Mutagenic analysis of the decarboxylases and hydratases in parallel meta-fission pathways
| dc.contributor.advisor | Whitman, Christian P. | en |
| dc.creator | Miller, Scott Garrett | en |
| dc.date.accessioned | 2012-09-20T17:27:30Z | en |
| dc.date.available | 2012-09-20T17:27:30Z | en |
| dc.date.issued | 2008-08 | en |
| dc.description | text | en |
| dc.description.abstract | The catechol meta-fission pathway, a degradation pathway for simple aromatic compounds, is rich in enzyme chemistry and replete with structural and evolutionary diversity. Vinyl pyruvate hydratase (VPH) and MhpD catalyze the same reaction in this pathway, but in different bacterial species. These metal ion-dependent enzymes reportedly catalyze a 1,5-keto-enol tautomerization reaction followed by a Michael addition of water. MhpD, and most likely VPH, are members of the fumarylacetoacetate hydrolase (FAH) superfamily. The crystal structure of MhpD and the sequence of VPH identified four potential active site residues, Lys-60, Leu-72, Asp-78, and Ser-160 (Ser-161 in VPH). The K60A and D78N mutants of VPH and MhpD had the most damaging effects on catalysis. Moreover, the K60A mutant seemingly uncoupled tautomerization from hydration and provided evidence for an [alpha, beta]-unsaturated ketone in the reaction. The effects of the L72A and S160A (S161A in VPH) mutants were smaller, suggesting less important roles in the mechanism. 5-(carboxymethyl)-2-Oxo-3-hexene-1,6-dioate decarboxylase (COHED) is a metal ion-dependent enzyme in the homoprotocatechuate (HPC) pathway, a chromosomally encoded meta-fission pathway from Escherichia coli C that parallels the catechol meta-fission pathway. COHED is also a member of the FAH superfamily. It is a monomeric protein with two domains. It is postulated that the C-terminal domain catalyzes the decarboxylation reaction and the N-terminal domain carries out the 1,3-keto-enol tautomerization reaction. Site-directed mutagenesis, NMR, and kinetic analysis with different substrates and inhibitors have identified three potential active-site residues Glu-276, Glu-278 (in the C-terminal domain), and Lys-110 (in the N-terminal domain). Replacement of either glutamate with a glutamine eliminated both the decarboxylase and tautomerase activities. The K110A mutant also diminished both activities, but more importantly eliminated the C-3 proton/deuteron exchange reaction observed for substrate analogs. The enzymes of the catechol and homoprotocatechuate pathways provide examples of enzyme optimization toward a specific substrate even among related compounds, as reflected by the FAH superfamily. Hence, the results of these studies add to the growing body of information about how enzymes evolve and how pathways are assembled. | en |
| dc.description.department | Pharmaceutical Sciences | en |
| dc.format.medium | electronic | en |
| dc.identifier.uri | http://hdl.handle.net/2152/17940 | en |
| dc.language.iso | eng | en |
| dc.rights | Copyright is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works. | en |
| dc.subject.lcsh | Hydrolases | en |
| dc.subject.lcsh | Mutation (Biology) | en |
| dc.subject.lcsh | Decarboxylases | en |
| dc.subject.lcsh | Decarboxylation | en |
| dc.subject.lcsh | Catalysis | en |
| dc.subject.lcsh | Escherichia coli | en |
| dc.title | Mutagenic analysis of the decarboxylases and hydratases in parallel meta-fission pathways | en |
| thesis.degree.department | Pharmacy | en |
| thesis.degree.discipline | Pharmacy | en |
| thesis.degree.grantor | The University of Texas at Austin | en |
| thesis.degree.level | Doctoral | en |
| thesis.degree.name | Doctor of Philosophy | en |