Health, Safety & Environment

MOLYBDENUM COFACTOR

Molybdenum Cofactor

Cleavage of molybdopterin synthase MoaD-MoaE linear fusion by JAMM/MPN(+) domain containing metalloprotease DR0402 from Deinococcus radiodurans

Molybdenum cofactor (Moco), molybdopterin (MPT) complexed with molybdenum, is an essential cofactor required for the catalytic center of diverse enzymes in all domains of life. Since Moco cannot be taken up as a nutrient unlike many other cofactors, Moco requires de novo biosynthesis. During the synthesis of MPT, the sulfur atom on the C-terminus of MoaD is transferred to cyclic pyranopterin monophosphate (cPMP) which is bound in the substrate pocket of MoaE. MoaD is a ubiquitin-like (Ubl) protein and has a C-terminal di-Gly motif which is a common feature of Ubl proteins. Despite the importance of free C terminal di-Gly motif of MoaD as a sulfur carrier, some bacteria encode a fused MPT synthase in which MoaD- and MoaE-like domains are located on a single peptide. Although it has recently been reported that the fused MPT synthase MoaX from Mycobacterium tuberculosis is posttranslationally cleaved into functional MoaD and MoaE in M. smegmatis, the protease responsible for the cleavage of MoaD-MoaE fusion protein has remained unknown to date. Here we report that the JAMM/MPN(+) domain containing metalloprotease DR0402 (JAMMDR) from Deinococcus radiodurans can cleave the MoaD-MoaE fusion protein DR2607, the sole MPT synthase in D. radiodurans, generating the MoaD having a C-terminal di-Gly motif. Furthermore, JAMMDR can also cleave off the MoaD from MoaD-eGFP fusion protein suggesting that JAMMDR recognizes the MoaD region rather than MoaE region in the cleaving process of MoaD-MoaE fusion protein.

Y. M. Yang, Y. B. Won, C. J. Ji, J. H. Kim, S. H. Ryu, Y. H. Ok, and J. W. Lee,Cleavage of molybdopterin synthase MoaD-MoaE linear fusion by JAMM/MPN(+) domain containing metalloprotease DR0402 from Deinococcus radiodurans, Biochemical and biophysical research communications, 2018, 502, 48-54.

Moco

The functional principle of eukaryotic molybdenum insertases

The molybdenum cofactor (Moco) is a redox-active prosthetic group found in the active site of Moco-dependent enzymes, which are vitally important for life. Moco biosynthesis involves several enzymes that catalyze the subsequent conversion of GTP into cyclic pyranopterin monophosphate (cPMP), molybdopterin (MPT), adenylated MPT (MPT-AMP), and finally Moco. While the underlying principles of cPMP, MPT, and MPT-AMP formation are well understood, the molybdenum insertase (Mo-insertase)-catalyzed final Moco maturation step is not. In the present study, we analyzed high-resolution X-ray datasets of the plant Mo-insertase Cnx1 E that revealed two molybdate-binding sites within the active site, hence improving the current view on Cnx1 E functionality. The presence of molybdate anions in either of these sites is tied to a distinctive backbone conformation, which we suggest to be essential for Mo-insertase molybdate selectivity and insertion efficiency.

J. Krausze, T. W. Hercher, D. Zwerschke, M. L. Kirk, W. Blankenfeldt, R. R. Mendel, and T. Kruse,The functional principle of eukaryotic molybdenum insertases, Biochemical Journal, 2018, 475, 1739-1753.

 

Molybdenum cofactor synthesis

97 Synthetic studies on the molybdenum cofactor: Total synthesis of pterindithiolenes by direct dithiolene formation from suitable pterin alkynes

A novel, efficient total synthesis of a series of pterindithiolenes (15, 16, 17 and 18) [(5,6-dihydro-[1,4]dithiin or 6,7-dihydro-5H-[1,4]dithiepin systems respectively for six and seven membered dithiolenes] has been reported. The six membered quinoxaline thioketal 9 and seven membered quinoxaline dithiolene 11 have also been synthesized from quinoxaline acetylenic alcohol 5 and the corresponding acetylenic ketone 10 respectively. The synthesis of five membered pterin thioketals 12 and 13 along with the conversion of 13 to the dithiolene 14 by the reaction with NBS is also reported. [GRAPHICS] .

A. C. Maity, M. K. Das, S. Maity, and S. Goswami,Synthetic studies on the molybdenum cofactor: Total synthesis of pterindithiolenes by direct dithiolene formation from suitable pterin alkynes, Synthetic Communications, 2018, 48, 1629-1639.

 

MOLYBDENUM COFACTOR DEFICIENCY

Molybdenum cofactor deficiency type A: Prenatal monitoring using MRI

Molybdenum cofactor deficiency type A (MoCD-A) is an inborn error of metabolism presenting early after birth with severe seizures. Recently, experimental substitution treatment with cyclic pyranopterin monophosphate (cPMP) has become available. Because prenatal data is scarce, we report data of prenatal Magnetic Resonance Imaging (MRI) in two cases with MoCD-A demonstrating signs of possible early brain injury. Prenatal MRI can be used for monitoring in MoCD-A to guide decision-making in timing of delivery. (C) 2017 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

C. M. A. Lubout, T. G. J. Derks, L. Meiners, J. J. Erwich, K. A. Bergman, R. J. Lunsing, G. Schwarz, A. Veldman, and F. J. van Spronsen,Molybdenum cofactor deficiency type A: Prenatal monitoring using MRI, European Journal of Paediatric Neurology, 2018, 22, 536-540.

 

MOLYBDENUM COFACTOR

The functional principle of eukaryotic molybdenum insertases

The molybdenum cofactor (Moco) is a redox-active prosthetic group found in the active site of Moco-dependent enzymes, which are vitally important for life. Moco biosynthesis involves several enzymes that catalyze the subsequent conversion of GTP into cyclic pyranopterin monophosphate (cPMP), molybdopterin (MPT), adenylated MPT (MPT-AMP), and finally Moco. While the underlying principles of cPMP, MPT, and MPT-AMP formation are well understood, the molybdenum insertase (Mo-insertase)-catalyzed final Moco maturation step is not. In the present study, we analyzed high-resolution X-ray datasets of the plant Mo-insertase Cnx1E that revealed two molybdate-binding sites within the active site, hence improving the current view on Cnx1E functionality. The presence of molybdate anions in either of these sites is tied to a distinctive backbone conformation, which we suggest to be essential for Mo-insertase molybdate selectivity and insertion efficiency.

J. Krausze, T. W. Hercher, D. Zwerschke, M. L. Kirk, W. Blankenfeldt, R. R. Mendel, and T. Kruse,The functional principle of eukaryotic molybdenum insertases, The Biochemical journal, 2018, 475, 1739-1753.

 

Cysteine Metabolism in Neuronal Redox Homeostasis

Besides its essential role in protein synthesis, cysteine plays vital roles in redox homeostasis, being a component of the major antioxidant glutathione (GSH) and a potent antioxidant by itself. In addition, cysteine undergoes a variety of post-translational modifications that modulate several physiological processes. It is becoming increasingly clear that redox-modulated events play important roles not only in peripheral tissues but also in the brain where cysteine disposition is central to these pathways. Dysregulated cysteine metabolism is associated with several neurodegenerative disorders. Accordingly, restoration of cysteine balance has therapeutic benefits. This review discusses metabolic signaling pathways pertaining to cysteine disposition in the brain under normal and pathological conditions, highlighting recent findings on cysteine metabolism during aging and in neurodegenerative conditions such as Huntington's disease (HD) and molybdenum cofactor (MoCo) deficiency (MoCD) among others.

B. D. Paul, J. I. Sbodio, and S. H. Snyder,Cysteine Metabolism in Neuronal Redox Homeostasis, Trends in pharmacological sciences, 2018, 39, 513-524.

 

Molybdenum and iron mutually impact their homeostasis in cucumber (Cucumis sativus) plants

Molybdenum (Mo) and iron (Fe) are essential micronutrients required for crucial enzyme activities in plant metabolism. Here we investigated the existence of a mutual control of Mo and Fe homeostasis in cucumber (Cucumis sativus). Plants were grown under single or combined Mo and Fe starvation. Physiological parameters were measured, the ionomes of tissues and the ionomes and proteomes of root mitochondria were profiled, and the activities of molybdo-enzymes and the synthesis of molybdenum cofactor (Moco) were evaluated. Fe and Mo were found to affect each other's total uptake and distribution within tissues and at the mitochondrial level, with Fe nutritional status dominating over Mo homeostasis and affecting Mo availability for molybdo-enzymes in the form of Moco. Fe starvation triggered Moco biosynthesis and affected the molybdo-enzymes, with its main impact on nitrate reductase and xanthine dehydrogenase, both being involved in nitrogen assimilation and mobilization, and on the mitochondrial amidoxime reducing component. These results, together with the identification of >100 proteins differentially expressed in root mitochondria, highlight the central role of mitochondria in the coordination of Fe and Mo homeostasis and allow us to propose the first model of the molecular interactions connecting Mo and Fe homeostasis.

G. Vigani, D. Di Silvestre, A. M. Agresta, S. Donnini, P. Mauri, C. Gehl, F. Bittner, and I. Murgia,Molybdenum and iron mutually impact their homeostasis in cucumber (Cucumis sativus) plants, New Phytologist, 2017, 213, 1222-1241.

 

[Ionome. All the inorganic ions present in an organism.]

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.

Levillain, F., Poquet, Y., Mallet, L., Mazeres, S., Marceau, M., Brosch, R., Bange, F. C., Supply, P., Magalon, A., and Neyrolles, O.,Horizontal acquisition of a hypoxia-responsive molybdenum cofactor biosynthesis pathway contributed to Mycobacterium tuberculosis pathoadaptation, PLoS pathogens, 2017, 13, e1006752.

The Molybdenum Cofactor Biosynthesis Network: In vivo Protein-Protein Interactions of an Actin Associated Multi-Protein Complex

Survival of plants and nearly all organisms depends on the pterin based molybdenum cofactor (Moco) as well as its effective biosynthesis and insertion into apo-enzymes. To this end, both the central Moco biosynthesis enzymes are characterized and the conserved four-step reaction pathway for Moco biosynthesis is well-understood. However, protection mechanisms to prevent degradation during biosynthesis as well as transfer of the highly oxygen sensitive Moco and its intermediates are not fully enlightened. The formation of protein complexes involving transient protein-protein interactions is an efficient strategy for protected metabolic channelling of sensitive molecules. In this review, Moco biosynthesis and allocation network is presented and discussed. This network was intensively studied based on two in vivo interaction methods: bimolecular fluorescence complementation (BiFC) and split-luciferase. Whereas BiFC allows localisation of interacting partners, split-luciferase assay determines interaction strengths in vivo. Results demonstrate (i) interaction of Cnx2 and Cnx3 within the mitochondria and (ii) assembly of a biosynthesis complex including the cytosolic enzymes Cnx5, Cnx6, Cnx7, and Cnxl, which enables a protected transfer of intermediates. The whole complex is associated with actin filaments via Cnxl as anchor protein. After biosynthesis, Moco needs to be handed over to the specific apo-enzymes. A potential pathway was discovered. Molybdenum-containing enzymes of the sulphite oxidase family interact directly with Cnxl. In contrast, the xanthine oxidoreductase family acquires Moco indirectly via a Moco binding protein (MoBP2) and Moco sulphurase ABA3. In summary, the uncovered interaction matrix enables an efficient transfer for intermediate and product protection via micro-compartmentation.

Kaufholdt, D., Baillie, C. K., Meinen, R., Mendel, R. R., and Hansch, R.,The Molybdenum Cofactor Biosynthesis Network: In vivo Protein-Protein Interactions of an Actin Associated Multi-Protein Complex, Frontiers in Plant Science, 2017, 8.

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 , 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 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 characterise CNX2 and suggest that ATM3 is indirectly required for cPMP synthesis.

I. Kruse, A. Maclean, L. Hill, and J. Balk,Genetic dissection of cyclic pyranopterin monophosphate biosynthesis in plant mitochondria, The Biochemical journal, 2017.

 

Shared Sulfur Mobilization Routes for tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Prokaryotes and Eukaryotes

Modifications of transfer RNA (tRNA) have been shown to play critical roles in the biogenesis, metabolism, structural stability and function of RNA molecules, and the specific modifications of nucleobases with sulfur atoms in tRNA are present in pro- and eukaryotes. Here, especially the thiomodifications xm(5)s(2)U at the wobble position 34 in tRNAs for Lys, Gln and Glu, were suggested to have an important role during the translation process by ensuring accurate deciphering of the genetic code and by stabilization of the tRNA structure. The trafficking and delivery of sulfur nucleosides is a complex process carried out by sulfur relay systems involving numerous proteins, which not only deliver sulfur to the specific tRNAs but also to other sulfur-containing molecules including iron-sulfur clusters, thiamin, biotin, lipoic acid and molybdopterin (MPT). Among the biosynthesis of these sulfur-containing molecules, the biosynthesis of the molybdenum cofactor (Moco) and the synthesis of thio-modified tRNAs in particular show a surprising link by sharing protein components for sulfur mobilization in pro- and eukaryotes.

S. Leimkuhler, M. Buhning, and L. Beilschmidt,Shared Sulfur Mobilization Routes for tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Prokaryotes and Eukaryotes, Biomolecules, 2017, 7.

The Molybdenum Cofactor Biosynthesis Network: In vivo Protein-Protein Interactions of an Actin Associated Multi-Protein Complex

Survival of plants and nearly all organisms depends on the pterin based molybdenum cofactor (Moco) as well as its effective biosynthesis and insertion into apo-enzymes. To this end, both the central Moco biosynthesis enzymes are characterized and the conserved four-step reaction pathway for Moco biosynthesis is well-understood. However, protection mechanisms to prevent degradation during biosynthesis as well as transfer of the highly oxygen sensitive Moco and its intermediates are not fully enlightened. The formation of protein complexes involving transient protein-protein interactions is an efficient strategy for protected metabolic channelling of sensitive molecules. In this review, Moco biosynthesis and allocation network is presented and discussed. This network was intensively studied based on two in vivo interaction methods: bimolecular fluorescence complementation (BiFC) and split-luciferase. Whereas BiFC allows localisation of interacting partners, split-luciferase assay determines interaction strengths in vivo. Results demonstrate (i) interaction of Cnx2 and Cnx3 within the mitochondria and (ii) assembly of a biosynthesis complex including the cytosolic enzymes Cnx5, Cnx6, Cnx7, and Cnxl, which enables a protected transfer of intermediates. The whole complex is associated with actin filaments via Cnxl as anchor protein. After biosynthesis, Moco needs to be handed over to the specific apo-enzymes. A potential pathway was discovered. Molybdenum-containing enzymes of the sulphite oxidase family interact directly with Cnxl. In contrast, the xanthine oxidoreductase family acquires Moco indirectly via a Moco binding protein (MoBP2) and Moco sulphurase ABA3. In summary, the uncovered interaction matrix enables an efficient transfer for intermediate and product protection via micro-compartmentation.

D. Kaufholdt, C. K. Baillie, R. Meinen, R. R. Mendel, and R. Hansch,The Molybdenum Cofactor Biosynthesis Network: In vivo Protein-Protein Interactions of an Actin Associated Multi-Protein Complex, Frontiers in Plant Science, 2017, 8.

 

Pterin function in bacteria

Pterins are widely conserved biomolecules that play essential roles in diverse organisms. First described as enzymatic cofactors in eukaryotic systems, bacterial -pterins were discovered in cyanobacteria soon after. Several pterin structures unique to bacteria have been described, with conjugation to glycosides and nucleotides commonly observed. Despite this significant structural diversity, relatively few biological functions have been elucidated. Molybdopterin, the best studied bacterial pterin, plays an essential role in the function of the Moco cofactor. Moco is an essential component of molybdoenzymes such as sulfite oxidase, nitrate reductase, and dimethyl sulfoxide reductase, all of which play important roles in bacterial metabolism and global nutrient cycles. Outside of the molybdoenzymes, pterin cofactors play important roles in bacterial cyanide utilization and aromatic amino acid metabolism. Less is known about the roles of pterins in nonenzymatic processes. Cyanobacterial pterins have been implicated in phenotypes related to UV protection and phototaxis. Research describing the pterin-mediated control of cyclic nucleotide metabolism, and their influence on virulence and attachment, points to a possible role for pterins in regulation of bacterial behavior. In this review, we describe the variety of pterin functions in bacteria, compare and contrast structural and mechanistic differences, and illuminate promising avenues of future research.

N. Feirer, and C. Fuqua,Pterin function in bacteria, Pteridines, 2017, 28, 23-36.

 

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.

Biosynthesis

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.