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Mechanistic, inhibitory, and mutagenic studies of inositol dehydrogenase from Bacillus subtilis

dc.contributor.advisorDr. David R. J. Palmeren_US
dc.contributor.committeeMemberDr. Stephen G. Urquharten_US
dc.contributor.committeeMemberDr. David A. R. Sandersen_US
dc.contributor.committeeMemberDr. Dale E. Warden_US
dc.contributor.committeeMemberDr. Jian Yangen_US
dc.contributor.committeeMemberDr. Jeffrey W. Keilloren_US
dc.creatorZheng, Hongyanen_US
dc.date.accessioned2010-06-02T11:12:29Zen_US
dc.date.accessioned2013-01-04T04:34:41Z
dc.date.available2012-06-18T08:00:00Zen_US
dc.date.available2013-01-04T04:34:41Z
dc.date.created2010-06en_US
dc.date.issued2010-06-17en_US
dc.date.submittedJune 2010en_US
dc.description.abstractInositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis catalyzes the reversible NAD+-dependent oxidation of the axial hydroxyl group of myo-inositol to form 2-keto-myo-inositol, NADH and H+. IDH is the first enzyme in catabolism of myo-inositol, and Bacillus subtilis is able to grow on myo-inositol as the sole carbon source. Our laboratory has previously shown that this enzyme has an unusual active site that can accommodate large hydrophobic substituents at 1L-4-position of myo-inositol. In this dissertation, the further characterization of this IDH is described, with focus on the mechanism, inhibition, kinetics, substrate binding, and alteration of substrate specificity. A kinetic isotope effect study revealed that the chemical step of the reaction was not rate-limiting. In order to probe the inositol-binding site, five inositol analogues were synthesized and evaluated as competitive inhibitors. Recently the crystal structures of the apo-IDH, holo-IDH and ternary complex have been solved. Using structural information, as well as modeling and sequence alignment approaches, we predicted the active site structure of the enzyme. On the basis of these predictions, coenzyme specificity was converted from entirely NAD+-dependent to 6-fold preference for NADP+ over NAD+ by site-directed mutagenesis. The critical residues for coenzyme recognition were therefore identified. Besides coenzyme specificity alteration, eleven amino acid residues in and around the proposed myo-inositol active site were also modified to test their roles in order to improve our understanding of substrate binding and activation.en_US
dc.identifier.urihttp://hdl.handle.net/10388/etd-06022010-111229en_US
dc.language.isoen_USen_US
dc.subjectkineticsen_US
dc.subjectinositol dehydrogenaseen_US
dc.subjectmechanismen_US
dc.subjectinhibitionen_US
dc.subjectmutagenesisen_US
dc.titleMechanistic, inhibitory, and mutagenic studies of inositol dehydrogenase from Bacillus subtilisen_US
dc.type.genreThesisen_US
dc.type.materialtexten_US
thesis.degree.departmentChemistryen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.grantorUniversity of Saskatchewanen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophy (Ph.D.)en_US

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