nice framework for further exploration of the "black box" of nitrogenase cofactor assembly and function.

Genetic dissection of chlorate respiration in Pseudomonas stutzeri PDA reveals syntrophic (per)chlorate reduction.

Genes important for growth of Pseudomonas stutzeri PDA on chlorate were identified using a randomly DNA bar-coded transposon mutant library. During chlorate reduction, mutations in genes encoding the chlorate reductase clrABC, predicted molybdopterin cofactor chaperon clrD, molybdopterin biosynthesis and two genes of unknown function (clrE, clrF) had fitness defects in pooled mutant assays (Bar-seq). Markerless in-frame deletions confirmed that clrA, clrB and clrC were essential for chlorate reduction, while clrD, clrE and clrF had less severe growth defects. Interestingly, the key detoxification gene cld was essential for chlorate reduction in isogenic pure culture experiments, but showed only minor fitness defects in Bar-seq experiments. We hypothesized this was enabled through chlorite dismutation by the community, as most strains in the Bar-seq library contained an intact cld. In support of this, Δcld grew with wild-type PDA or ΔclrA, and purified Cld also restored growth to the Δcld mutant. Expanding on this, wild-type PDA and a Δcld mutant of the perchlorate reducer Azospira suillum PS grew on perchlorate in co-culture, but not individually. These results demonstrate that co-occurrence of cld and a chloroxyanion reductase within a single organism is not necessary and raises the possibility of syntrophic (per)chlorate respiration in the environment.

The Structure of Escherichia coli TcdA (Also Known As CsdL) Reveals a Novel Topology and Provides Insight into the tRNA Binding Surface Required for N6-Threonylcarbamoyladenosine Dehydratase Activity

Escherichia coli TcdA (also known as CsdL) was previously shown to catalyze the ATP-dependent dehydration/cyclization of hypermodified tRNA N6-threonylcarbamoyladenosine into further cyclic N6-threonylcarbamoyladenosine. In this study, we report the X-ray crystal structures of E. coli TcdA with either AMP or ATP bound. The AMP/ATP-bound N-terminal sub-domain of TcdA resembles the ATP-binding Rossmann fold of E. coli ThiF and MoeB that are enzymes respectively taking part in the biosynthesis of thiamine and molybdopterin; however, the remaining C-terminal sub-domain of TcdA adopts a structure unrelated to any other known folds. In TcdA, the ATP-utilizing adenylation of tRNA N6-threonylcarbamoyladenosine and a subsequent thioester formation via an active cysteine, similar to the mechanisms in ThiF and MoeB, could take place for the dehydratase function. Analysis of the structure with sequence alignment suggests the disordered Cys234 of TcdA as the most likely catalytic residue. The structure further indicates that the C-terminal sub-domain can provide a binding interface for the tRNA substrate. Binding study using the surface mutants of TcdA and tRNA reveals that the positively charged regions of mainly the C-terminal sub-domain are important for the tRNA recognition.

Molybdopterin biosynthesis: trapping of intermediates for the MoaA-catalyzed reaction using 2'-deoxyGTP and 2'-chloroGTP as substrate analogues.

MoaA is a radical S-adenosylmethionine (AdoMet) enzyme that catalyzes a complex rearrangement of guanosine-5'-triphosphate (GTP) in the first step of molybdopterin biosynthesis. In this paper, we provide additional characterization of the MoaA reaction product, describe the use of 2′-chloroGTP to trap the GTP C3′ radical, generated by hydrogen atom transfer to the 5′-deoxyadenosyl radical, and the use of 2′-deoxyGTP to block a late step in the reaction sequence. These probes, coupled with the previously reported trapping of an intermediate in which C3′ of the ribose is linked to C8 of the purine, allow us to propose a plausible mechanism for the MoaA-catalyzed reaction.

Cysteine desulphurase plays an important role in environmental adaptation of the hyperthermophilic archaeon Thermococcus kodakarensis.

The sulphur atoms of sulphur-containing cofactors that are essential for numerous cellular functions in living organisms originate from L-cysteine via cysteine desulphurase (CSD) activity. However, many (hyper)thermophilic archaea, which thrive in solfataric fields and are positioned near the root of the evolutionary tree of life, lack CSD orthologues. The existence of CSD orthologues in a subset of (hyper)thermophilic archaea is of interest with respect to the evolution of sulphur-trafficking systems for the cofactors. This study demonstrates that the disruption of the csd gene of Thermococcus kodakarensis, a facultative elemental sulphur (S(0))-reducing hyperthermophilic archaeon, encoding Tk-CSD, conferred a growth defect evident only in the absence of S(0), and that growth can be restored by the addition of S(0), but not sulphide. We show that the csd gene is not required for biosynthesis of thiamine pyrophosphate or molybdopterin, irrespective of the presence or absence of S(0), but is necessary for iron-sulphur cluster biosynthesis in the absence of S(0). Recombinant form of Tk-CSD expressed in Escherichia coli was obtained and it was found to catalyse the desulphuration of L-cysteine. The obtained data suggest that hyperthermophiles might benefit from a capacity for CSD-dependent iron-sulphur cluster biogenesis, which allows them to thrive outside solfataric environments.

Prokaryotic ubiquitin-like protein modification.

Prokaryotes form ubiquitin (Ub)-like isopeptide bonds on the lysine residues of proteins by at least two distinct pathways that are reversible and regulated. In mycobacteria, the C-terminal Gln of Pup (prokaryotic ubiquitin-like protein) is deamidated and isopeptide linked to proteins by a mechanism distinct from ubiquitylation in enzymology yet analogous to ubiquitylation in targeting proteins for destruction by proteasomes. Ub-fold proteins of archaea (SAMPs, small archaeal modifier proteins) and Thermus (TtuB, tRNA-two-thiouridine B) that differ from Ub in amino acid sequence, yet share a common β-grasp fold, also form isopeptide bonds by a mechanism that appears streamlined compared with ubiquitylation. SAMPs and TtuB are found to be members of a small group of Ub-fold proteins that function not only in protein modification but also in sulfur-transfer pathways associated with tRNA thiolation and molybdopterin biosynthesis. These multifunctional Ub-fold proteins are thought to be some of the most ancient of Ub-like protein modifiers.

Ubiquitin‐fold protein and JAMM/MPN+ metalloprotease constitute a regulating system for enzyme inhibition and activation (931.1)

Molybdenum cofactor (Moco) is essential for diverse enzymes to form the catalytic center. Moco biosynthesis is a conserved pathway existent in all three life domains, and Moco biosynthesis deficiency in humans causes serious disease. Molybdopterin (MPT) synthase catalyzes the biosynthesis of MPT, a precursor of Moco. Crystal structures of MPT synthase have been reported for over 10 years; however, its post‐translational regulation is poorly known. Here we report a MPT synthase inhibiting and activating mechanism by using the archaeon Haloferax volcanii as a model. We provide strong evidence that the MPT synthase activity is inhibited by SAMP1 (an ubiquitin‐fold protein homolog of the small subunit of MPT synthase, MoaD) covalent linkage to MoaE (a homolog of the large subunit of MPT synthase). Furthermore, we show that the JAMM/MPN+ metalloprotease HvJAMM1 is required to cleave covalently linked inactive SAMP1‐MoaE to free SAMP1 and MoaE, which suggests activation of MPT synthase. Since SAMP1 is an ubiquitin‐fold small protein, our research also indicates a broad idea that ubiquitin‐fold proteins can inhibit an enzyme by linking to its active site and that a protease, like HvJAMM1, can cleave this linkage and reactivate the enzyme.Grant Funding Source: NIH R01 GM057498 and DOE DE‐FG02‐05ER15650

New challenges in structural biology: catching the complexity of dynamic nanomachines.

New challenges in structural biology: catching the complexity of dynamic nanomachines.

Influence of the molybdenum cofactor biosynthesis on anaerobic respiration, biofilm formation and motility in Burkholderia thailandensis

Burkholderia thailandensis is closely related to Burkholderia pseudomallei, a bacterial pathogen and the causative agent of melioidosis. B. pseudomallei can survive and persist within a hypoxic environment for up to one year and has been shown to grow anaerobically in the presence of nitrate. Currently, little is known about the role of anaerobic respiration in pathogenesis of melioidosis. Using B. thailandensis as a model, a library of 1344 transposon mutants was created to identify genes required for anaerobic nitrate respiration. One transposon mutant (CA01) was identified with an insertion in BTH_I1704 (moeA), a gene required for the molybdopterin biosynthetic pathway. This pathway is involved in the synthesis of a molybdopterin cofactor required for a variety of molybdoenzymes, including nitrate reductase. Disruption of molybdopterin biosynthesis prevented growth under anaerobic conditions, when using nitrate as the sole terminal electron acceptor. Defects in anaerobic respiration, nitrate reduction, motility and biofilm formation were observed for CA01. Mutant complementation with pDA-17:BTH_I1704 was able to restore anaerobic growth on nitrate, nitrate reductase activity and biofilm formation, but did not restore motility. This study highlights the potential importance of molybdoenzyme-dependent anaerobic respiration in the survival and virulence of B. thailandensis.

Simultaneous Involvement of a Tungsten-Containing Aldehyde:Ferredoxin Oxidoreductase and a Phenylacetaldehyde Dehydrogenase in Anaerobic Phenylalanine Metabolism

Anaerobic phenylalanine metabolism in the denitrifying betaproteobacterium Aromatoleum aromaticum is initiated by conversion of phenylalanine to phenylacetate, which is further metabolized via benzoyl-coenzyme A (CoA). The formation of phenylacetate is catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, and a phenylacetaldehyde-oxidizing enzyme. The presence of these enzymes was detected in extracts of cells grown with phenylalanine and nitrate. We found that two distinct enzymes are involved in the oxidation of phenylacetaldehyde to phenylacetate, an aldehyde:ferredoxin oxidoreductase (AOR) and a phenylacetaldehyde dehydrogenase (PDH). Based on sequence comparison, growth studies with various tungstate concentrations, and metal analysis of the enriched enzyme, AOR was shown to be a tungsten-containing enzyme, necessitating specific cofactor biosynthetic pathways for molybdenum- and tungsten-dependent enzymes simultaneously. We predict from the genome sequence that most enzymes of molybdopterin biosynthesis are shared, while the molybdate/tungstate uptake systems are duplicated and specialized paralogs of the sulfur-inserting MoaD and the metal-inserting MoeA proteins seem to be involved in dedicating biosynthesis toward molybdenum or tungsten cofactors. We also characterized PDH biochemically and identified both NAD(+) and NADP(+) as electron acceptors. We identified the gene coding for the enzyme and purified a recombinant Strep-tagged PDH variant. The homotetrameric enzyme is highly specific for phenylacetaldehyde, has cooperative kinetics toward the substrate, and shows considerable substrate inhibition. Our data suggest that A. aromaticum utilizes PDH as the primary enzyme during anaerobic phenylalanine degradation, whereas AOR is not essential for the metabolic pathway. We hypothesize a function as a detoxifying enzyme if high aldehyde concentrations accumulate in the cytoplasm, which would lead to substrate inhibition of PDH.

Genetic Evidence for a Molybdopterin-Containing Tellurate Reductase

The genetic identity and cofactor composition of the bacterial tellurate reductase are currently unknown. In this study, we examined the requirement of molybdopterin biosynthesis and molybdate transporter genes for tellurate reduction in Escherichia coli K-12. The results show that mutants deleted of the moaA, moaB, moaE, or mog gene in the molybdopterin biosynthesis pathway lost the ability to reduce tellurate. Deletion of the modB or modC gene in the molybdate transport pathway also resulted in complete loss of tellurate reduction activity. Genetic complementation by the wild-type sequences restored tellurate reduction activity in the mutant strains. These findings provide genetic evidence that tellurate reduction in E. coli involves a molybdoenzyme.

Catalysis of a New Ribose Carbon-Insertion Reaction by the Molybdenum Cofactor Biosynthetic Enzyme MoaA

MoaA, a radical S-adenosylmethionine enzyme, catalyzes the first step in molybdopterin biosynthesis. This reaction involves a complex rearrangement in which C8 of guanosine triphosphate is inserted between C2' and C3' of the ribose. This study identifies the site of initial hydrogen atom abstraction by the adenosyl radical and advances a mechanistic proposal for this unprecedented reaction.