This work describes the crystallographic studies of two enzymes and provides mechanistic insights into their respective catalytic processes. The first study investigates the molecular basis of the fructose-2,6-bisphosphatase reaction of the inducible isoform of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). The bifunctional enzyme is solely responsible for the cellular concentration of a regulator of glucose metabolism, fructose-2,6-bisphosphate. PFKB3 was investigated using the crystal structures of the enzyme in a phospho-enzyme intermediate state (PFKFB3-PF-6-P), in a transition state-analogous complex (PFKFB3AlF4), and in a complex with pyrophosphate (PFKFB3PPi). With these structures, the structures of the Michaelis complex and the transition state were extrapolated. Additionally the C-terminal domain (residues 440-446) was rearranged in PFKFB3PPi, implying that this domain plays a critical role in binding of substrate to, and release of product from, the bisphosphatase catalytic pocket. These findings provide a new insight into the understanding of the phosphoryl transfer reaction.
The second study investigates the molecular basis of the reaction catalyzed by the pseudo-glycosyltransferase, VldE. VldE catalyzes non-glycosidic C-N coupling between an unsaturated cyclitol and a saturated aminocyclitol with the conservation of the anomeric configuration to form validoxylamine A 7´-phosphate, the biosynthetic precursor of the antibiotic validamycin A. To study the molecular basis of its mechanism, the three-dimensional structures
of VldE from Streptomyces hygroscopicus subsp. limoneus was determined in apo form, in complex with GDP, in complex with GDP and validoxylamine A 7´-phosphate, and in complex with GDP and trehalose. The structures of VldE with the catalytic site in both an open and closed conformation are also described. With these structures, the preferred binding of the guanine moiety by VldE, rather than the uracil moiety as seen in OtsA, could be explained. The elucidation of the VldE structure in complex with the entirety of its products provides insight into the internal return mechanism by which catalysis occurs with a net retention of the anomeric configuration of the donated cyclitol.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-07272012-154005 |
| Date | 30 July 2012 |
| Creators | Cavalier, Michael Christopher |
| Contributors | Lee, Yong-Hwan, Waldrop, Grover, Newcomer, Marcia, Luo, Bing-Hao, Burba, Daniel |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-07272012-154005/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached herein 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 LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, 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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