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K. V. Rajagopalan - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of a molybdopterin synthase Precursor Z complex insight into its sulfur transfer mechanism and its role in molybdenum cofactor deficiency
Biochemistry, 2008Co-Authors: Juma N Daniels, K. V. Rajagopalan, Margot M Wuebbens, Hermann SchindelinAbstract:In almost all biological life forms, molybdenum and tungsten are coordinated by molybdopterin (MPT), a tricyclic pyranopterin containing a cis-dithiolene group. Together, the metal and the pterin moiety form the redox reactive molybdenum cofactor (Moco). Mutations in patients with deficiencies in Moco biosynthesis usually occur in the enZymes catalyZing the first and second steps of biosynthesis, leading to the formation of Precursor Z and MPT, respectively. The second step is catalyZed by the heterotetrameric MPT synthase protein consisting of two large (MoaE) and two small (MoaD) subunits with the MoaD subunits located at opposite ends of a central MoaE dimer. Previous studies have determined that the conversion of the sulfur- and metal-free Precursor Z to MPT by MPT synthase involves the transfer of sulfur atoms from a C-terminal MoaD thiocarboxylate to the C-1' and C-2' positions of Precursor Z. Here, we present the crystal structures of non-thiocarboxylated MPT synthase from Staphylococcus aureus in its apo form and in complex with Precursor Z. A comparison of the two structures reveals conformational changes in a loop that participates in interactions with Precursor Z. In the complex, Precursor Z is bound by strictly conserved residues in a pocket at the MoaE dimer interface in close proximity of the C-terminal glycine of MoaD. Biochemical evidence indicates that the first dithiolene sulfur is added at the C-2' position.
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crystal structure of a molybdopterin synthase Precursor Z complex insight into its sulfur transfer mechanism and its role in molybdenum cofactor deficiency
Biochemistry, 2008Co-Authors: Juma N Daniels, K. V. Rajagopalan, Margot M Wuebbens, Hermann SchindelinAbstract:In almost all biological life forms, molybdenum and tungsten are coordinated by molybdopterin (MPT), a tricyclic pyranopterin containing a cis-dithiolene group. Together, the metal and the pterin moiety form the redox reactive molybdenum cofactor (Moco). Mutations in patients with deficiencies in Moco biosynthesis usually occur in the enZymes catalyZing the first and second steps of biosynthesis, leading to the formation of Precursor Z and MPT, respectively. The second step is catalyZed by the heterotetrameric MPT synthase protein consisting of two large (MoaE) and two small (MoaD) subunits with the MoaD subunits located at opposite ends of a central MoaE dimer. Previous studies have determined that the conversion of the sulfur- and metal-free Precursor Z to MPT by MPT synthase involves the transfer of sulfur atoms from a C-terminal MoaD thiocarboxylate to the C-1‘ and C-2‘ positions of Precursor Z. Here, we present the crystal structures of non-thiocarboxylated MPT synthase from Staphylococcus aureus in ...
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evidence for the physiological role of a rhodanese like protein for the biosynthesis of the molybdenum cofactor in humans
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Andreas Matthies, Ralf R. Mendel, K. V. Rajagopalan, Silke LeimkühlerAbstract:Recent studies have identified the human genes involved in the biosynthesis of the molybdenum cofactor. The human MOCS3 protein contains an N-terminal domain similar to the Escherichia coli MoeB protein and a C-terminal segment displaying similarities to the sulfurtransferase rhodanese. The MOCS3 protein is believed to catalyZe both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of the smaller subunit of molybdopterin (MPT) synthase. The MOCS3 rhodanese-like domain (MOCS3-RLD) was purified after heterologous expression in E. coli and was shown to catalyZe the transfer of sulfur from thiosulfate to cyanide. In a defined in vitro system for the generation of MPT from Precursor Z, the sulfurated form of MOCS3-RLD was able to provide the sulfur for the thiocarboxylation of MOCS2A, the small MPT synthase subunit in humans. Mutation of the putative persulfide-forming active-site cysteine residue C412 abolished the sulfurtransferase activity of MOCS3-RLD completely, showing the importance of this cysteine residue for catalysis. In contrast to other mammalian rhodaneses, which are mostly localiZed within mitochondria, MOCS3 in addition to the subunits of MPT synthase are localiZed in the cytosol.
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mechanistic studies of human molybdopterin synthase reaction and characteriZation of mutants identified in group b patients of molybdenum cofactor deficiency
Journal of Biological Chemistry, 2003Co-Authors: Silke Leimkühler, K. V. Rajagopalan, Andrea Freuer, Jose Angel Santamaria Araujo, Ralf R. MendelAbstract:Biosynthesis of the molybdenum cofactor involves the initial formation of Precursor Z, its subsequent conversion to molybdopterin (MPT) by MPT synthase, and attachment of molybdenum to the dithiolene moiety of MPT. The sulfur used for the formation of the dithiolene group of MPT exists in the form of a thiocarboxylate group at the C terminus of the smaller subunit of MPT synthase. Human MPT synthase contains the MOCS2A and MOCS2B proteins that display homology to the Escherichia coli proteins MoaD and MoaE, respectively. MOCS2A and MOCS2B were purified after heterologous expression in E. coli, and the separately purified subunits readily assemble into a functional MPT synthase tetramer. The rate of conversion of Precursor Z to MPT by the human enZyme is slower than that of the eubacterial homologue. To obtain insights into the molecular mechanism leading to human molybdenum cofactor deficiency, site-specific mutations identified in patients showing symptoms of molybdenum cofactor deficiency were generated. CharacteriZation of a V7F substitution in MOCS2A, identified in a patient with an unusual mild form of the disease, showed that the mutation weakens the interaction between MOCS2A and MOCS2B, whereas a MOCS2B-E168K mutation identified in a severely affected patient attenuates binding of Precursor Z.
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A sulfurtransferase is required in the transfer of cysteine sulfur in the in vitro synthesis of molybdopterin from Precursor Z in Escherichia coli.
Journal of Biological Chemistry, 2001Co-Authors: Silke Leimkühler, K. V. RajagopalanAbstract:Abstract It has been shown that conversion of Precursor Z to molybdopterin (MPT) by Escherichia coli MPT synthase entails the transfer of the sulfur atom of the C-terminal thiocarboxylate from the small subunit of the synthase to generate the dithiolene group of MPT and that the moeB mutant ofE. coli contains inactive MPT synthase devoid of the thiocarboxylate. The data presented here demonstrate thatl-cysteine can serve as the source of the sulfur for the biosynthesis of MPT in vitro but only in the presence of a persulfide-containing sulfurtransferase such as IscS, cysteine sulfinate desulfinase (CSD), or CsdB. A fully defined in vitro system has been developed in which an inactive form of MPT synthase can be activated by incubation with MoeB, Mg-ATP,l-cysteine, and one of the NifS-like sulfurtransferases, and the addition of Precursor Z to the in vitro system gives rise to MPT formation. The use of radiolabeledl-[35S]cysteine has demonstrated that both sulfurs of the dithiolene group of MPT originate froml-cysteine. It was found that MPT can be produced from Precursor Z in an E. coli iscS mutant strain, indicating that IscS is not required for the in vivo sulfuration of MPT synthase. A comparison of the ability of the three sulfurtransferases to provide the sulfur for MPT formation showed the highest activity for CSD in the in vitro system.
Hermann Schindelin - One of the best experts on this subject based on the ideXlab platform.
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endor spectroscopy shows that guanine n1 binds to 4fe 4s cluster ii of the s adenosylmethionine dependent enZyme moaa mechanistic implications
Journal of the American Chemical Society, 2009Co-Authors: Nicholas S Lees, Petra Hanzelmann, Heather L Hernandez, Sowmya Subramanian, Hermann Schindelin, Michael K Johnson, Brian M HoffmanAbstract:The S-adenosylmethionine-dependent enZyme MoaA, in concert with MoaC, catalyZes the first step of molybdenum cofactor biosynthesis, the conversion of guanosine 5′-triphosphate (5′-GTP) into Precursor Z. A published X-ray crystal structure of MoaA with the substrate 5′-GTP revealed that the substrate might be bound to the unique iron of one of two 4Fe−4S clusters through either or both the amino and N1 nitrogen nuclei. Use of 35 GHZ continuous-wave ENDOR spectroscopy of MoaA with unlabeled and 15N-labeled substrate and a reduced [4Fe−4S]+ cluster now demonstrates that only one nitrogen nucleus is bound to the cluster. Experiments with the substrate analogue inosine 5′-triphosphate further demonstrate that it is the N1 nitrogen that binds. Two of the more distant nitrogen nuclei have also been detected by 35 GHZ pulsed ENDOR spectroscopy, allowing a rough approximation of their distances from the cluster to be calculated. Combining this information with the crystal structure, we propose that the guanine bas...
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crystal structure of a molybdopterin synthase Precursor Z complex insight into its sulfur transfer mechanism and its role in molybdenum cofactor deficiency
Biochemistry, 2008Co-Authors: Juma N Daniels, K. V. Rajagopalan, Margot M Wuebbens, Hermann SchindelinAbstract:In almost all biological life forms, molybdenum and tungsten are coordinated by molybdopterin (MPT), a tricyclic pyranopterin containing a cis-dithiolene group. Together, the metal and the pterin moiety form the redox reactive molybdenum cofactor (Moco). Mutations in patients with deficiencies in Moco biosynthesis usually occur in the enZymes catalyZing the first and second steps of biosynthesis, leading to the formation of Precursor Z and MPT, respectively. The second step is catalyZed by the heterotetrameric MPT synthase protein consisting of two large (MoaE) and two small (MoaD) subunits with the MoaD subunits located at opposite ends of a central MoaE dimer. Previous studies have determined that the conversion of the sulfur- and metal-free Precursor Z to MPT by MPT synthase involves the transfer of sulfur atoms from a C-terminal MoaD thiocarboxylate to the C-1' and C-2' positions of Precursor Z. Here, we present the crystal structures of non-thiocarboxylated MPT synthase from Staphylococcus aureus in its apo form and in complex with Precursor Z. A comparison of the two structures reveals conformational changes in a loop that participates in interactions with Precursor Z. In the complex, Precursor Z is bound by strictly conserved residues in a pocket at the MoaE dimer interface in close proximity of the C-terminal glycine of MoaD. Biochemical evidence indicates that the first dithiolene sulfur is added at the C-2' position.
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crystal structure of a molybdopterin synthase Precursor Z complex insight into its sulfur transfer mechanism and its role in molybdenum cofactor deficiency
Biochemistry, 2008Co-Authors: Juma N Daniels, K. V. Rajagopalan, Margot M Wuebbens, Hermann SchindelinAbstract:In almost all biological life forms, molybdenum and tungsten are coordinated by molybdopterin (MPT), a tricyclic pyranopterin containing a cis-dithiolene group. Together, the metal and the pterin moiety form the redox reactive molybdenum cofactor (Moco). Mutations in patients with deficiencies in Moco biosynthesis usually occur in the enZymes catalyZing the first and second steps of biosynthesis, leading to the formation of Precursor Z and MPT, respectively. The second step is catalyZed by the heterotetrameric MPT synthase protein consisting of two large (MoaE) and two small (MoaD) subunits with the MoaD subunits located at opposite ends of a central MoaE dimer. Previous studies have determined that the conversion of the sulfur- and metal-free Precursor Z to MPT by MPT synthase involves the transfer of sulfur atoms from a C-terminal MoaD thiocarboxylate to the C-1‘ and C-2‘ positions of Precursor Z. Here, we present the crystal structures of non-thiocarboxylated MPT synthase from Staphylococcus aureus in ...
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binding of 5 gtp to the c terminal fes cluster of the radical s adenosylmethionine enZyme moaa provides insights into its mechanism
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Petra Hanzelmann, Hermann SchindelinAbstract:The first step in molybdenum cofactor biosynthesis, the conversion of 5′-GTP to Precursor Z, an oxygen-sensitive tetrahydropyranopterin is catalyZed by the S-adenosylmethionine (SAM)-dependent enZyme MoaA and the accessory protein MoaC. This reaction involves the radical-initiated intramolecular rearrangement of the guanine C8 atom. MoaA harbors an N-terminal [4Fe–4S] cluster, which is involved in the reductive cleavage of SAM and generates a 5′-deoxyadenosyl radical (5′-dA•), and a C-terminal [4Fe–4S] cluster presumably involved in substrate binding and/or activation. Biochemical studies identified residues involved in 5′-GTP binding and the determinants of nucleotide specificity. The crystal structure of MoaA in complex with 5′-GTP confirms the biochemical data and provides valuable insights into the subsequent radical reaction. MoaA binds 5′-GTP with high affinity and interacts through its C-terminal [4Fe–4S] cluster with the guanine N1 and N2 atoms, in a yet uncharacteriZed binding mode. The tightly anchored triphosphate moiety prevents the escape of radical intermediates. This structure also visualiZes the l-Met and 5′-dA cleavage products of SAM. Rotation of the 5′-dA ribose and/or conformational changes of the guanosine are proposed to bring the 5′-deoxyadenosyl radical into close proximity of either the ribose C2′ and C3′ or the guanine C8 carbon atoms leading to hydrogen abstraction.
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crystal structure of the s adenosylmethionine dependent enZyme moaa and its implications for molybdenum cofactor deficiency in humans
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Petra Hanzelmann, Hermann SchindelinAbstract:The MoaA and MoaC proteins catalyZe the first step during molybdenum cofactor biosynthesis, the conversion of a guanosine derivative to Precursor Z. MoaA belongs to the S-adenosylmethionine (SAM)-dependent radical enZyme superfamily, members of which catalyZe the formation of protein and/or substrate radicals by reductive cleavage of SAM by a [4Fe–4S] cluster. A defined in vitro system is described, which generates Precursor Z and led to the identification of 5′-GTP as the substrate. The structures of MoaA in the apo-state (2.8 A) and in complex with SAM (2.2 A) provide valuable insights into its mechanism and help to define the defects caused by mutations in the human ortholog of MoaA that lead to molybdenum cofactor deficiency, a usually fatal disease accompanied by severe neurological symptoms. The central core of each subunit of the MoaA dimer is an incomplete triosephosphate isomerase barrel formed by the N-terminal part of the protein, which contains the [4Fe–4S] cluster typical for SAM-dependent radical enZymes. SAM is the fourth ligand to the cluster and binds to its unique Fe as an N/O chelate. The lateral opening of the incomplete triosephosphate isomerase barrel is covered by the C-terminal part of the protein containing an additional [4Fe–4S] cluster, which is unique to MoaA proteins. Both FeS clusters are separated by ≈17 A, with a large active site pocket between. The noncysteinyl-ligated unique Fe site of the C-terminal [4Fe–4S] cluster is proposed to be involved in the binding and activation of 5′-GTP.
Silke Leimkühler - One of the best experts on this subject based on the ideXlab platform.
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Site-directed mutagenesis of the active site loop of the rhodanese-like domain of the human molybdopterin synthase sulfurase MOCS3. Major differences in substrate specificity between eukaryotic and bacterial homologs.
The FEBS journal, 2007Co-Authors: Karsten Krepinsky, Silke LeimkühlerAbstract:Sequence alignments of human molybdopterin synthase sulfurase, MOCS3, showed that the N-terminal domain is homologous to Escherichia coli MoeB, whereas the C-terminal domain is homologous to rhodanese-like proteins. Previous studies showed that the activity of the separately purified rhodanese-like domain of MOCS3 displayed 1000-fold lower activity in comparison to bovine rhodanese with thiosulfate as sulfur source. When the six amino acid active site loop of MOCS3 rhodanese-like domain was exchanged with the loop found in bovine rhodanese, thiosulfate:cyanide sulfurtransferase activity was increased 165-fold. Site-directed mutagenesis of each individual residue of the active site loop of the MOCS3 rhodanese-like domain showed that the charge of the last amino acid determines thiosulfate sulfurtransferase activity. Replacing Asp417 by threonine resulted in 90-fold increased activity, whereas replacing it by arginine increased the activity 470-fold. Using a fully defined in vitro system containing Precursor Z, MOCS2A, E. coli MoaE, E. coli MoeB, Mg-ATP, MOCS3 rhodanese-like domain, and thiosulfate, it was shown that sulfur transfer to MOCS2A was also affected by the alterations, but not as drastically. Our studies revealed that in humans and most eukaryotes thiosulfate is not the physiologic sulfur donor for MOCS3, whereas in bacterial homologs, which have an arginine at the last position of the active site loop, thiosulfate can be used as a sulfur source for molybdenum cofactor biosynthesis. The phylogenetic analysis of MoeB homologs showed that eukaryotic homologs are of bacterial origin. Furthermore, it could be shown that an MoeB homolog named MoeZ, where the dual CXXC Zinc-binding motif of the MoeB domain is not present, arose independently several times during evolution.
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Ten novel mutations in the molybdenum cofactor genes MOCS1 and MOCS2 and in vitro characteriZation of a MOCS2 mutation that abolishes the binding ability of molybdopterin synthase
Human genetics, 2005Co-Authors: Silke Leimkühler, Mathilde Charcosset, Philippe Latour, Claude Dorche, Soledad Kleppe, Fernando Scaglia, Irmina Szymczak, Petra Schupp, Rita Hahnewald, Jochen ReissAbstract:Molybdenum cofactor deficiency (MIM#252150) is a severe autosomal-recessive disorder with a devastating outcome. The cofactor is the product of a complex biosynthetic pathway involving four different genes (MOCS1, MOCS2, MOCS3 and GEPH). This disorder is caused almost exclusively by mutations in the MOCS1 or MOCS2 genes. Mutations affecting this biosynthetic pathway result in a lethal phenotype manifested by progressive neurological damage via the inactivation of the molybdenum cofactor-dependent enZyme, sulphite oxidase. Here we describe a total of ten novel disease-causing mutations in the MOCS1 and MOCS2 genes. Nine out of these ten mutations were classified as pathogenic in nature, since they create a stop codon, affect constitutive splice site positions, or change strictly conserved motifs. The tenth mutation abolishes the stop codon of the MOCS2B gene, thus elongating the corresponding protein. The mutation was expressed in vitro and was found to abolish the binding affinities of the large subunit of molybdopterin synthase (MOCS2B) for both Precursor Z and the small subunit of molybdopterin synthase (MOCS2A).
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molybdenum cofactor biosynthesis in humans identification of a persulfide group in the rhodanese like domain of mocs3 by mass spectrometry
Biochemistry, 2005Co-Authors: Andreas Matthies, Manfred Nimtz, Silke LeimkühlerAbstract:The human MOCS3 protein contains an N-terminal domain similar to the Escherichia coli MoeB protein and a C-terminal segment displaying similarities to the sulfurtransferase rhodanese. MOCS3 is proposed to catalyZe both the adenylation and the subsequent generation of a thiocarboxylate group at the C-terminus of the smaller subunit of molybdopterin (MPT) synthase during Moco biosynthesis in humans. Recent studies have shown that the MOCS3 rhodanese-like domain (MOCS3-RLD) catalyZes the transfer of sulfur from thiosulfate to cyanide and is also able to provide the sulfur for the thiocarboxylation of MOCS2A in a defined in vitro system for the generation of MPT from Precursor Z. MOCS3-RLD contains four cysteine residues of which only C412 in the six amino acid active loop is conserved in homologous proteins from other organisms. ESI-MS/MS studies gave direct evidence for the formation of a persulfide group that is exclusively formed on C412. Simultaneous mutagenesis of the remaining three cysteine residues s...
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evidence for the physiological role of a rhodanese like protein for the biosynthesis of the molybdenum cofactor in humans
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Andreas Matthies, Ralf R. Mendel, K. V. Rajagopalan, Silke LeimkühlerAbstract:Recent studies have identified the human genes involved in the biosynthesis of the molybdenum cofactor. The human MOCS3 protein contains an N-terminal domain similar to the Escherichia coli MoeB protein and a C-terminal segment displaying similarities to the sulfurtransferase rhodanese. The MOCS3 protein is believed to catalyZe both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of the smaller subunit of molybdopterin (MPT) synthase. The MOCS3 rhodanese-like domain (MOCS3-RLD) was purified after heterologous expression in E. coli and was shown to catalyZe the transfer of sulfur from thiosulfate to cyanide. In a defined in vitro system for the generation of MPT from Precursor Z, the sulfurated form of MOCS3-RLD was able to provide the sulfur for the thiocarboxylation of MOCS2A, the small MPT synthase subunit in humans. Mutation of the putative persulfide-forming active-site cysteine residue C412 abolished the sulfurtransferase activity of MOCS3-RLD completely, showing the importance of this cysteine residue for catalysis. In contrast to other mammalian rhodaneses, which are mostly localiZed within mitochondria, MOCS3 in addition to the subunits of MPT synthase are localiZed in the cytosol.
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mechanistic studies of human molybdopterin synthase reaction and characteriZation of mutants identified in group b patients of molybdenum cofactor deficiency
Journal of Biological Chemistry, 2003Co-Authors: Silke Leimkühler, K. V. Rajagopalan, Andrea Freuer, Jose Angel Santamaria Araujo, Ralf R. MendelAbstract:Biosynthesis of the molybdenum cofactor involves the initial formation of Precursor Z, its subsequent conversion to molybdopterin (MPT) by MPT synthase, and attachment of molybdenum to the dithiolene moiety of MPT. The sulfur used for the formation of the dithiolene group of MPT exists in the form of a thiocarboxylate group at the C terminus of the smaller subunit of MPT synthase. Human MPT synthase contains the MOCS2A and MOCS2B proteins that display homology to the Escherichia coli proteins MoaD and MoaE, respectively. MOCS2A and MOCS2B were purified after heterologous expression in E. coli, and the separately purified subunits readily assemble into a functional MPT synthase tetramer. The rate of conversion of Precursor Z to MPT by the human enZyme is slower than that of the eubacterial homologue. To obtain insights into the molecular mechanism leading to human molybdenum cofactor deficiency, site-specific mutations identified in patients showing symptoms of molybdenum cofactor deficiency were generated. CharacteriZation of a V7F substitution in MOCS2A, identified in a patient with an unusual mild form of the disease, showed that the mutation weakens the interaction between MOCS2A and MOCS2B, whereas a MOCS2B-E168K mutation identified in a severely affected patient attenuates binding of Precursor Z.
Ralf R. Mendel - One of the best experts on this subject based on the ideXlab platform.
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characteriZation of mocs1a an oxygen sensitive iron sulfur protein involved in human molybdenum cofactor biosynthesis
Journal of Biological Chemistry, 2004Co-Authors: Petra Hanzelmann, Ralf R. Mendel, Heather L Hernandez, Michael K Johnson, Christian Menzel, Ricardo Garciaserres, Boi Hanh Huynh, Hermann SchindelinAbstract:The human proteins MOCS1A and MOCS1B catalyZe the conversion of a guanosine derivative to Precursor Z during molybdenum cofactor biosynthesis. MOCS1A shares homology with S-adenosylmethionine (AdoMet)-dependent radical enZymes, which catalyZe the formation of protein and/or substrate radicals by reductive cleavage of AdoMet through a [4Fe-4S] cluster. Sequence analysis of MOCS1A showed two highly conserved cysteine motifs, one near the N terminus and one near the C terminus. MOCS1A was heterologously expressed in Escherichia coli and purified under aerobic and anaerobic conditions. Individual mutations of the conserved cysteines to serine revealed that all are essential for synthesis of Precursor Z in vivo. The type and properties of the iron-sulfur (FeS) clusters were investigated using a combination of UV-visible absorption, variable temperature magnetic circular dichroism, resonance Raman, Mossbauer, and EPR spectroscopies coupled with iron and acid-labile sulfide analyses. The results indicated that anaerobically purified MOCS1A is a monomeric protein containing two oxygen-sensitive FeS clusters, each coordinated by only three cysteine residues. A redox-active [4Fe-4S](2+,+) cluster is ligated by an N-terminal CX(3)CX(2)C motif as is the case with all other AdoMet-dependent radical enZymes investigated thus far. A C-terminal CX(2)CX(13)C motif that is unique to MOCS1A and its orthologs primarily ligates a [3Fe-4S](0) cluster. However, MOCS1A could be reconstituted in vitro under anaerobic conditions to yield a form containing two [4Fe-4S](2+) clusters. The N-terminal [4Fe-4S](2+) cluster was rapidly degraded by oxygen via a semistable [2Fe-2S](2+) cluster intermediate, and the C-terminal [4Fe-4S](2+) cluster was rapidly degraded by oxygen to yield a semistable [3Fe-4S](0) cluster intermediate.
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rescue of lethal molybdenum cofactor deficiency by a biosynthetic Precursor from escherichia coli
Human Molecular Genetics, 2004Co-Authors: Gunter Schwarz, Tanja Otte, Petra Hanzelmann, Jose Angel Santamariaaraujo, Stefan Wolf, Ibrahim M Adham, Hermann Josef Grone, Herbert Schwegler, Jorn Oliver Sass, Ralf R. MendelAbstract:Substitution therapies for orphan genetic diseases, including enZyme replacement methods, are frequently hampered by the limited availability of the required therapeutic substance. We describe the isolation of a pterin intermediate from bacteria that was successfully used for the therapy of a hitherto incurable and lethal disease. Molybdenum cofactor (Moco) deficiency is a pleiotropic genetic disorder characteriZed by the loss of the molybdenum-dependent enZymes sulphite oxidase, xanthine oxidoreductase and aldehyde oxidase due to mutations in Moco biosynthesis genes. An intermediate of this pathway—‘Precursor Z’—is more stable than the cofactor itself and has an identical structure in all phyla. Thus, it was overproduced in the bacterium Escherichia coli, purified and used to inject Precursor Z-deficient knockout mice that display a phenotype which resembles that of the human deficiency state. Precursor Z-substituted mice reach adulthood and fertility. Biochemical analyses further suggest that the described treatment can lead to the alleviation of most symptoms associated with human Moco deficiency.
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evidence for the physiological role of a rhodanese like protein for the biosynthesis of the molybdenum cofactor in humans
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Andreas Matthies, Ralf R. Mendel, K. V. Rajagopalan, Silke LeimkühlerAbstract:Recent studies have identified the human genes involved in the biosynthesis of the molybdenum cofactor. The human MOCS3 protein contains an N-terminal domain similar to the Escherichia coli MoeB protein and a C-terminal segment displaying similarities to the sulfurtransferase rhodanese. The MOCS3 protein is believed to catalyZe both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of the smaller subunit of molybdopterin (MPT) synthase. The MOCS3 rhodanese-like domain (MOCS3-RLD) was purified after heterologous expression in E. coli and was shown to catalyZe the transfer of sulfur from thiosulfate to cyanide. In a defined in vitro system for the generation of MPT from Precursor Z, the sulfurated form of MOCS3-RLD was able to provide the sulfur for the thiocarboxylation of MOCS2A, the small MPT synthase subunit in humans. Mutation of the putative persulfide-forming active-site cysteine residue C412 abolished the sulfurtransferase activity of MOCS3-RLD completely, showing the importance of this cysteine residue for catalysis. In contrast to other mammalian rhodaneses, which are mostly localiZed within mitochondria, MOCS3 in addition to the subunits of MPT synthase are localiZed in the cytosol.
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The Tetrahydropyranopterin Structure of the Sulfur-free and Metal-free Molybdenum Cofactor Precursor
Journal of Biological Chemistry, 2004Co-Authors: Jose Angel Santamaria-araujo, Tanja Otte, Berthold Fischer, Manfred Nimtz, Victor Wray, Ralf R. Mendel, Gunter SchwarzAbstract:Abstract The molybdenum cofactor (Moco), a highly conserved pterin compound coordinating molybdenum (Mo), is required for the activity of all Mo-dependent enZymes with the exception of nitrogenase. Moco is synthesiZed by a unique and evolutionary old multi-step pathway with two intermediates identified so far, the sulfur-free and metal-free pterin derivative Precursor Z and molybdopterin, a pterin with an enedithiolate function essential for Mo ligation. The latter pterin component is believed to form a tetrahydropyranopterin similar to the one found for Moco in the crystal structure of Mo as well as tungsten (W) enZymes. Here we report the spectroscopic characteriZation and structure elucidation of Precursor Z purified from Escherichia coli overproducing MoaA and MoaC, two proteins essential for bacterial Precursor Z synthesis. We have shown that purified Precursor Z is as active as Precursor Z present in E. coli cell extracts, demonstrating that no modifications during the purification procedure have occurred. High resolution electrospray ioniZation mass spectrometry afforded a [M + H]+ ion compatible with a molecular formula of C10H15N5O8P. Consequently 1H NMR spectroscopy not allowed structural characteriZation of the molecule but confirmed that this intermediate undergoes direct oxidation to the previously well characteriZed non-productive follow-up product compound Z. The 1H chemical shift and coupling constant data are incompatible with previous structural proposals and indicate that Precursor Z already is a tetrahydropyranopterin system and carries a geminal diol function in the C1′ position.
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mechanistic studies of human molybdopterin synthase reaction and characteriZation of mutants identified in group b patients of molybdenum cofactor deficiency
Journal of Biological Chemistry, 2003Co-Authors: Silke Leimkühler, K. V. Rajagopalan, Andrea Freuer, Jose Angel Santamaria Araujo, Ralf R. MendelAbstract:Biosynthesis of the molybdenum cofactor involves the initial formation of Precursor Z, its subsequent conversion to molybdopterin (MPT) by MPT synthase, and attachment of molybdenum to the dithiolene moiety of MPT. The sulfur used for the formation of the dithiolene group of MPT exists in the form of a thiocarboxylate group at the C terminus of the smaller subunit of MPT synthase. Human MPT synthase contains the MOCS2A and MOCS2B proteins that display homology to the Escherichia coli proteins MoaD and MoaE, respectively. MOCS2A and MOCS2B were purified after heterologous expression in E. coli, and the separately purified subunits readily assemble into a functional MPT synthase tetramer. The rate of conversion of Precursor Z to MPT by the human enZyme is slower than that of the eubacterial homologue. To obtain insights into the molecular mechanism leading to human molybdenum cofactor deficiency, site-specific mutations identified in patients showing symptoms of molybdenum cofactor deficiency were generated. CharacteriZation of a V7F substitution in MOCS2A, identified in a patient with an unusual mild form of the disease, showed that the mutation weakens the interaction between MOCS2A and MOCS2B, whereas a MOCS2B-E168K mutation identified in a severely affected patient attenuates binding of Precursor Z.
Gunter Schwarz - One of the best experts on this subject based on the ideXlab platform.
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molybdenum cofactor biosynthesis and deficiency
Cellular and Molecular Life Sciences, 2005Co-Authors: Gunter SchwarzAbstract:The molybdenum cofactor (Moco) forms the active site of all molybdenum (Mo) enZymes, except nitrogenase. Mo enZymes catalyZe important redox reactions in global metabolic cycles. Moco consists of Mo covalently bound to one or two dithiolates attached to a unique tricyclic pterin moiety commonly referred to as molybdopterin (MPT). Moco is synthesiZed by an ancient and conserved biosynthetic pathway that can be divided into four steps, according to the biosynthetic intermediates Precursor Z (cyclic pyranopterin monophosphate), MPT and adenylated MPT. In a fifth step modifications such as attachment of nucleotides, sulfuration or bond formation between Mo and the protein result in different catalytic Mo centers. A defect in any of the steps of Moco biosynthesis results in the pleiotropic loss of all Mo enZyme activities. Human Moco deficiency is a hereditary metabolic disorder characteriZed by severe neurodegeneration resulting in early childhood death. Recently, a first substitution therapy was established.
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rescue of lethal molybdenum cofactor deficiency by a biosynthetic Precursor from escherichia coli
Human Molecular Genetics, 2004Co-Authors: Gunter Schwarz, Tanja Otte, Petra Hanzelmann, Jose Angel Santamariaaraujo, Stefan Wolf, Ibrahim M Adham, Hermann Josef Grone, Herbert Schwegler, Jorn Oliver Sass, Ralf R. MendelAbstract:Substitution therapies for orphan genetic diseases, including enZyme replacement methods, are frequently hampered by the limited availability of the required therapeutic substance. We describe the isolation of a pterin intermediate from bacteria that was successfully used for the therapy of a hitherto incurable and lethal disease. Molybdenum cofactor (Moco) deficiency is a pleiotropic genetic disorder characteriZed by the loss of the molybdenum-dependent enZymes sulphite oxidase, xanthine oxidoreductase and aldehyde oxidase due to mutations in Moco biosynthesis genes. An intermediate of this pathway—‘Precursor Z’—is more stable than the cofactor itself and has an identical structure in all phyla. Thus, it was overproduced in the bacterium Escherichia coli, purified and used to inject Precursor Z-deficient knockout mice that display a phenotype which resembles that of the human deficiency state. Precursor Z-substituted mice reach adulthood and fertility. Biochemical analyses further suggest that the described treatment can lead to the alleviation of most symptoms associated with human Moco deficiency.
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The Tetrahydropyranopterin Structure of the Sulfur-free and Metal-free Molybdenum Cofactor Precursor
Journal of Biological Chemistry, 2004Co-Authors: Jose Angel Santamaria-araujo, Tanja Otte, Berthold Fischer, Manfred Nimtz, Victor Wray, Ralf R. Mendel, Gunter SchwarzAbstract:Abstract The molybdenum cofactor (Moco), a highly conserved pterin compound coordinating molybdenum (Mo), is required for the activity of all Mo-dependent enZymes with the exception of nitrogenase. Moco is synthesiZed by a unique and evolutionary old multi-step pathway with two intermediates identified so far, the sulfur-free and metal-free pterin derivative Precursor Z and molybdopterin, a pterin with an enedithiolate function essential for Mo ligation. The latter pterin component is believed to form a tetrahydropyranopterin similar to the one found for Moco in the crystal structure of Mo as well as tungsten (W) enZymes. Here we report the spectroscopic characteriZation and structure elucidation of Precursor Z purified from Escherichia coli overproducing MoaA and MoaC, two proteins essential for bacterial Precursor Z synthesis. We have shown that purified Precursor Z is as active as Precursor Z present in E. coli cell extracts, demonstrating that no modifications during the purification procedure have occurred. High resolution electrospray ioniZation mass spectrometry afforded a [M + H]+ ion compatible with a molecular formula of C10H15N5O8P. Consequently 1H NMR spectroscopy not allowed structural characteriZation of the molecule but confirmed that this intermediate undergoes direct oxidation to the previously well characteriZed non-productive follow-up product compound Z. The 1H chemical shift and coupling constant data are incompatible with previous structural proposals and indicate that Precursor Z already is a tetrahydropyranopterin system and carries a geminal diol function in the C1′ position.
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functionality of alternative splice forms of the first enZymes involved in human molybdenum cofactor biosynthesis
Journal of Biological Chemistry, 2002Co-Authors: Petra Hanzelmann, Gunter Schwarz, Ralf R. MendelAbstract:Abstract In humans, genetic deficiencies of enZymes involved in molybdenum cofactor biosynthesis trigger an autosomal recessive and usually fatal disease with severe mostly neurological symptoms. In each of the three biosynthesis steps, at least two proteins or domains are linked for catalysis. For steps 1 and 2, bicistronic mocs (molybdenumcofactor synthesis) mRNAs were found (mocs1 and mocs2) that have been proposed to encode two separate proteins (A and B). In both cases, the A proteins share a highly conserved ubiquitin-like double glycine motif, which is functionally important at least for the small subunit of molybdopterin (MPT) synthase (MOCS2A). Besides the bicistronic form ofmocs1, two alternative splice transcripts were found, resulting in the expression of multidomain proteins embodying both MOCS1A, but without the double glycine motif, and the entire MOCS1B. Here we describe the first functional characteriZation of the human proteins MOCS1A and MOCS1B as well as the MOCS1A-MOCS1B fusion proteins that catalyZe the formation of Precursor Z, a 6-alkyl pterin with a cyclic phosphate, the immediate Precursor of MPT in molybdenum cofactor biosynthesis. High level expression of MOCS1A and MOCS1B inEscherichia coli resulted in the formation and accumulation of Precursor Z that was subsequently converted to MPT. We showed that for catalytic activity MOCS1A needs an accessible C-terminal double glycine motif. In the MOCS1A-MOCS1B fusion proteins lacking the MOCS1A double glycines, only MOCS1B activity could be detected. No evidence was found for an expression of MOCS1B from the bicistronicmocs1A-mocs1B splice type I cDNA, indicating that MOCS1B is only expressed as a fusion to an inactive MOCS1A. Comparative mutational studies of MOCS1A and the small subunit of the E. coli MPT synthase (MoaD) indicate a different function of the double glycine motifs in both proteins.
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functionality of alternative splice forms of the first enZymes involved in human molybdenum cofactor biosynthesis
Journal of Biological Chemistry, 2002Co-Authors: Petra Hanzelmann, Gunter Schwarz, Ralf R. MendelAbstract:Abstract In humans, genetic deficiencies of enZymes involved in molybdenum cofactor biosynthesis trigger an autosomal recessive and usually fatal disease with severe mostly neurological symptoms. In each of the three biosynthesis steps, at least two proteins or domains are linked for catalysis. For steps 1 and 2, bicistronic mocs (molybdenumcofactor synthesis) mRNAs were found (mocs1 and mocs2) that have been proposed to encode two separate proteins (A and B). In both cases, the A proteins share a highly conserved ubiquitin-like double glycine motif, which is functionally important at least for the small subunit of molybdopterin (MPT) synthase (MOCS2A). Besides the bicistronic form ofmocs1, two alternative splice transcripts were found, resulting in the expression of multidomain proteins embodying both MOCS1A, but without the double glycine motif, and the entire MOCS1B. Here we describe the first functional characteriZation of the human proteins MOCS1A and MOCS1B as well as the MOCS1A-MOCS1B fusion proteins that catalyZe the formation of Precursor Z, a 6-alkyl pterin with a cyclic phosphate, the immediate Precursor of MPT in molybdenum cofactor biosynthesis. High level expression of MOCS1A and MOCS1B inEscherichia coli resulted in the formation and accumulation of Precursor Z that was subsequently converted to MPT. We showed that for catalytic activity MOCS1A needs an accessible C-terminal double glycine motif. In the MOCS1A-MOCS1B fusion proteins lacking the MOCS1A double glycines, only MOCS1B activity could be detected. No evidence was found for an expression of MOCS1B from the bicistronicmocs1A-mocs1B splice type I cDNA, indicating that MOCS1B is only expressed as a fusion to an inactive MOCS1A. Comparative mutational studies of MOCS1A and the small subunit of the E. coli MPT synthase (MoaD) indicate a different function of the double glycine motifs in both proteins.