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Bavesh D. Kana - One of the best experts on this subject based on the ideXlab platform.
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cleavage of the moax encoded fused Molybdopterin synthase from mycobacterium tuberculosis is necessary for activity
BMC Microbiology, 2015Co-Authors: Nicole C Narrandes, Edith E Machowski, Valerie Mizrahi, Bavesh D. KanaAbstract:Background Molybdopterin Cofactor (MoCo) biosynthesis in Mycobacterium tuberculosis is associated with a multiplicity of genes encoding several enzymes in the pathway, including the Molybdopterin (MPT) synthase, a hetero tetramer comprising two MoaD and two MoaE subunits. In addition to moaD1, moaD2, moaE1, moaE2, the M. tuberculosis genome also contains a moaX gene which encodes an MPT-synthase in which the MoaD and MoaE domains are located on a single polypeptide. In this study, we assessed the requirement for post-translational cleavage of MoaX for functionality of this novel, fused MPT synthase and attempted to establish a functional hierarchy for the various MPT-synthase encoding genes in M. tuberculosis.
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Cleavage of the moaX-encoded fused Molybdopterin synthase from Mycobacterium tuberculosis is necessary for activity
BMC Microbiology, 2015Co-Authors: Nicole C Narrandes, Edith E Machowski, Valerie Mizrahi, Bavesh D. KanaAbstract:Background Molybdopterin Cofactor (MoCo) biosynthesis in Mycobacterium tuberculosis is associated with a multiplicity of genes encoding several enzymes in the pathway, including the Molybdopterin (MPT) synthase, a hetero tetramer comprising two MoaD and two MoaE subunits. In addition to moaD1 , moaD2 , moaE1 , moaE2 , the M. tuberculosis genome also contains a moaX gene which encodes an MPT-synthase in which the MoaD and MoaE domains are located on a single polypeptide. In this study, we assessed the requirement for post-translational cleavage of MoaX for functionality of this novel, fused MPT synthase and attempted to establish a functional hierarchy for the various MPT-synthase encoding genes in M. tuberculosis. Results Using a heterologous Mycobacterium smegmatis host and the activity of the MoCo-dependent nitrate reductase, we confirmed that moaD2 and moaE2 from M. tuberculosis together encode a functional MPT synthase. In contrast, moaD1 displayed no functionality in this system, even in the presence of the MoeBR sulphurtransferase, which contains the rhodansese-like domain, predicted to activate MoaD subunits. We demonstrated that cleavage of MoaX into its constituent MoaD and MoaE subunits was required for MPT synthase activity and confirmed that cleavage occurs between the Gly82 and Ser83 residues in MoaX. Further analysis of the Gly81-Gly82 motif confirmed that both of these residues are necessary for catalysis and that the Gly81 was required for recognition/cleavage of MoaX by an as yet unidentified protease. In addition, the MoaE component of MoaX was able to function in conjunction with M. smegmatis MoaD2 suggesting that cleavage of MoaX renders functionally interchangeable subunits. Expression of MoaX in E. coli revealed that incorrect post-translational processing is responsible for the lack of activity of MoaX in this heterologous host. Conclusions There is a degree of functional interchangeability between the MPT synthase subunits of M. tuberculosis . In the case of MoaX, post-translational cleavage at the Gly82 residue is required for function.
Emilio Fernández - One of the best experts on this subject based on the ideXlab platform.
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The Chlamydomonas reinhardtii MoCo carrier protein is multimeric and stabilizes Molybdopterin Cofactor in a molybdate charged form
FEBS letters, 1998Co-Authors: Claus-peter Witte, Gunter Schwarz, Ralf R. Mendel, M. Isabel Igeño, Emilio FernándezAbstract:In Chlamydomonas reinhardtii, Molybdopterin Cofactor (MoCo) able to reconstitute active nitrate reductase (NR) with apoenzyme from the Neurospora crassa mutant nit-1 was found mostly bound to a carrier protein (CP). This protein is scarce in the algal free extracts and has been purified 520-fold. MoCoCP is a protein of 64 kDa with subunits of 16.5 kDa and an isoelectric point of 4.5. In contrast to free MoCo, MoCo bound to CP was remarkably protected against inactivation under both aerobic conditions and basic pH. MocoCP transferred active MoCo to apoNR in vitro without addition of molybdate, though reconstituted activity was 20% higher in the presence of molybdate. Incubation with tungstate specifically inhibited MoCoCP activity but had no effect on the activity of free MoCo released from milk xanthine oxidase. MoCoCP did not charge molybdate unless in the presence of N. crassa extracts. Our data support that MoCoCP stabilizes MoCo in an active form charged with molybdate to provide MoCo to apomolybdoenzymes.
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Direct transfer of Molybdopterin Cofactor to aponitrate reductase from a carrier protein in Chlamydomonas reinhardtii.
FEBS letters, 1992Co-Authors: Miguel Aguilar, Jacobo Cárdenas, Kyrill L. Kalakoutskii, Emilio FernándezAbstract:A Chlamydomonas reinhardtii molybdenum Cofactor (MoCo)-carrier protein (CP), capable of reconstituting nitrate reductase activity with apoprotein from the Neurospora crassa mutant nit-1, was subjected to experiments of diffusion through a dialysis membrane and gel filtration. CP bonded firmly MoCo and did not release it efficiently unless aponitrate reductase was present in the incubation mixture, Stability of MoCo bound to CP against air and heat was very similar to that of free-MoCo released from milk xanthine oxidase. Our data strongly suggest that MoCo is directly transferred from CP to aponitrate reductase to form an active enzyme.
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nit 7: A New Locus for Molybdopterin Cofactor Biosynthesis in the Green Alga Chlamydomonas reinhardtii
Plant physiology, 1992Co-Authors: Miguel Aguilar, Rafael Prieto, Jacobo Cárdenas, Emilio FernándezAbstract:Two new nitrate reductase-deficient mutants from Chlamydomonas reinhardtii have been genetically and biochemically characterized. Both H1 and F23 mutants carry single recessive allelic mutations that map at a new locus designated nit-7. This locus is unlinked to the other six nit loci related to the nitrate assimilation pathway in C. reinhardtii. Both mutant alleles H1 and F23 lack an active Molybdopterin Cofactor, the activity of which is restored neither in vitro nor in vivo by high concentrations of molybdate. Nitrate reductase subunits in these mutants seem to assemble, although not in a stable form, in a high molecular weight complex and, as in other molybdenum Cofactor-defective mutants of C. reinhardtii, they cannot reconstitute nitrate reductase activity with an active molybdenum Cofactor source from extracts of ammonium-grown cells. The results suggest that nit-7 mutants are defective in Molybdopterin biosynthesis. They do produce some precursor(s) that are capable of binding to nitrate reductase subunits.
Nicole C Narrandes - One of the best experts on this subject based on the ideXlab platform.
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cleavage of the moax encoded fused Molybdopterin synthase from mycobacterium tuberculosis is necessary for activity
BMC Microbiology, 2015Co-Authors: Nicole C Narrandes, Edith E Machowski, Valerie Mizrahi, Bavesh D. KanaAbstract:Background Molybdopterin Cofactor (MoCo) biosynthesis in Mycobacterium tuberculosis is associated with a multiplicity of genes encoding several enzymes in the pathway, including the Molybdopterin (MPT) synthase, a hetero tetramer comprising two MoaD and two MoaE subunits. In addition to moaD1, moaD2, moaE1, moaE2, the M. tuberculosis genome also contains a moaX gene which encodes an MPT-synthase in which the MoaD and MoaE domains are located on a single polypeptide. In this study, we assessed the requirement for post-translational cleavage of MoaX for functionality of this novel, fused MPT synthase and attempted to establish a functional hierarchy for the various MPT-synthase encoding genes in M. tuberculosis.
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Cleavage of the moaX-encoded fused Molybdopterin synthase from Mycobacterium tuberculosis is necessary for activity
BMC Microbiology, 2015Co-Authors: Nicole C Narrandes, Edith E Machowski, Valerie Mizrahi, Bavesh D. KanaAbstract:Background Molybdopterin Cofactor (MoCo) biosynthesis in Mycobacterium tuberculosis is associated with a multiplicity of genes encoding several enzymes in the pathway, including the Molybdopterin (MPT) synthase, a hetero tetramer comprising two MoaD and two MoaE subunits. In addition to moaD1 , moaD2 , moaE1 , moaE2 , the M. tuberculosis genome also contains a moaX gene which encodes an MPT-synthase in which the MoaD and MoaE domains are located on a single polypeptide. In this study, we assessed the requirement for post-translational cleavage of MoaX for functionality of this novel, fused MPT synthase and attempted to establish a functional hierarchy for the various MPT-synthase encoding genes in M. tuberculosis. Results Using a heterologous Mycobacterium smegmatis host and the activity of the MoCo-dependent nitrate reductase, we confirmed that moaD2 and moaE2 from M. tuberculosis together encode a functional MPT synthase. In contrast, moaD1 displayed no functionality in this system, even in the presence of the MoeBR sulphurtransferase, which contains the rhodansese-like domain, predicted to activate MoaD subunits. We demonstrated that cleavage of MoaX into its constituent MoaD and MoaE subunits was required for MPT synthase activity and confirmed that cleavage occurs between the Gly82 and Ser83 residues in MoaX. Further analysis of the Gly81-Gly82 motif confirmed that both of these residues are necessary for catalysis and that the Gly81 was required for recognition/cleavage of MoaX by an as yet unidentified protease. In addition, the MoaE component of MoaX was able to function in conjunction with M. smegmatis MoaD2 suggesting that cleavage of MoaX renders functionally interchangeable subunits. Expression of MoaX in E. coli revealed that incorrect post-translational processing is responsible for the lack of activity of MoaX in this heterologous host. Conclusions There is a degree of functional interchangeability between the MPT synthase subunits of M. tuberculosis . In the case of MoaX, post-translational cleavage at the Gly82 residue is required for function.
Bruno Guigliarelli - One of the best experts on this subject based on the ideXlab platform.
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DFT Investigation of the Molybdenum Cofactor in Periplasmic Nitrate Reductases: Structure of the Mo(V) EPR-Active Species
2016Co-Authors: Frédéric Biaso, Bénédicte Burlat, Bruno GuigliarelliAbstract:The periplasmic nitrate reductase NAP belongs to the DMSO reductase family that regroups molybdoenzymes housing a bis-Molybdopterin Cofactor as the active site. Several forms of the Mo(V) state, an intermediate redox state in the catalytic cycle of the enzyme, have been evidenced by EPR spectroscopy under various conditions, but their structure and catalytic relevance are not fully understood. On the basis of structural data available from the literature, we built several models that reproduce the first coordination sphere of the molybdenum Cofactor and used DFT methods to make magneto-structural correlations on EPR-detected species. “High-g” states, which are the most abundant Mo(V) species, are characterized by a low-anisotropy g tensor and a high gmin value. We assign this signature to a six-sulfur coordination sphere in a pseudotrigonal prismatic geometry with a partial disulfide bond. The “very high-g” species is well described with a sulfido ion as the sixth ligand. The “low-g” signal can be successfully associated to a Mo(V) sulfite–oxidase-type active site with only one pterin moiety coordinated to the molybdenum ion with an oxo or sulfido axial ligand. For all these species we investigate their catalytic activity using a thermodynamic point of view on the molybdenum coordination sphere. Beyond the periplasmic nitrate reductase case, this work provides useful magneto-structural correlations to characterize EPR-detected species in mononuclear molybdoenzymes
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DFT investigation of the molybdenum Cofactor in periplasmic nitrate reductases: structure of the Mo(V) EPR-active species.
Inorganic chemistry, 2012Co-Authors: Frédéric Biaso, Bénédicte Burlat, Bruno GuigliarelliAbstract:The periplasmic nitrate reductase NAP belongs to the DMSO reductase family that regroups molybdoenzymes housing a bis-Molybdopterin Cofactor as the active site. Several forms of the Mo(V) state, an...
Valerie Mizrahi - One of the best experts on this subject based on the ideXlab platform.
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bis-Molybdopterin Guanine Dinucleotide Is Required for Persistence of Mycobacterium tuberculosis in Guinea Pigs
2016Co-Authors: Valerie Mizrahi, Bavesh B D. KanaeAbstract:Mycobacterium tuberculosis is able to synthesize Molybdopterin Cofactor (MoCo), which is utilized by numerous enzymes that catalyze redox reactions in carbon, nitrogen, and sulfur metabolism. In bacteria, MoCo is further modified through the activity of a guanylyltransferase, MobA, which converts MoCo to bis-Molybdopterin guanine dinucleotide (bis-MGD), a form of the co-factor that is required by the dimethylsulfoxide (DMSO) reductase family of enzymes, which includes the nitrate reductase NarGHI. In this study, the functionality of themobA homolog inM. tuberculosiswas confirmed by demonstrating the loss of assimilatory and respiratory nitrate reductase activity in amobA deletion mutant. This mutant displayed no survival defects in humanmonocytes or mouse lungs but failed to persist in the lungs of guinea pigs. These results implicate one or more bis-MGD-dependent enzymes in the persistence ofM. tuberculosis in guinea pig lungs and underscore the applicability of this animal model for assessing the role of molybdoenzymes in this pathogen. Comparative genomics suggest an association between molyb-denum Cofactor (MoCo) and pathogenesis inMycobacterium tuberculosis, the causative agent of tuberculosis (TB) (1). Consis
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cleavage of the moax encoded fused Molybdopterin synthase from mycobacterium tuberculosis is necessary for activity
BMC Microbiology, 2015Co-Authors: Nicole C Narrandes, Edith E Machowski, Valerie Mizrahi, Bavesh D. KanaAbstract:Background Molybdopterin Cofactor (MoCo) biosynthesis in Mycobacterium tuberculosis is associated with a multiplicity of genes encoding several enzymes in the pathway, including the Molybdopterin (MPT) synthase, a hetero tetramer comprising two MoaD and two MoaE subunits. In addition to moaD1, moaD2, moaE1, moaE2, the M. tuberculosis genome also contains a moaX gene which encodes an MPT-synthase in which the MoaD and MoaE domains are located on a single polypeptide. In this study, we assessed the requirement for post-translational cleavage of MoaX for functionality of this novel, fused MPT synthase and attempted to establish a functional hierarchy for the various MPT-synthase encoding genes in M. tuberculosis.
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Cleavage of the moaX-encoded fused Molybdopterin synthase from Mycobacterium tuberculosis is necessary for activity
BMC Microbiology, 2015Co-Authors: Nicole C Narrandes, Edith E Machowski, Valerie Mizrahi, Bavesh D. KanaAbstract:Background Molybdopterin Cofactor (MoCo) biosynthesis in Mycobacterium tuberculosis is associated with a multiplicity of genes encoding several enzymes in the pathway, including the Molybdopterin (MPT) synthase, a hetero tetramer comprising two MoaD and two MoaE subunits. In addition to moaD1 , moaD2 , moaE1 , moaE2 , the M. tuberculosis genome also contains a moaX gene which encodes an MPT-synthase in which the MoaD and MoaE domains are located on a single polypeptide. In this study, we assessed the requirement for post-translational cleavage of MoaX for functionality of this novel, fused MPT synthase and attempted to establish a functional hierarchy for the various MPT-synthase encoding genes in M. tuberculosis. Results Using a heterologous Mycobacterium smegmatis host and the activity of the MoCo-dependent nitrate reductase, we confirmed that moaD2 and moaE2 from M. tuberculosis together encode a functional MPT synthase. In contrast, moaD1 displayed no functionality in this system, even in the presence of the MoeBR sulphurtransferase, which contains the rhodansese-like domain, predicted to activate MoaD subunits. We demonstrated that cleavage of MoaX into its constituent MoaD and MoaE subunits was required for MPT synthase activity and confirmed that cleavage occurs between the Gly82 and Ser83 residues in MoaX. Further analysis of the Gly81-Gly82 motif confirmed that both of these residues are necessary for catalysis and that the Gly81 was required for recognition/cleavage of MoaX by an as yet unidentified protease. In addition, the MoaE component of MoaX was able to function in conjunction with M. smegmatis MoaD2 suggesting that cleavage of MoaX renders functionally interchangeable subunits. Expression of MoaX in E. coli revealed that incorrect post-translational processing is responsible for the lack of activity of MoaX in this heterologous host. Conclusions There is a degree of functional interchangeability between the MPT synthase subunits of M. tuberculosis . In the case of MoaX, post-translational cleavage at the Gly82 residue is required for function.