title: Metabolic fate of unsaturated glucuronic/iduronic acids from glycosaminoglycans: molecular identification and structure determination of streptococcal isomerase and dehydrogenase.
abstract: Glycosaminoglycans in mammalian extracellular matrices are degraded to their constituents, unsaturated uronic (glucuronic/iduronic) acids and amino sugars, through successive reactions of bacterial polysaccharide lyase and unsaturated glucuronyl hydrolase. Genes coding for glycosaminoglycan-acting lyase, unsaturated glucuronyl hydrolase, and the phosphotransferase system are assembled into a cluster in the genome of pathogenic bacteria, such as streptococci and clostridia. Here, we studied the streptococcal metabolic pathway of unsaturated uronic acids and the structure/function relationship of its relevant isomerase and dehydrogenase. Two proteins (gbs1892 and gbs1891) of Streptococcus agalactiae strain NEM316 were overexpressed in Escherichia coli, purified, and characterized. 4-Deoxy-l-threo-5-hexosulose-uronate (Dhu) nonenzymatically generated from unsaturated uronic acids was converted to 2-keto-3-deoxy-d-gluconate via 3-deoxy-d-glycero-2,5-hexodiulosonate through successive reactions of gbs1892 isomerase (DhuI) and gbs1891 NADH-dependent reductase/dehydrogenase (DhuD). DhuI and DhuD enzymatically corresponded to 4-deoxy-l-threo-5-hexosulose-uronate ketol-isomerase (KduI) and 2-keto-3-deoxy-d-gluconate dehydrogenase (KduD), respectively, involved in pectin metabolism, although no or low sequence identity was observed between DhuI and KduI or between DhuD and KduD, respectively. Genes for DhuI and DhuD were found to be included in the streptococcal genetic cluster, whereas KduI and KduD are encoded in clostridia. Tertiary and quaternary structures of DhuI and DhuD were determined by x-ray crystallography. Distinct from KduI β-barrels, DhuI adopts an α/β/α-barrel structure as a basic scaffold similar to that of ribose 5-phosphate isomerase. The structure of DhuD is unable to accommodate the substrate/cofactor, suggesting that conformational changes are essential to trigger enzyme catalysis. This is the first report on the bacterial metabolism of glycosaminoglycan-derived unsaturated uronic acids by isomerase and dehydrogenase.
authors.affiliation: From the Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, Graduate School of Agriculture, and.
authors.forename: Yukie
authors.initials: Y
authors.lastname: Maruyama
authors.affiliation: From the Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, Graduate School of Agriculture, and.
authors.forename: Sayoko
authors.initials: S
authors.lastname: Oiki
authors.affiliation: From the Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, Graduate School of Agriculture, and.
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Takase
authors.affiliation: the Laboratory of Applied Structural Biology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Bunzo
authors.initials: B
authors.lastname: Mikami
authors.affiliation: From the Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, Graduate School of Agriculture, and.
authors.forename: Kousaku
authors.initials: K
authors.lastname: Murata
authors.affiliation: From the Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, Graduate School of Agriculture, and whasimot@kais.kyoto-u.ac.jp.
authors.forename: Wataru
authors.initials: W
authors.lastname: Hashimoto
mesh_terms: D018360:Crystallography, X-Ray; D005109:Extracellular Matrix / Q000737:chemistry / Q000378:metabolism; D005965:Glucuronates / Q000737:chemistry / Q000378:metabolism; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism; D007067:Iduronic Acid / Q000737:chemistry / Q000378:metabolism; D007535:Isomerases / Q000737:chemistry* / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D013290:Streptococcal Infections / Q000201:enzymology* / Q000473:pathology; D013292:Streptococcus agalactiae / Q000737:chemistry / Q000201:enzymology* / Q000472:pathogenicity; D013379:Substrate Specificity; D014574:Uronic Acids / Q000737:chemistry / Q000378:metabolism
chemical_list: D005965:Glucuronates; D006025:Glycosaminoglycans; D014574:Uronic Acids; D007067:Iduronic Acid; D010088:Oxidoreductases; D007535:Isomerases
keywords: Bacterial Metabolism; Dehydrogenase; Glycosaminoglycan; Streptococcus; X-ray Crystallography