Molecular identification of unsaturated uronate reductase prerequisite for alginate metabolism in Sphingomonas sp. A1

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• 作者：Ryuichi Takase ; Akihito Ochiai ; Bunzo Mikami ; Wataru Hashimoto ; Kousaku Murata
• 关键词：Alginate ; Crystal structure ; 2-Keto-3-deoxy-d-gluconic acid ; Short-chain dehydrogenase/reductase ; Sphingomonas
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：2010
• 出版时间：September 2010
• 年：2010
• 卷：1804
• 期：9
• 页码：1925-1936
• 全文大小：2363 K

文摘

In Sphingomonas sp. A1, alginate is degraded by alginate lyases to its constituent monosaccharides, which are nonenzymatically converted to an α-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 °C. Crystal structures of A1-R and its complex with NADP were determined at around 1.6 Å resolution by X-ray crystallography. The enzyme consists of three layers (α/β/α), 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.