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Title: Studies on the adenosylcobalamin-dependent glutamate mutase
Author: Chen, H.-P. H.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1998
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Glutamate mutase is one of a group of adenosylcobalamin(AdoCbl)-dependent carbon skeleton mutases which catalyzes the interconversion of L-glutamate and threo-β-methyl asparate. It is comprised of two weakly-associating subunits, S and E, which combine with AdoCbl to form the active holo-enzyme. The genes encoding glutamate mutase were designated mut and glm genes in Clostridium tetamorphum and Clostridim cochlearium respectively. Both of them have been cloned, sequenced and over-expressed in E. coli. The crystal structures of MeCbl-dependent methionine synthase and AdoCbl-dependent MMCM serve as a model for how glutamate mutase, which contains the same "DXHXX cobalamin-binding motif, binds AdoCbl. To test this model and to understand more about the role of Asp-His-Co "triad" in catalysis and coenzyme-binding, five mutants, MutS-D14A, MutS-D14N, MutS-D14E, MutS-H16G and MutS-H16Q, were made using the technique of site-directed mutagenesis. Mutations of either Asp14 or His16 result in the decrease of kcat by 1000-fold. The u.v.-visible spectra of the wild-type and mutant holoenzyme indicated that the mutant enzymes coordinate cobalt less well. The apparent Kd for AdoCbl is raised by about 50-fold when His16 is mutated and by 5-10-fold when Asp14 is mutated. These results suggest that both His16 and Asp14 are important in AdoCbl-binding and catalysis. The association of AdoCbl, E and S subunit to form a holoenzyme complex is a kinetically complicated process, which leads to the variation of kcat and the apparent Km and Kd for AdoCbl with protein concentration. To facilitate mechanistic and structure studies on glutamate mutase, the S subunit was genetically fused to the C-terminus of the E subunit through a 11 amino acid linker. This fusion protein, GlmEs, was over-expressed in E.coli and purified to homogeneity. It binds AdoCbl with the same affinity as wild-type enzyme when MutS is presented saturating conditions. More importantly, turnover is no longer protein concentration-dependent.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available