Mechanistic Insights into the Diverged Enzymes of the Amidohydrolase Superfamily
| dc.contributor.advisor | Raushel, Frank M. | |
| dc.contributor.committeeMember | Lindahl, Paul A. | |
| dc.contributor.committeeMember | Reinhart, Gregory D. | |
| dc.contributor.committeeMember | Watanabe, Coran H. | |
| dc.creator | Nguyen, Tinh T. | |
| dc.date.accessioned | 2011-02-22T22:23:29Z | |
| dc.date.accessioned | 2011-02-22T23:44:02Z | |
| dc.date.available | 2011-02-22T22:23:29Z | |
| dc.date.available | 2011-02-22T23:44:02Z | |
| dc.date.created | 2009-12 | |
| dc.date.issued | 2011-02-22 | |
| dc.date.submitted | December 2009 | |
| dc.description.abstract | The amidohydrolase superfamily is a functionally diverse set of enzymes that catalyzes predominantly hydrolysis reactions involving sugars, nucleic acids, amino acids, and organophosphate esters. A more divergent member of this superfamily, URI (uronate isomerase) from Escherichia coli, catalyzes the isomerization of D-glucuronate to D-fructuronate and D-galacturonate to D-tagaturonate. In Bacillus halodurans, two distinct operons were identified for the metabolism of D-glucuronate and D-galacturonate based on kinetics and genomic context. The canonical uronate isomerase is encoded by the gene Bh0705. A second URI in this organism, Bh0493, is the outlier of the group in terms of sequence similarity. Kinetic evidences indicate that Bh0705 is relatively specific for the isomerization of D-glucuronate, while Bh0493 is specific for the Dgalacturonate pathway. Bell-shaped pH-rate profiles were observed for the wild type URI from Escherichia coli. Primary isotope effects with [2-2H]-D-glucuronate and solvent viscosity studies are consistent with product release as the rate limiting step. X-ray structure of Bh0493 was determined in the presence of D-glucuronate. A chemical mechanism is proposed that utilizes a proton transfer from C-2 of D-glucuronate to C-1 that is initiated by the combined actions of Asp-355 and the C-5 hydroxyl of the substrate that is bound to the metal ion. The formation of the cis-enediol intermediate is further facilitated by the shuttling of the proton between the C-2 and C-1 oxygens by the conserved Tyr-50 and/or Arg-357. Another divergent member of the AHS is the enzyme renal dipeptidase. Structural studies of the enzyme from Streptomyces coelicolor (Sco3058) demonstrate that the active site consists of a binuclear metal center. Bell-shaped pH-rate profiles are observed for both Zn2+ and Cd2+ enzymes. A chemical mechanism for renal dipeptidase is proposed based on structural analysis of the enzyme-inhibitor complex. The reaction is initiated by the polarization of the amide bond by the B-metal. Asp-320 activates the bridging hydroxide for nucleophilic attack at the peptide carbon center, forming a tetrahedral intermediate that is stabilized by the metal center and His-150. The protonated Asp-320 donates the proton to the a-amino group of the leaving group, causing the collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogen bond. | en |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | https://hdl.handle.net/1969.1/ETD-TAMU-2009-12-7237 | |
| dc.language.iso | en_US | |
| dc.subject | Amidohydrolase superfamily | en |
| dc.subject | uronate isomerase | en |
| dc.subject | renal dipeptidase | en |
| dc.title | Mechanistic Insights into the Diverged Enzymes of the Amidohydrolase Superfamily | en |
| dc.type | Book | en |
| dc.type | Thesis | en |
| dc.type.genre | Electronic Dissertation | en |
| dc.type.material | text | en |
| thesis.degree.department | Chemistry | en |
| thesis.degree.discipline | Chemistry | en |
| thesis.degree.grantor | Texas A&M University | en |
| thesis.degree.level | Doctoral | en |
| thesis.degree.name | Doctor of Philosophy | en |