1. Molecular identification of unsaturated uronate reductase prerequisite for alginate metabolism in Sphingomonas sp. A1
- Author
-
Takase, Ryuichi, Ochiai, Akihito, Mikami, Bunzo, Hashimoto, Wataru, and Murata, Kousaku
- Subjects
- *
SPHINGOMONAS , *NUCLEAR magnetic resonance , *THIN layer chromatography , *ALGINATES , *X-ray crystallography , *URONIC acids , *ELECTROSPRAY ionization mass spectrometry , *MATRIX-assisted laser desorption-ionization - Abstract
Abstract: 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 1.6Å 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. [Copyright &y& Elsevier]
- Published
- 2010
- Full Text
- View/download PDF