Cell biology of molybdenum in plants

The transition element molybdenum (Mo) is of essential importance for (nearly) all biological systems as it is required by enzymes catalyzing important reactions within the cell. The metal itself is biologically inactive unless it is complexed by a special cofactor. With the exception of bacterial nitrogenase, where Mo is a constituent of the FeMo-cofactor, Mo is bound to a pterin, thus forming the molybdenum cofactor (Moco) which is the active compound at the catalytic site of all other Mo-enzymes. In plants, the most prominent Mo-enzymes are nitrate reductase, sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase, and the mitochondrial amidoxime reductase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also includes iron as well as copper in an indispensable way. After its synthesis, Moco is distributed to the apoproteins of Mo-enzymes by Moco-carrier/binding proteins that also participate in Moco-insertion into the cognate apoproteins. Xanthine dehydrogenase and aldehyde oxidase, but not the other Mo-enzymes, require a final step of posttranslational activation of their catalytic Mo-center for becoming active.     

Cell biology of molybdenum in plants and humans

The transition element molybdenum (Mo) needs to be complexed by a special cofactor in order to gain catalytic activity. With the exception of bacterial Mo-nitrogenase, where Mo is a constituent of the FeMo-cofactor, Mo is bound to a pterin, thus forming the molybdenum cofactor Moco, which in different variants is the active compound at the catalytic site of all other Mo-containing enzymes. In eukaryotes, the most prominent Mo-enzymes are nitrate reductase, sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase, and the mitochondrial amidoxime reductase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also requires iron, ATP and copper. After its synthesis, Moco is distributed to the apoproteins of Mo-enzymes by Moco-carrier/binding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms. In humans, Moco deficiency is a severe inherited inborn error in metabolism resulting in severe neurodegeneration in newborns and causing early childhood death. This article is part of a Special Issue entitled: Cell Biology of Metals.     

Molybdoenzymes and molybdenum cofactor in plants

The transition element molybdenum (Mo) is essential for (nearly) all organisms and occurs in more than 40 enzymes catalysing diverse redox reactions, however, only four of them have been found in plants. (1) Nitrate reductase catalyses the key step in inorganic nitrogen assimilation, (2) aldehyde oxidase(s) have been shown to catalyse the last step in the biosynthesis of the phytohormone abscisic acid, (3) xanthine dehydrogenase is involved in purine catabolism and stress reactions, and (4) sulphite oxidase is probably involved in detoxifying excess sulphite. Among Mo-enzymes, the alignment of amino acid sequences permits domains that are well conserved to be defined. With the exception of bacterial nitrogenase, Mo-enzymes share a similar pterin compound at their catalytic sites, the molybdenum cofactor. Mo itself seems to be biologically inactive unless it is complexed by the cofactor. This molybdenum cofactor combines with diverse apoproteins where it is responsible for the correct anchoring and positioning of the Mo-centre within the holo-enzyme so that the Mo-centre can interact with other components of the enzyme's electron transport chain. A model for the three-step biosynthesis of Moco involving the complex interaction of six proteins will be described. A putative Moco-storage protein distributing Moco to the apoproteins of Mo-enzymes will be discussed. After insertion, xanthine dehydrogenase and aldehyde oxidase, but not nitrate reductase and sulphite oxidase, require the addition of a terminal sulphur ligand to their Mo-site, which is catalysed by the sulphur transferase ABA3.     

Molybdoenzymes and Molybdenum Cofactor in Plants

The transition element molybdenum is essential for (nearly) all organisms and occurs in more than 30 enzymes catalyzing diverse redox reactions; however, only three Mo-enzymes have been found in plants so far. (1) Nitrate reductase catalyzes the key step in inorganic nitrogen assimilation, (2) aldehyde oxidase(s) recently have been shown to catalyze the last step in the biosynthesis of the phytohormones indole acetic acid and abscisic acid, respectively, and (3) xanthine dehydrogenase is involved in purine catabolism. These enzymes are homodimeric proteins harboring an electron transport chain that involves different prosthetic groups (FAD, heme, or Fe-S, Mo). Among different Mo-enzymes, the alignment of amino acid sequences helps to define regions that are well conserved (domains) and other regions that are highly variable in sequence (interdomain hinge regions). The existence of additional plant Mo-enzymes (like sulfite oxidase) also has to be considered. In this review we focus on structure-function relationships and stress the functional importance of the enzymes for the plant. With the exception of bacterial nitrogenase, Mo-enzymes share a similar pterin compound at their catalytic sites, the molybdenum cofactor. Molybdenum itself seems to be biologically inactive unless it is complexed by the cofactor. This molybdenum cofactor combines with diverse apoproteins where it is responsible for the correct anchoring and positioning of the Mo-center within the holo-enzyme so that the Mo-center can interact with other components of the enzyme's electron transport chain. The organic moiety of the molybdenum cofactor is a unique pterin named molybdopterin. The core structure of molybdopterin is conserved in all organisms. Accordingly, its biosynthetic pathway seems to be conserved because a similar set of cofactor genes has been found in bacteria and higher plants. We describe a model for the biosynthesis of the plant molybdenum cofactor involving the complex interaction of seven proteins.     

Molybdenum metabolism in the alga Chlamydomonas stands at the crossroad of those in Arabidopsis and humans

Molybdenum (Mo) is a very scarce element whose function is fundamental in living beings within the active site of Mo-oxidoreductases, playing key roles in the metabolism of N, S, purines, hormone biosynthesis, transformation of drugs and xenobiotics, etc. In eukaryotes, each step from Mo acquisition until its incorporation into a biologically active molybdenum cofactor (Moco) together with the assembly of this Moco in Mo-enzymes is almost understood. The deficiency in function of a particular molybdoenzyme can be critical for the survival of the organism dependent on the pathway involved. However, incapacity in forming a functional Moco has a pleiotropic effect in the different processes involving this cofactor. A detailed overview of Mo metabolism: (a) specific transporters for molybdate, (b) the universal biosynthesis pathway for Moco from GTP, (c) Moco-carrier and Moco-binding proteins for Moco transfer and (d) Mo-enzymes, is analyzed in light of recent findings and three systems are compared, the unicellular microalga Chlamydomonas, the plant Arabidopsis and humans.     

A Novel Role for Arabidopsis Mitochondrial ABC Transporter ATM3 in Molybdenum Cofactor Biosynthesis

The molybdenum cofactor (Moco) is a prosthetic group required by a number of enzymes, such as nitrate reductase, sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. Its biosynthesis in eukaryotes can be divided into four steps, of which the last three are proposed to occur in the cytosol. Here, we report that the mitochondrial ABC transporter ATM3, previously implicated in the maturation of extramitochondrial iron-sulfur proteins, has a crucial role also in Moco biosynthesis. In ATM3 insertion mutants of Arabidopsis thaliana, the activities of nitrate reductase and sulfite oxidase were decreased to ∼50%, whereas the activities of xanthine dehydrogenase and aldehyde oxidase, whose activities also depend on iron-sulfur clusters, were virtually undetectable. Moreover, atm3 mutants accumulated cyclic pyranopterin monophosphate, the first intermediate of Moco biosynthesis, but showed decreased amounts of Moco. Specific antibodies against the Moco biosynthesis proteins CNX2 and CNX3 showed that the first step of Moco biosynthesis is localized in the mitochondrial matrix. Together with the observation that cyclic pyranopterin monophosphate accumulated in purified mitochondria, particularly in atm3 mutants, our data suggest that mitochondria and the ABC transporter ATM3 have a novel role in the biosynthesis of Moco.     

Plant molybdoenzymes and their response to stress

Molybdenum-containing enzymes catalyse basic reactions in the nitrogen, sulphur and carbon metabolism. Mo-enzymes contain at their catalytic sites an organometallic structure termed the molybdenum cofactor or Moco. In higher plants, Moco is incorporated into the apoproteins of four enzymes: nitrate reductase (EC 1.6.6.1-3; NR), xanthine dehydrogenase (EC 1.1.1.204; XDH), aldehyde oxidase (EC 1.2.3.1; AO) and sulphite oxidase (EC1.8.3.1; SO). Molybdoenzymes in plants are key enzymes in nitrate assimilation, purine metabolism, hormone biosynthesis, and most probably in sulphite detoxification. They are considered to be involved in stress acclimation processes and, therefore, elucidation of the mechanisms of their response to environmental stress conditions is of agricultural importance for the improvement of plant stress tolerance. Here we would like to give a brief functional and biochemical characteristic of the four plant molybdoenzymes and to focus mainly on their sensitivity to environmental stress factors.     

Molybdenum Metabolism in Plants

Abstract: Among the micronutrients essential for plant growth and for microsymbionts, Mo is required in minute amounts. However, since Mo is often sequestered by Fe- or Al-oxihydrox-ides, especially in acidic soils, the concentration of the water-soluble molybdate anion available for uptake by plants may be limiting for the plant, even when the total Mo content of the soil is sufficient. In contrast to bacteria, no specific molybdenum uptake system is known for plants, but since molybdate and sulfate behave similarly and have similar structure, uptake of molybdate could be mediated unspecifically by one of the sulfate transporters. Transport into the different plant organs proceeds via xylem and phloem. A pterin-bound molybdenum is the cofactor of important plant enzymes involved in redox processes: nitrate reductase, xanthine dehydrogenase, aIdehyde oxidase, and probably sulfite oxidase. Biosynthesis of the molybdenum cofactor (Moco) starts with a guanosine-X-phos-phate. Subsequently, a sulfur-free pterin is synthesized, sulfur is added, and finally molybdenum is incorporated. In addition to the molybdopterin enzymes, small molybdopterin binding proteins without catalytic function are known and are probably involved in the storage of Moco. In symbiotic systems the nitrogen supply of the host plant is strongly influenced by the availability of Mo in soil, since both bacterial nitrogenase and NADPH-dependent nitrate reductase of mycorrhizal fungi are Mo enzymes.     

Identification of a Rhodobacter capsulatus L-cysteine desulfurase that sulfurates the molybdenum cofactor when bound to XdhC and before its insertion into xanthine dehydrogenase

The molybdenum cofactor (Moco) containing enzymes aldehyde oxidase and xanthine dehydrogenase (XDH) require for activity a sulfuration step that inserts a terminal sulfur ligand into Moco. XdhC was shown to be essential for the production of active XDH in Rhodobacter capsulatus but is itself not a subunit of the purified enzyme. XdhC binds stoichiometric amounts of Moco and is further able to transfer its bound Moco to XDH. Previous work suggested that XdhC particularly stabilizes the sulfurated form of Moco before the insertion into XDH. In this work, we identify an R. capsulatus l-cysteine desulfurase, NifS4, which is involved in the formation of the Mo=S ligand of Moco. We show that NifS4 interacts with XdhC and not with XDH. NifS4 mobilizes sulfur from l-cysteine by formation of a protein-bound persulfide intermediate and transfers this sulfur further to Moco. This reaction was shown to be more effective than the chemical sulfuration of Moco using sulfide as sulfur source. Further studies clearly showed that Moco is sulfurated before the insertion into XDH, while it is bound to XdhC. Conclusively, XdhC has a versatile role in R. capsulatus: binding of Moco, interaction with NifS4 for the sulfuration of Moco, protection of sulfurated Moco from oxidation, and further transfer to XDH.     

Structure-based alteration of substrate specificity and catalytic activity of sulfite oxidase from sulfite oxidation to nitrate reduction

Eukaryotic sulfite oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study, we describe human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction. On the basis of sequence alignments and the known crystal structure of chicken sulfite oxidase, two residues are conserved in nitrate reductases that align with residues in the active site of sulfite oxidase. On the basis of the crystal structure of yeast nitrate reductase, both positions were mutated in human sulfite oxidase and chicken sulfite oxidase. The resulting double-mutant variants demonstrated a marked decrease in sulfite oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore, the triple variant was also produced. The nitrate reducing ability of the human sulfite oxidase triple mutant was nearly 3-fold greater than that of the double mutant. To obtain detailed structural data for the active site of these variants, we introduced the analogous mutations into chicken sulfite oxidase to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were determined to 2.4 and 2.1 ? resolution, respectively.     

In vitro incorporation of nascent molybdenum cofactor into human sulfite oxidase

We were able to reconstitute molybdopterin (MPT)-free sulfite oxidase in vitro with the molybdenum cofactor (Moco) synthesized de novo from precursor Z and molybdate. MPT-free human sulfite oxidase apoprotein was obtained by heterologous expression in an Escherichia coli mutant with a defect in the early steps of MPT biosynthesis. In vitro reconstitution of the purified apoprotein was achieved using an incubation mixture containing purified precursor Z, purified MPT synthase, and sodium molybdate. In vitro synthesized MPT generated from precursor Z by MPT synthase remains bound to the synthase. Surprisingly, MPT synthase was found capable of donating bound MPT to MPT-free sulfite oxidase. MPT was not released from MPT synthase when either bovine serum albumin or Moco-containing sulfite oxidase was used in place of aposulfite oxidase. After the inclusion of sodium molybdate in the reconstitution mixture, active sulfite oxidase was obtained, revealing that in vitro MPT synthase and aposulfite oxidase are sufficient for the insertion of MPT into sulfite oxidase and the conversion of MPT into Moco in the presence of high concentrations of molybdate. The conversion of MPT into Moco by molybdate chelation apparently occurs concomitantly with the insertion of MPT into sulfite oxidase.     

Molybdenum cofactor deficiency in a patient previously characterized as deficient in sulfite oxidase

The metabolic status of a patient previously characterized as deficient in sulfite oxidase was reexamined applying new methodology which has been developed to distinguish between a defect specific to the sulfite oxidase protein and sulfite oxidase deficiency which arises as a result of molybdenum cofactor deficiency. Urothione, the metabolic degradation product of the molybdenum cofactor, was undetectable in urine samples from the patient. Analysis of molybdenum cofactor levels in fibroblasts by monitoring reconstitution of apo nitrate reductase in extracts of the Neurospora crassa mutant nit-1 revealed that cells from the patient were severely depleted. Quantitation of urinary oxypurines showed that hypoxanthine and xanthine were highly elevated while uric acid remained in the normal range. These results were interpreted to indicate a severe but incomplete deficiency of the molybdenum cofactor. The presence of very low levels of active cofactor, supporting the synthesis of low levels of active sulfite oxidase and xanthine dehydrogenase, could explain the metabolic patterns of sulfur and purine products and the relatively mild clinical symptoms in this individual.     

Activation of nit-1 nitrate reductase by W-formate dehydrogenase

Formate dehydrogenase ( FDH ) from Clostridium thermoaceticum is a known tungsten enzyme. FDH was tested for the presence of nitrogenase-type cofactor and nitrate reductase-type cofactor by the Azotobacter vinelandii UW-45 and Neurospora crassa nit-1 reconstitution assays, respectively. Tungsten formate dehydrogenase (W- FDH ), containing only a small Mo impurity, activated the nit-1 nitrate reductase extracts when molybdate was also added, but not when tungstate was added. These results show W- FDH contains the cofactor common to all known Mo-enzymes except nitrogenase. The difference between the redox chemistries of W- FDH and W-substituted sulfite oxidase appears to relate to differences in tungsten ligation other than that donated by the cofactor or to variations in the protein environment surrounding the tungsten active site.     

Synthesis of adenylated molybdopterin: an essential step for molybdenum insertion

The molybdenum cofactor (Moco) is part of the active site of all molybdenum (Mo)-dependent enzymes, except nitrogenase. Moco consists of molybdopterin (MPT), a phosphorylated pyranopterin with an enedithiolate coordinating Mo and it is synthesized by an evolutionary old multistep pathway. The plant protein Cnx1 from Arabidopsis thaliana catalyzes with its two domains (E and G) the terminal step of Moco biosynthesis, the insertion of Mo into MPT. Recently, the high-resolution MPT-bound structure of the Cnx1 G domain (Cnx1G) has been determined (Kuper, J., Llamas, A., Hecht, H. J., Mendel, R. R., and Schwarz, G. (2004) Nature 430, 803-806). Besides defining the MPT-binding site a novel and unexpected modification of MPT has been identified, adenylated MPT. Here we demonstrate that it is Cnx1G that catalyzes the adenylation of MPT. In vitro synthesized MPT was quantitatively transferred from Escherichia coli MPT synthase to Cnx1G. The subsequent adenylation reaction by Cnx1G was Mg(2+)- and ATP-dependent. Whereas Mn(2+) could partially replace Mg(2+), ATP was the only nucleotide accepted by Cnx1G. Consequently the formation of pyrophosphate was demonstrated, which was dependent on the ability of Cnx1G to bind MPT. Pyrophosphate, either formed in the reaction or added externally, inhibited the Cnx1G-catalyzed MPT adenylation reaction. Catalytically inactive Cnx1G mutant variants showed impaired MPT adenylation confirming that MPT-AMP is the reaction product of Cnx1G. Therefore Cnx1G is a MPT adenylyltransferase catalyzing the activation of MPT, a universal reaction in the Moco synthetic pathway because Cnx1G is able to reconstitute also bacterial and mammalian Moco biosynthesis.     

The crystal structure of plant sulfite oxidase provides insights into sulfite oxidation in plants and animals

The molybdenum cofactor (Moco) containing sulfite oxidase (SO) from Arabidopsis thaliana has recently been identified and biochemically characterized. The enzyme is found in peroxisomes and believed to detoxify excess sulfite that is produced during sulfur assimilation, or due to air pollution. Plant SO (PSO) is homodimeric and homologous to animal SO, but contains only a single Moco domain without an additional redox center. Here, we present the first crystal structure of a plant Moco enzyme, the apo-state of Arabidopsis SO at 2.6 A resolution. The overall fold and coordination of the Moco are similar to chicken SO (CSO). Comparisons of conserved surface residues and the charge distribution in PSO and CSO reveal major differences near the entrance to both active sites reflecting different electron acceptors. Arg374 has been identified as an important substrate binding residue due to its conformational change when compared to the sulfate bound structure of CSO.     

Splice-specific functions of gephyrin in molybdenum cofactor biosynthesis

Gephyrin is a multifunctional protein involved in the clustering of inhibitory neuroreceptors. In addition, gephyrin catalyzes the last step in molybdenum cofactor (Moco) biosynthesis essential for the activities of Mo-dependent enzymes such as sulfite oxidase and xanthine oxidoreductase. Functional complexity and diversity of gephyrin is believed to be regulated by alternative splicing in a tissue-specific manner. Here, we investigated eight gephyrin variants with combinations of seven alternatively spliced exons located in the N-terminal G domain, the central domain, and the C-terminal E domain. Their activity in Moco synthesis was analyzed in vivo by reconstitution of gephyrin-deficient L929 cells, which were found to be defective in the G domain of gephyrin. Individual domain functions were assayed in addition and confirmed that variants containing either an additional C5 cassette or missing the C6 cassette are inactive in Moco synthesis. In contrast, different alterations within the central domain retained the Moco synthetic activity of gephyrin. The recombinant gephyrin G domain containing the C5 cassette forms dimers in solution, binds molybdopterin, but is unable to catalyze molybdopterin (MPT) adenylylation. Determination of Moco and MPT content in different tissues showed that besides liver and kidney, brain was capable of synthesizing Moco most efficiently. Subsequent analysis of cultured neurons and glia cells demonstrated glial Moco synthesis due to the expression of gephyrins containing the cassettes C2 and C6 with and without C3.1.     

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.     

A novel role for human Nfs1 in the cytoplasm: Nfs1 acts as a sulfur donor for MOCS3, a protein involved in molybdenum cofactor biosynthesis

The human MOCS3 gene encodes a protein involved in activation and sulfuration of the C terminus of MOCS2A, the smaller subunit of the molybdopterin (MPT) synthase. MPT synthase catalyzes the formation of the dithiolene group of MPT that is required for the coordination of the molybdenum atom in the last step of molybdenum cofactor (Moco) biosynthesis. The two-domain protein MOCS3 catalyzes both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of MOCS2A by its C-terminal rhodanese-like domain (RLD). The low activity of MOCS3-RLD with thiosulfate as sulfur donor and detailed mutagenesis studies showed that thiosulfate is most likely not the physiological sulfur source for Moco biosynthesis in eukaryotes. It was suggested that an l-cysteine desulfurase might be involved in the sulfuration of MOCS3 in vivo. In this report, we investigated the involvement of the human l-cysteine desulfurase Nfs1 in sulfur transfer to MOCS3-RLD. A variant of Nfs1 was purified in conjunction with Isd11 in a heterologous expression system in Escherichia coli, and the kinetic parameters of the purified protein were determined. By studying direct protein-protein interactions, we were able to show that Nfs1 interacted specifically with MOCS3-RLD and that sulfur is transferred from l-cysteine to MOCS3-RLD via an Nfs1-bound persulfide intermediate. Because MOCS3 was shown to be located in the cytosol, our results suggest that cytosolic Nfs1 has an important role in sulfur transfer for the biosynthesis of Moco.     

Biology of the molybdenum cofactor

The transition element molybdenum (Mo) is an essential micronutrient for plants where it is needed as a catalytically active metal during enzyme catalysis. Four plant enzymes depend on molybdenum: nitrate reductase, sulphite oxidase, xanthine dehydrogenase, and aldehyde oxidase. However, in order to gain biological activity and fulfil its function in enzymes, molybdenum has to be complexed by a pterin compound thus forming the molybdenum cofactor. In this article, the path of molybdenum from its uptake into the cell, via formation of the molybdenum cofactor and its storage, to the final modification of the molybdenum cofactor and its insertion into apo-metalloenzymes will be reviewed.     

The identification of a novel protein involved in molybdenum cofactor biosynthesis in Escherichia coli

In the second step of the molybdenum cofactor (Moco) biosynthesis in Escherichia coli, the l-cysteine desulfurase IscS was identified as the primary sulfur donor for the formation of the thiocarboxylate on the small subunit (MoaD) of MPT synthase, which catalyzes the conversion of cyclic pyranopterin monophosphate to molybdopterin (MPT). Although in Moco biosynthesis in humans, the thiocarboxylation of the corresponding MoaD homolog involves two sulfurtransferases, an l-cysteine desulfurase, and a rhodanese-like protein, the rhodanese-like protein in E. coli remained enigmatic so far. Using a reverse approach, we identified a so far unknown sulfurtransferase for the MoeB-MoaD complex by protein-protein interactions. We show that YnjE, a three-domain rhodanese-like protein from E. coli, interacts with MoeB possibly for sulfur transfer to MoaD. The E. coli IscS protein was shown to specifically interact with YnjE for the formation of the persulfide group on YnjE. In a defined in vitro system consisting of MPT synthase, MoeB, Mg-ATP, IscS, and l-cysteine, YnjE was shown to enhance the rate of the conversion of added cyclic pyranopterin monophosphate to MPT. However, YnjE was not an enhancer of the cysteine desulfurase activity of IscS. This is the first report identifying the rhodanese-like protein YnjE as being involved in Moco biosynthesis in E. coli. We believe that the role of YnjE is to make the sulfur transfer from IscS for Moco biosynthesis more specific because IscS is involved in a variety of different sulfur transfer reactions in the cell.