Health, Safety & Environment

Molybdopterin cofactor

Functional Complementation Studies Reveal Different Interaction Partners of Escherichia coil 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 similar to 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 mcm(5)s(2)U34 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.

Buhning, M., Friemel, M., and Leimkuhler, S.,Functional Complementation Studies Reveal Different Interaction Partners of Escherichia coil IscS and Human NFS1, Biochemistry, 2017, 56, 4592-4605.

Molybdenum cofactor deficiency
Molybdenum cofactor and isolated sulphite oxidase deficiencies: Clinical and molecular spectrum among Egyptian patients

AIM: Molybdenum cofactor deficiency (MoCD) and Sulfite oxidase deficiency (SOD) are rare autosomal recessive conditions of sulfur-containing amino acid metabolism with overlapping clinical features and emerging therapies. The clinical phenotype is indistinguishable and they can only be differentiated biochemically. MOCS1, MOCS2, MOCS3, and GPRN genes contribute to the synthesis of molybdenum cofactor, and SUOX gene encodes sulfite oxidase. The aim of this study was to elucidate the clinical, radiological, biochemical and molecular findings in patients with SOD and MoCD.

METHODS: Detailed clinical and radiological assessment of 9 cases referred for neonatal encephalopathy with hypotonia, microcephaly, and epilepsy led to a consideration of disorders of sulfur-containing amino acid metabolism. The diagnosis of six with MoCD and three with SOD was confirmed by biochemical tests, targeted sequencing, and whole exome sequencing where suspicion of disease was lower.

RESULTS: Novel SUOX mutations were detected in 3 SOD cases and a novel MOCS2 mutation in 1 MoCD case. Most patients presented in the first 3 months of life with intractable tonic-clonic seizures, axial hypotonia, limb hypertonia, exaggerated startle response, feeding difficulties, and progressive cystic encephalomalacia on brain imaging. A single patient with MoCD had hypertrophic cardiomyopathy, hitherto unreported with these diseases.

INTERPRETATION: Our results emphasize that intractable neonatal seizures, spasticity, and feeding difficulties can be important early signs for these disorders. Progressive microcephaly, intellectual disability and specific brain imaging findings in the first year were additional diagnostic aids. These clinical cues can be used to minimize delays in diagnosis, especially since promising treatments are emerging for MoCD type A.

Zaki, M. S., Selim, L., El-Bassyouni, H. T., Issa, M. Y., Mahmoud, I., Ismail, S., Girgis, M., Sadek, A. A., Gleeson, J. G., and Abdel Hamid, M. S.,Molybdenum cofactor and isolated sulphite oxidase deficiencies: Clinical and molecular spectrum among Egyptian patients, European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society, 2016, 20, 714-22.

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.

Hoffmann, M. C., Ali, K., Sonnenschein, M., Robrahn, L., Strauss, D., Narberhaus, F., and Masepohl, B.,Molybdate uptake by Agrobacterium tumefaciens correlates with the cellular molybdenum cofactor status, Molecular microbiology, 2016, 101, 809-22.

Designing the molybdopterin core through regioselective coupling of building blocks

Molybdopterin is an essential cofactor for all forms of life. The cofactor is composed of a pterin moiety appended to a dithiolene-functionalized pyran ring, and through the dithiolene moiety it binds metal ions. Different synthetic strategies for dithiolene-functionalized pyran precursors that have been designed and synthesized are discussed. These precursors also harbor 1,2-diketone or osone functionality that has been condensed with 1,2-diaminobenzene or other heterocycles resulting in several quinoxaline or pterin derivatives. Use of additives improves the regioselectivity of the complexes. The molecules have been characterized by H-1 and C-13 NMR and IR spectroscopies, as well as by mass spectrometry. In addition, several compounds have been crystallographically characterized. The geometries of the synthesized molecules are more planar than the geometry of the cofactor found in proteins.

Pimkov, I. V., Serli-Mitasev, B., Peterson, A. A., Ratvasky, S. C., Hammann, B., and Basu, P.,Designing the Molybdopterin Core through Regioselective Coupling of Building Blocks, Chemistry-a European Journal, 2015, 21, 17057-17072.

Molybdoenzyme cofactor biosythesis

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.

Hover, B. M., Tonthat, N. K., Schumacher, M. A., and Yokoyama, K.,Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis, Proc Natl Acad Sci U S A, 2015, 112, 6347.

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.

Black, K. A., and Dos Santos, P. C.,Shared-intermediates in the biosynthesis of thio-cofactors: Mechanism and functions of cysteine desulfurases and sulfur acceptors, Biochim Biophys Acta, 2015, 1853, 1470

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.

Yokoyama, K., and Leimkuhler, S.,The role of FeS clusters for molybdenum cofactor biosynthesis and molybdoenzymes in bacteria, Biochim Biophys Acta, 2015, 1853, 1335.

The molybdopterin cofactor is required for the activity of a variety of oxidoreductases. The xanthine oxidase class of molybdoenzymes requires the molybdopterin cofactor to have a terminal, cyanolysable sulfur ligand. In the sulfite oxidase/nitrate reductase class, an oxygen is present in the same position.


The biosynthesis of the molybdenum cofactors (Moco) is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum. Moco is the essential component of a group of redox enzymes, which are diverse in terms of their phylogenetic distribution and their architectures, both at the overall level and in their catalytic geometry. A wide variety of transformations are catalyzed by these enzymes at carbon, sulfur and nitrogen atoms, which include the transfer of an oxo group or two electrons to or from the substrate. More than 50 molybdoenzymes were identified to date. In all molybdoenzymes except nitrogenase, molybdenum is coordinated to a dithiolene group on the 6-alkyl side chain of a pterin called molybdopterin (MPT). The biosynthesis of Moco can be divided into three general steps, with a fourth one present only in bacteria and archaea: (1) formation of the cyclic pyranopterin monophosphate, (2) formation of MPT, (3) insertion of molybdenum into molybdopterin to form Moco, and (4) additional modification of Moco in bacteria with the attachment of a nucleotide to the phosphate group of MPT, forming the dinucleotide variant of Moco. This review will focus on the biosynthesis of Moco in bacteria, humans and plants.

Mendel, R. R. and Leimkuhler, S., The biosynthesis of the molybdenum cofactors, Journal of Biological Inorganic Chemistry, 2015, 20, 337-347.

Structure review

Over the past two decades, a plethora of crystal structures of molybdenum enzymes has appeared in the literature providing a clearer picture of the enzymatic active sites and increasing the challenge to chemists to develop accurate models for those sites. In this minireview we discuss the most recent model studies aimed to reproduce detailed features of the pterin-dithiolene ligand, both as the uncoordinated form and as a chelate coordinated to molybdenum.

Basu, P. and Burgmayer, S. J. N., Recent developments in the study of molybdoenzyme models, Journal of Biological Inorganic Chemistry, 2015, 20, 373-383.

Mutations in both the ma-l gene of Drosophila melanogaster and the hxB gene of Aspergillus nidulans cause loss of activities of those molybdoenzymes that require a cyanolysable sulfur in the active centre. The ma-l and hxB genes encode highly similar proteins containing domains common to pyridoxal phosphate-dependent cysteine transulphurases, including the cofactor binding site and a conserved cysteine, which is the putative sulfur donor.
Key similarities were found with NifS, the enzyme involved in the generation of the iron-sulphur centres in nitrogenase. These similarities suggest an analogous mechanism for the generation of the terminal molybdenum-bound sulfur ligand. Putative homologues have been identified of these genes in organisms, including humans.

Amrani, L., Primus, J., Glatigny, A., Arcangeli, L., Scazzocchio, C., and Finnerty, V., Comparison of the sequences of the aspergillus nidulans hxB and drosophila melanogaster ma-l genes with nifS from azotobacter vinelandii suggests a mechanism for the insertion of the terminal sulfur atom in the molybdopterin cofactor, Mol.Microbiol., 2000, 38, 114-125.

All mononuclear molybdoenzymes bind molybdenum in a complex with an organic cofactor termed molybdopterin. In many bacteria, including Escherichia coli, molybdopterin can be further modified by attachment of a guanine monophosphate group to the terminal phosphate of molybdopterin to form molybdopterin guanine dinucleotide. This modification reaction is required for the functioning of many bacterial molybdoenzymes, including the nitrate reductases, dimethylsulfoxide and trimethylamine-N-oxide reductases, and formate dehydrogenases. The guanine monophosphate attachment step is catalyzed by the cellular enzyme MobA. The crystal structure of the 21.6 kDa E. coli MobA has been determined.

Stevenson, C.E.M., Sargent, F., Buchanan, G., Palmer, T., and Lawson, D. M., Crystal structure of the molybdenum cofactor biosynthesis protein MobA from Escherichia coli at near-atomic resolution, Structure, 2000, 8, 1115-1125.

Molybdenum cofactor biosynthesis

Background: Localization and identification of interaction partners of two splice variants of the human 3-mercaptopyruvate sulfurtransferase TUM1.

Results: We show that TUM1 interacts with proteins involved in Moco and FeS cluster biosynthesis.

Conclusion: Human TUM1 is a dual localized protein in the cytosol and mitochondria with distinct roles in sulfur transfer and interaction partners.

Significance: The study contributes to the sulfur transfer pathway for the biosynthesis of sulfur-containing biofactors. The human tRNA thiouridine modification protein (TUM1), also designated as 3-mercaptopyruvate sulfurtransferase (MPST), has been implicated in a wide range of physiological processes in the cell. The roles range from an involvement in thiolation of cytosolic tRNAs to the generation of H2S as signaling molecule both in mitochondria and the cytosol. TUM1 is a member of the sulfurtransferase family and catalyzes the conversion of 3-mercaptopyruvate to pyruvate and protein-bound persulfide. Here, we purified and characterized two novel TUM1 splice variants, designated as TUM1-Iso1 and TUM1-Iso2. The purified proteins showed similar kinetic behavior and comparable pH and temperature dependence. Cellular localization studies, however, showed a different localization pattern between the isoforms. TUM1-Iso1 is exclusively localized in the cytosol, whereas TUM1-Iso2 showed a dual localization both in the cytosol and mitochondria. Interaction studies were performed with the isoforms both in vitro using the purified proteins and in vivo by fluorescence analysis in human cells, using the split-EGFP system. The studies showed that TUM1 interacts with the l-cysteine desulfurase NFS1 and the rhodanese-like protein MOCS3, suggesting a dual function of TUM1 both in sulfur transfer for the biosynthesis of the molybdenum cofactor, and for the thiolation of tRNA. Our studies point to distinct roles of each TUM1 isoform in the sulfur transfer processes in the cell, with different compartmentalization of the two splice variants of TUM1.

Frasdorf, B., Radon, C., and Leimkuhler, S., Characterization and Interaction Studies of Two Isoforms of the Dual Localized 3-Mercaptopyruvate Sulfurtransferase TUM1 from Humans, Journal of Biological Chemistry, 2014, 289, 34543-34556.