In this research project, structural aspects of monomeric NADP+-dependent isocitrate dehydrogenase from Corynebacterium glutamicum (CgIDH) are investigated together with site-directed mutagenesis and fluorescence spectroscopy studies. CgIDH, one of the enzymes of the Krebs cycle, catalyzes the decarboxylation of isocitrate into α-ketoglutarate, which in some bacteria and plants regulates the flow of carbon into either the Krebs cycle or the glyoxylate bypass depending on the available carbon source. The structure of CgIDH complexed with Mg2+ has been determined at 1.75 Å resolution using X-ray crystallography. In contrast to the closed conformation of published structures of monomeric NADP+-dependent IDH from <i> Azotobactor vinelandii </i> complexed with either isocitrate-Mn2+ or NADP+, the structure of CgIDH complexed with Mg2+ demonstrates the open conformation. The superimposed structure of CgIDH complexed with Mg2+ onto the structures of AvIDH complexes reveals that Domain II is rotated ~24° or ~35º, respectively, relative to Domain I when isocitrate-Mn2+ or NADP+ is bound, resulting in the closure of the active site between the two domains. Fluorescence spectroscopic studies support the proposal that the presence of isocitrate or NADP+ could mediate the conformational changes in CgIDH. <p>In addition, three CgIDH mutants (S130D, K253Q, and Y416T) were created based on the structural analysis and previous mutagenesis studies of homodimeric NADP+-dependent IDH. Both the specific activities and the fluorescence spectra of these CgIDH mutants elucidate the roles of these active site residues in CgIDH catalysis. It has been suggested that the conformational changes observed in the presence of the substrate(s) may regulate enzymatic activity in CgIDH, in contrast to homodimeric NADP+-dependent IDH in Escherichia coli, where the phosphorylation cycle controls activity. It is also presumed that both Lys253 and Tyr416 may play critical roles in CgIDH activity, as do the equivalent residues in homodimeric IDH from porcine heart mitochondria. Similar structural features and conformational changes among monomeric CgIDH and homodimeric NADP+-dependent IDH enzymes suggest the phylogenetic relationships among various monomeric and homodimeric NADP+-dependent IDH from different sources.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-09132004-124844 |
| Date | 17 September 2004 |
| Creators | Imabayashi, Fumie |
| Contributors | Khandelwal, Ramji L., Delbaere, Louis T. J., Napper, Scott |
| 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-09132004-124844/ |
| 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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