| Type of Submission |
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Submission Type: |
Poster Presentation |
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Subject Category: |
Applied Microbiology |
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| Session Information |
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Presentation Date: |
May 26, 2003 |
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Abstract ID: |
A11 |
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Session: |
Poster 1 |
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Time: |
14:00 |
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| Presenting Author |
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| Other Authors |
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H. LEE, University of Guelph
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| Title |
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Aspartate-44 and Histidine-111 are Essential to Catalysis in the Saccharomyces cerevisiae Xylose Reductase |
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| Abstract Text |
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Xylose reductase (XR) catalyzes the NAD(P)H-dependent reduction of xylose to xylitol, the initial step in the xylose metabolic pathway. Saccharomyces cerevisiae, the preferred ethanol-producing microorganism, is unable to utilize xylose as a sole substrate despite possessing a functional NADPH-dependent XR (SCXR). SCXR was expressed and purified to homogeneity using affinity chromatography. Primary sequence comparisons of yeast XRs and human aldose reductase (HAR) revealed conservation of amino acids involved in catalysis. Conserved Asp-44 and His-111 residues were identified, and the SCXR mutants D44A and H111A were created and their kinetic properties characterized. The H111A mutant had negligible activity and kinetic constants could not be determined. The D44A mutant exhibited lower specific activity, but higher Km for xylose and NADPH than the wild-type. Treatment of SCXR with the histidine-specific modifier diethylpyrocarbonate (DEPC) suggested that a histidine residue was modified resulting in inactivation. Intrinsic enzyme fluorescence spectroscopic data strongly indicate that D44A and H111A mutants continue to bind NADPH as well as the wild-type. This suggests that D44 and H111 are involved in interaction with the substrate and not the cosubstrate during catalysis. |
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