Inositol dehydrogenase (IDH, EC 1.1.1.18) from <i>Bacillus subtilis</i> catalyzes the reversible NAD<sup>+</sup>-dependent oxidation of the axial hydroxyl group of <i>myo</i>-inositol to form 2-keto-<i>myo</i>-inositol, NADH and H<sup>+</sup>. IDH is the first enzyme in catabolism of myo-inositol, and <i>Bacillus subtilis</i> is able to grow on <i>myo</i>-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 <i>myo</i>-inositol.<p>
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 <i>apo</i>-IDH, <i>holo</i>-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<sup>+</sup>-dependent to 6-fold preference for NADP<sup>+</sup> over NAD<sup>+</sup> 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 <i>myo</i>-inositol active site were also modified to test their roles in order to improve our understanding of substrate binding and activation.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-06022010-111229 |
| Date | 18 June 2010 |
| Creators | Zheng, Hongyan |
| Contributors | Dr. David R. J. Palmer, Dr. Stephen G. Urquhart, Dr. David A. R. Sanders, Dr. Dale E. Ward, Dr. Jian Yang, Dr. Jeffrey W. Keillor |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-06022010-111229/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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