Poster: Enzymology
Abs #
245: Expression of Arabidopsis b-Glucosidases in Pichia pastoris and Characterization of Their Substrate Specificities.
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Presenter: |
Poulton, Jonathan E, jepoultn@blue.weeg.uiowa.edu |
Authors | Poulton, Jonathan E (A) Xu, Zhiwei (A) Escamilla-Trevino, Luis L (A) Zeng, Lihui (A) Shih, Ming-Che (A) Cheng, Chi-Lien (A) Mohamed, Ali (B) Kishimoto, Tadashi (C) Turan, Yusuf (C) Zheng, Meiying (C) Winkel-Shirley, Brenda (C) Bevan, David R (C) Esen, Asim (C) | | Affiliations: |
(A): The University of Iowa
(B): Virginia State University
(C): Virginia Polytechnic Institute & State University
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| Web Site: | http://www.biology.uiowa.edu/arabidopsis/ | |
b-O-Glucosidases (EC 3.2.1.21) are ubiquitous enzymes that catalyze the hydrolysis of aryl and alkyl b-D-glucosides as well as b-linked oligosaccharides. In plants, these enzymes have been implicated in such processes as defense against pests, phytohormone activation, lignification, and cellulose degradation. The Arabidopsis genome has 46 putative b-glucosidase genes (40 b-O-glucosidases and 6 b-S-glucosidases) which can be divided into 8-10 subfamilies by phylogenetic analysis. We are using a functional genomic approach to discover the biological roles played by each member of the Arabidopsis b-glucosidase gene family. Using the Pichia pastoris expression system, we have expressed hydrolases encoded by several members of this family. Four of these enzymes, encoded by At2g44450, At3g18080, At5g44640, and At5g36890, have been purified to homogeneity. To gain insights into the biological roles played by these enzymes, two approaches are being taken. We are determining the aglycone and sugar specificities of each recombinant enzyme in vitro. As expected, all four enzymes efficiently hydrolyze para-nitrophenyl-b-D-glucoside (PNPG). For At2g44450 and At5g44640, cellobiose and laminarin are the most rapidly hydrolyzed of the natural substrates tested. Interestingly, the hydrolase encoded by At3g18080 cleaves para-nitrophenyl-b-D-mannoside twice as fast as PNPG. Metabolic profiling using HPLC is also being employed to identify the natural substrates of each enzyme. The purified At3g18080-encoded hydrolase degrades two compounds in leaf extracts and three in silique extracts; their chemical nature is under investigation.
