The biosynthesis of the molybdenum cofactors in Escherichia coli.

The biosynthesis of the molybdenum cofactor (Moco) is highly conserved among all kingdoms of life. In all molybdoenzymes containing Moco, the molybdenum atom is coordinated to a dithiolene group present in the pterin-based 6-alkyl side chain of molybdopterin (MPT). In general, the biosynthesis of Moco can be divided into four steps in in bacteria: (i) the starting point is the formation of the cyclic pyranopterin monophosphate (cPMP) from 5'-GTP, (ii) in the second step the two sulfur atoms are inserted into cPMP leading to the formation of MPT, (iii) in the third step the molybdenum atom is inserted into MPT to form Moco and (iv) in the fourth step bis-Mo-MPT is formed and an additional modification of Moco is possible with the attachment of a nucleotide (CMP or GMP) to the phosphate group of MPT, forming the dinucleotide variants of Moco. This review presents an update on the well-characterized Moco biosynthesis in the model organism Escherichia coli including novel discoveries from the recent years.

The impact of a His-tag on DNA binding by RNA polymerase alpha-C-terminal domain from Helicobacter pylori.

Polyhistidine tags (His-tags) are commonly employed in protein purification strategies due to the high affinity and specificity for metal-NTA columns, the relative simplicity of such protocols, and the assumption that His-tags do not affect the native activities of proteins. However, there is a growing body of evidence that such tags can modulate protein structure and function. In this study, we demonstrate that a His-tag impacts DNA complex formation by the C-terminal domain of the α-subunit (αCTD) of Helicobacter pylori RNA polymerase in a metal-dependent fashion. The αCTD was purified with a cleavable His-tag, and complex formation between αCTD, the nickel-responsive metalloregulator HpNikR, and DNA was investigated using electrophoretic mobility shift assays. An interaction between His-tagged αCTD (HisαCTD) and the HpNikR-DNA complex was observed; however, this interaction was not observed upon removal of the His-tag. Further analysis revealed that complex formation between HisαCTD and DNA is non-specific and dependent on the type of metal ions present. Overall, the results indicate that a histidine tag is able to modulate DNA-binding activity and suggests that the impact of metal affinity tags should be considered when analyzing the in vitro biomolecular interactions of metalloproteins.

Iron-Dependent Regulation of Molybdenum Cofactor Biosynthesis Genes in Escherichia coli.

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 [4Fe-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.

Horizontal acquisition of a hypoxia-responsive molybdenum cofactor biosynthesis pathway contributed to Mycobacterium tuberculosis pathoadaptation.

The unique ability of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, to persist for long periods of time in lung hypoxic lesions chiefly contributes to the global burden of latent TB. We and others previously reported that the M. tuberculosis ancestor underwent massive episodes of horizontal gene transfer (HGT), mostly from environmental species. Here, we sought to explore whether such ancient HGT played a part in M. tuberculosis evolution towards pathogenicity. We were interested by a HGT-acquired M. tuberculosis-specific gene set, namely moaA1-D1, which is involved in the biosynthesis of the molybdenum cofactor. Horizontal acquisition of this gene set was striking because homologues of these moa genes are present all across the Mycobacterium genus, including in M. tuberculosis. Here, we discovered that, unlike their paralogues, the moaA1-D1 genes are strongly induced under hypoxia. In vitro, a M. tuberculosis moaA1-D1-null mutant has an impaired ability to respire nitrate, to enter dormancy and to survive in oxygen-limiting conditions. Conversely, heterologous expression of moaA1-D1 in the phylogenetically closest non-TB mycobacterium, Mycobacterium kansasii, which lacks these genes, improves its capacity to respire nitrate and grants it with a marked ability to survive oxygen depletion. In vivo, the M. tuberculosis moaA1-D1-null mutant shows impaired survival in hypoxic granulomas in C3HeB/FeJ mice, but not in normoxic lesions in C57BL/6 animals. Collectively, our results identify a novel pathway required for M. tuberculosis resistance to host-imposed stress, namely hypoxia, and provide evidence that ancient HGT bolstered M. tuberculosis evolution from an environmental species towards a pervasive human-adapted pathogen.

Identification and characterization of the rice pre-harvest sprouting mutants involved in molybdenum cofactor biosynthesis.

In cereal crops, ABA deficiency during seed maturation phase causes pre-harvest sprouting (PHS), and molybdenum cofactor (MoCo) is required for ABA biosynthesis. Here, two rice PHS mutants F254 and F5-1 were characterized. In addition to the PHS, these mutants showed pleiotropic phenotypes such as twisting and slender leaves, and then died when the seedling developed to four or five leaves. Map-based cloning showed that OsCNX6 and OsCNX1 encoding homologs of MoaE and MoeA were responsible for F254 and F5-1 mutants, respectively. Genetic complementation indicated that OsCNX6 not only rescued the PHS and seedling lethal phenotype of the cnx6 mutant, but also recovered the MoCo-dependent enzyme activities such as xanthine dehydrogenase (XDH), aldehyde oxidase (AO), nitrate reductase (NR) and sulfite oxidase (SO). Expression pattern showed that OsCNX6 was richly expressed in seed during embryo maturation by quantitative reverse transcriptase PCR and RNA in situ hybridization. Furthermore, the OsCNX6 overexpression plants can significantly enhance the MoCo-dependent enzyme activities, and improved the osmotic and salt stress tolerance without unfavorable phenotypes. Collectively, these data indicated that OsCNX6 participated in MoCo biosynthesis, and is essential for rice development, especially for seed dormancy and germination, and OsCNX6 could be an effective target for improving abiotic stress tolerance in rice.

Recombinant expression and purification of a functional bacterial metallo-chaperone PbrD-fusion construct as a potential biosorbent for Pb(II).

PbrD is a lead (II) binding protein encoded by the pbr lead resistance operon found exclusively in Cupriavidus metallidurans CH34. Its ability to sequester Pb(II) shows potential for it to be developed as a biosorbent for Pb in the bioremediation of contaminated wastewaters. In this study the pbrD gene from C. metallidurans CH34 was transformed and overexpressed in Escherichia coli BL21 (DE3) using the pET32 Xa/Lic vector. Optimal expression of recombinant (r)PbrD (∼50 kDa) was achieved post-induction with IPTG within inclusion bodies (IBs). Inclusion bodies were solubilised by denaturation and purified by Ni-NTA affinity chromatography. The purified denatured protein containing the N-terminal Trx•Tag™, His•Tag® and S®Tag™ was refolded in vitro via dialysis to a biologically functional form. Circular dichroism spectra of refolded rPbrD-fusion protein indicated a high degree of turns, ß-sheets and 310 helices content and tryptophan fluorescence showed a structural conformational change in the presence of Pb(II). Refolded rPbrD-fusion protein bound 99.7% of Pb(II) when mixed with lead nitrate in ten-fold increasing concentrations. Adsorption isotherms including Langmuir, Freundlich, Temkin and Dubinin-Radushkevich models were applied to determine the biosorption mechanism. A biologically functional rPbrD-fusion protein has potential application in the development of a biosorbent for remediation of Pb(II) from wastewater.

Genetic dissection of cyclic pyranopterin monophosphate biosynthesis in plant mitochondria.

Mitochondria play a key role in the biosynthesis of two metal cofactors, iron-sulfur (FeS) clusters and molybdenum cofactor (Moco). The two pathways intersect at several points, but a scarcity of mutants has hindered studies to better understand these links. We screened a collection of sirtinol-resistant Arabidopsis thaliana mutants for lines with decreased activities of cytosolic FeS enzymes and Moco enzymes. We identified a new mutant allele of ATM3 (ABC transporter of the mitochondria 3), encoding the ATP-binding cassette transporter of the mitochondria 3 (systematic name ABCB25), confirming the previously reported role of ATM3 in both FeS cluster and Moco biosynthesis. We also identified a mutant allele in CNX2, cofactor of nitrate reductase and xanthine dehydrogenase 2, encoding GTP 3',8-cyclase, the first step in Moco biosynthesis which is localized in the mitochondria. A single-nucleotide polymorphism in cnx2-2 leads to substitution of Arg88 with Gln in the N-terminal FeS cluster-binding motif. cnx2-2 plants are small and chlorotic, with severely decreased Moco enzyme activities, but they performed better than a cnx2-1 knockout mutant, which could only survive with ammonia as a nitrogen source. Measurement of cyclic pyranopterin monophosphate (cPMP) levels by LC-MS/MS showed that this Moco intermediate was below the limit of detection in both cnx2-1 and cnx2-2, and accumulated more than 10-fold in seedlings mutated in the downstream gene CNX5 Interestingly, atm3-1 mutants had less cPMP than wild type, correlating with previous reports of a similar decrease in nitrate reductase activity. Taken together, our data functionally characterize CNX2 and suggest that ATM3 is indirectly required for cPMP synthesis.

Radical Breakthroughs in Natural Product and Cofactor Biosynthesis.

The radical SAM (S-adenosyl-l-methionine) superfamily is one of the largest group of enzymes with >113000 annotated sequences [Landgraf, B. J., et al. (2016) Annu. Rev. Biochem. 85, 485-514]. Members of this superfamily catalyze the reductive cleavage of SAM using an oxygen sensitive 4Fe-4S cluster to transiently generate 5'-deoxyadenosyl radical that is subsequently used to initiate diverse free radical-mediated reactions. Because of the unique reactivity of free radicals, radical SAM enzymes frequently catalyze chemically challenging reactions critical for the biosynthesis of unique structures of cofactors and natural products. In this Perspective, I will discuss the impact of characterizing novel functions in radical SAM enzymes on our understanding of biosynthetic pathways and use two recent examples from my own group with a particular emphasis on two radical SAM enzymes that are responsible for carbon skeleton formation during the biosynthesis of a cofactor and natural products.

Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis.

The molybdenum cofactor (Moco) is essential for all kingdoms of life, plays central roles in various biological processes, and must be biosynthesized de novo. During Moco biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangement of guanosine 5'-triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC (molybdenum cofactor biosynthesis protein A and C, respectively). Conventionally, MoaA was considered to catalyze the majority of this transformation, with MoaC playing little or no role in the pyranopterin formation. Recently, this view was challenged by the isolation of 3',8-cyclo-7,8-dihydro-guanosine 5'-triphosphate (3',8-cH2GTP) as the product of in vitro MoaA reactions. To elucidate the mechanism of formation of Moco pyranopterin backbone, we performed biochemical characterization of 3',8-cH2GTP and functional and X-ray crystallographic characterizations of MoaC. These studies revealed that 3',8-cH2GTP is the only product of MoaA that can be converted to cPMP by MoaC. Our structural studies captured the specific binding of 3',8-cH2GTP in the active site of MoaC. These observations provided strong evidence that the physiological function of MoaA is the conversion of GTP to 3',8-cH2GTP (GTP 3',8-cyclase), and that of MoaC is to catalyze the rearrangement of 3',8-cH2GTP into cPMP (cPMP synthase). Furthermore, our structure-guided studies suggest that MoaC catalysis involves the dynamic motions of enzyme active-site loops as a way to control the timing of interaction between the reaction intermediates and catalytically essential amino acid residues. Thus, these results reveal the previously unidentified mechanism behind Moco biosynthesis and provide mechanistic and structural insights into how enzymes catalyze complex rearrangement reactions.

From the Eukaryotic Molybdenum Cofactor Biosynthesis to the Moonlighting Enzyme mARC.

All eukaryotic molybdenum (Mo) enzymes contain in their active site a Mo Cofactor (Moco), which is formed by a tricyclic pyranopterin with a dithiolene chelating the Mo atom. Here, the eukaryotic Moco biosynthetic pathway and the eukaryotic Moco enzymes are overviewed, including nitrate reductase (NR), sulfite oxidase, xanthine oxidoreductase, aldehyde oxidase, and the last one discovered, the moonlighting enzyme mitochondrial Amidoxime Reducing Component (mARC). The mARC enzymes catalyze the reduction of hydroxylated compounds, mostly N-hydroxylated (NHC), but as well of nitrite to nitric oxide, a second messenger. mARC shows a broad spectrum of NHC as substrates, some are prodrugs containing an amidoxime structure, some are mutagens, such as 6-hydroxylaminepurine and some others, which most probably will be discovered soon. Interestingly, all known mARC need the reducing power supplied by different partners. For the NHC reduction, mARC uses cytochrome b5 and cytochrome b5 reductase, however for the nitrite reduction, plant mARC uses NR. Despite the functional importance of mARC enzymatic reactions, the structural mechanism of its Moco-mediated catalysis is starting to be revealed. We propose and compare the mARC catalytic mechanism of nitrite versus NHC reduction. By using the recently resolved structure of a prokaryotic MOSC enzyme, from the mARC protein family, we have modeled an in silico three-dimensional structure of a eukaryotic homologue.

Functional Complementation Studies Reveal Different Interaction Partners of Escherichia coli IscS and Human NFS1.

The trafficking and delivery of sulfur to cofactors and nucleosides is a highly regulated and conserved process among all organisms. All sulfur transfer pathways generally have an l-cysteine desulfurase as an initial sulfur-mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of sulfur-containing biomolecules like iron-sulfur (Fe-S) clusters, thiamine, biotin, lipoic acid, the molybdenum cofactor (Moco), and thiolated nucleosides in tRNA. The human l-cysteine desulfurase NFS1 and the Escherichia coli homologue IscS share a level of amino acid sequence identity of ∼60%. While E. coli IscS has a versatile role in the cell and was shown to have numerous interaction partners, NFS1 is mainly localized in mitochondria with a crucial role in the biosynthesis of Fe-S clusters. Additionally, NFS1 is also located in smaller amounts in the cytosol with a role in Moco biosynthesis and mcm5s2U34 thio modifications of nucleosides in tRNA. NFS1 and IscS were conclusively shown to have different interaction partners in their respective organisms. Here, we used functional complementation studies of an E. coli iscS deletion strain with human NFS1 to dissect their conserved roles in the transfer of sulfur to a specific target protein. Our results show that human NFS1 and E. coli IscS share conserved binding sites for proteins involved in Fe-S cluster assembly like IscU, but not with proteins for tRNA thio modifications or Moco biosynthesis. In addition, we show that human NFS1 was almost fully able to complement the role of IscS in Moco biosynthesis when its specific interaction partner protein MOCS3 from humans was also present.

The Role of SufS Is Restricted to Fe-S Cluster Biosynthesis in Escherichia coli.

In Escherichia coli, two different systems that are important for the coordinate formation of Fe-S clusters have been identified, namely, the ISC and SUF systems. The ISC system is the housekeeping Fe-S machinery, which provides Fe-S clusters for numerous cellular proteins. The IscS protein of this system was additionally revealed to be the primary sulfur donor for several sulfur-containing molecules with important biological functions, among which are the molybdenum cofactor (Moco) and thiolated nucleosides in tRNA. Here, we show that deletion of central components of the ISC system in addition to IscS leads to an overall decrease in Fe-S cluster enzyme and molybdoenzyme activity in addition to a decrease in the number of Fe-S-dependent thiomodifications of tRNA, based on the fact that some proteins involved in Moco biosynthesis and tRNA thiolation are Fe-S-dependent. Complementation of the ISC deficient strains with the suf operon restored the activity of Fe-S-containing proteins, including the MoaA protein, which is involved in the conversion of 5'GTP to cyclic pyranopterin monophosphate in the fist step of Moco biosynthesis. While both systems share a high degree of similarity, we show that the function of their respective l-cysteine desulfurase IscS or SufS is specific for each cellular pathway. It is revealed that SufS cannot play the role of IscS in sulfur transfer for the formation of 2-thiouridine, 4-thiouridine, or the dithiolene group of molybdopterin, being unable to interact with TusA or ThiI. The results demonstrate that the role of the SUF system is exclusively restricted to Fe-S cluster assembly in the cell.

Molybdate uptake by Agrobacterium tumefaciens correlates with the cellular molybdenum cofactor status.

Many enzymes require the molybdenum cofactor, Moco. Under Mo-limiting conditions, the high-affinity ABC transporter ModABC permits molybdate uptake and Moco biosynthesis in bacteria. Under Mo-replete conditions, Escherichia coli represses modABC transcription by the one-component regulator, ModE, consisting of a DNA-binding and a molybdate-sensing domain. Instead of a full-length ModE protein, many bacteria have a shorter ModE protein, ModE(S) , consisting of a DNA-binding domain only. Here, we asked how such proteins sense the intracellular molybdenum status. We show that the Agrobacterium tumefaciens ModE(S) protein Atu2564 is essential for modABC repression. ModE(S) binds two Mo-boxes in the modA promoter as shown by electrophoretic mobility shift assays. Northern analysis revealed cotranscription of modE(S) with the upstream gene, atu2565, which was dispensable for ModE(S) activity. To identify genes controlling ModE(S) function, we performed transposon mutagenesis. Tn5 insertions resulting in derepressed modA transcription mapped to the atu2565-modE(S) operon and several Moco biosynthesis genes. We conclude that A. tumefaciens ModE(S) activity responds to Moco availability rather than to molybdate concentration directly, as is the case for E. coli ModE. Similar results in Sinorhizobium meliloti suggest that Moco dependence is a common feature of ModE(S) regulators.

Shared function and moonlighting proteins in molybdenum cofactor biosynthesis.

The biosynthesis of the molybdenum cofactor (Moco) is a highly conserved pathway in bacteria, archaea and eukaryotes. The molybdenum atom in Moco-containing enzymes is coordinated to the dithiolene group of a tricyclic pyranopterin monophosphate cofactor. The biosynthesis of Moco can be divided into three conserved steps, with a fourth present only in bacteria and archaea: (1) formation of cyclic pyranopterin monophosphate, (2) formation of molybdopterin (MPT), (3) insertion of molybdenum into MPT to form Mo-MPT, and (4) additional modification of Mo-MPT in bacteria with the attachment of a GMP or CMP nucleotide, forming the dinucleotide variants of Moco. While the proteins involved in the catalytic reaction of each step of Moco biosynthesis are highly conserved among the Phyla, a surprising link to other cellular pathways has been identified by recent discoveries. In particular, the pathways for FeS cluster assembly and thio-modifications of tRNA are connected to Moco biosynthesis by sharing the same protein components. Further, proteins involved in Moco biosynthesis are not only shared with other pathways, but additionally have moonlighting roles. This review gives an overview of Moco biosynthesis in bacteria and humans and highlights the shared function and moonlighting roles of the participating proteins.

Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization.

Molybdenum is an essential nutrient for metabolism in plant, bacteria, and animals. Molybdoenzymes are involved in nitrogen assimilation and oxidoreductive detoxification, and bioconversion reactions of environmental, industrial, and pharmaceutical interest. Molybdoenzymes contain a molybdenum cofactor (Moco), which is a pyranopterin heterocyclic compound that binds a molybdenum atom via a dithiolene group. Because Moco is a large and complex compound deeply buried within the protein, molybdoenzymes are accompanied by private chaperone proteins responsible for the cofactor's insertion into the enzyme and the enzyme's maturation. An efficient recombinant expression and purification of both Moco-free and Moco-containing molybdoenzymes and their chaperones is of paramount importance for fundamental and applied research related to molybdoenzymes. In this work, we focused on a D1 protein annotated as a chaperone of steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S. The D1 protein is presumably involved in the maturation of S25DH engaged in oxygen-independent oxidation of sterols. As this chaperone is thought to be a crucial element that ensures the insertion of Moco into the enzyme and consequently, proper folding of S25DH optimization of the chaperon's expression is the first step toward the development of recombinant expression and purification methods for S25DH. We have identified common E. coli strains and conditions for both expression and purification that allow us to selectively produce Moco-containing and Moco-free chaperones. We have also characterized the Moco-containing chaperone by EXAFS and HPLC analysis and identified conditions that stabilize both forms of the protein. The protocols presented here are efficient and result in protein quantities sufficient for biochemical studies.

Molybdopterin biosynthesis-Mechanistic studies on a novel MoaA catalyzed insertion of a purine carbon into the ribose of GTP.

The first step in the biosynthesis of the molybdopterin cofactor involves an unprecedented insertion of the purine C8 carbon between the C2' and C3' carbons of the ribose moiety of GTP. Here we review mechanistic studies on this remarkable transformation. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Shared-intermediates in the biosynthesis of thio-cofactors: Mechanism and functions of cysteine desulfurases and sulfur acceptors.

Cysteine desulfurases utilize a PLP-dependent mechanism to catalyze the first step of sulfur mobilization in the biosynthesis of sulfur-containing cofactors. Sulfur activation and integration into thiocofactors involve complex mechanisms and intricate biosynthetic schemes. Cysteine desulfurases catalyze sulfur-transfer reactions from l-cysteine to sulfur acceptor molecules participating in the biosynthesis of thio-cofactors, including Fe-S clusters, thionucleosides, thiamin, biotin, and molybdenum cofactor. The proposed mechanism of cysteine desulfurases involves the PLP-dependent cleavage of the C-S bond from l-cysteine via the formation of a persulfide enzyme intermediate, which is considered the hallmark step in sulfur mobilization. The subsequent sulfur transfer reaction varies with the class of cysteine desulfurase and sulfur acceptor. IscS serves as a mecca for sulfur incorporation into a network of intertwined pathways for the biosynthesis of thio-cofactors. The involvement of a single enzyme interacting with multiple acceptors, the recruitment of shared-intermediates partaking roles in multiple pathways, and the participation of Fe-S enzymes denote the interconnectivity of pathways involving sulfur trafficking. In Bacillus subtilis, the occurrence of multiple cysteine desulfurases partnering with dedicated sulfur acceptors partially deconvolutes the routes of sulfur trafficking and assigns specific roles for these enzymes. Understanding the roles of promiscuous vs. dedicated cysteine desulfurases and their partnership with shared-intermediates in the biosynthesis of thio-cofactors will help to map sulfur transfer events across interconnected pathways and to provide insight into the hierarchy of sulfur incorporation into biomolecules. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

The role of FeS clusters for molybdenum cofactor biosynthesis and molybdoenzymes in bacteria.

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.

Mouse model for molybdenum cofactor deficiency type B recapitulates the phenotype observed in molybdenum cofactor deficient patients.

Molybdenum cofactor (MoCo) deficiency is a rare, autosomal-recessive disorder, mainly caused by mutations in MOCS1 (MoCo deficiency type A) or MOCS2 (MoCo deficiency type B) genes; the absence of active MoCo results in a deficiency in all MoCo-dependent enzymes. Patients with MoCo deficiency present with neonatal seizures, feeding difficulties, severe developmental delay, brain atrophy and early childhood death. Although substitution therapy with cyclic pyranopterin monophosphate (cPMP) has been successfully used in both Mocs1 knockout mice and in patients with MoCo deficiency type A, there is currently no Mocs2 knockout mouse and no curative therapy for patients with MoCo deficiency type B. Therefore, we generated and characterized a Mocs2-null mouse model of MoCo deficiency type B. Expression analyses of Mocs2 revealed a ubiquitous expression pattern; however, at the cellular level, specific cells show prominent Mocs2 expression, e.g., neuronal cells in cortex, hippocampus and brainstem. Phenotypic analyses demonstrated that Mocs2 knockout mice failed to thrive and died within 11 days after birth. None of the tested MoCo-dependent enzymes were active in Mocs2-deficient mice, leading to elevated concentrations of purines, such as hypoxanthine and xanthine, and non-detectable levels of uric acid in the serum and urine. Moreover, elevated concentrations of S-sulfocysteine were measured in the serum and urine. Increased levels of xanthine resulted in bladder and kidney stone formation, whereas increased concentrations of toxic sulfite triggered neuronal apoptosis. In conclusion, Mocs2-deficient mice recapitulate the severe phenotype observed in humans and can now serve as a model for preclinical therapeutic approaches for MoCo deficiency type B.

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.