Journal
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
Volume 1804, Issue 9, Pages 1925-1936Publisher
ELSEVIER
DOI: 10.1016/j.bbapap.2010.05.010
Keywords
Alginate; Crystal structure; 2-Keto-3-deoxy-D-gluconic acid; Short-chain dehydrogenase/reductase; Sphingomonas
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Funding
- Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN) in Japan
- Japan Society for the Promotion of Science
- Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan
- Japan Society for the Promotion of Science for Young Scientists
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In Sphingomonas sp. A1, alginate is degraded by alginate lyases to its constituent monosaccharides, which are nonenzymatically converted to an alpha-keto acid, namely, 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). The properties of the DEH-metabolizing enzyme and its gene in strain A1 were characterized. In the presence of alginate, strain A1 cells inducibly produced an NADPH-dependent DEH reductase (A1-R) in their cytoplasm. Molecular cloning of the enzyme gene indicated that A1-R belonged to the short-chain dehydrogenase/reductase superfamily and catalyzed the conversion of DEH to 2-keto-3-deoxy-D-gluconic acid most efficiently at around pH 7.0 and 50 degrees C. Crystal structures of A1-R and its complex with NADP were determined at around 1.6 angstrom resolution by X-ray crystallography. The enzyme consists of three layers (alpha/beta/alpha) , with a coenzyme-binding Rossmann fold. NADP is surrounded by positively charged residues, and Gly-38 and Arg-39 are crucial for NADP binding. Site-directed mutagenesis studies suggest that Ser-150, Tyr-164, and Lys-168 located around the Rossmann fold constitute the catalytic triad. To our knowledge, this is the first report on molecular cloning and structure determination of a bacterial DEH reductase responsible for alginate metabolism. (C) 2010 Elsevier B.V. All rights reserved.
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