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.
Highlights
Some streptococci target host extracellular matrix glycosaminoglycans for infection
Two proteins 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 3deoxy-D-glycero-2,5-hexodiulosonate through successive reactions of gbs1892 isomerase (DhuI) and gbs1891 NADH-dependent reductase/dehydrogenase (DhuD)
We have identified unsaturated glucuronyl hydrolase (UGL), which is essential for degrading unsaturated disaccharides to constituent monosaccharides in bacteria, including streptococci [8, 9], and found that a genome segment termed the UGL genetic cluster is responsible for the depolymerization, import, and degradation of glycosaminoglycans in streptococci, such as Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes [10]
Summary
Some streptococci target host extracellular matrix glycosaminoglycans for infection. Results: The streptococcal metabolic pathway of glycosaminoglycan-derived unsaturated uronates was identified. 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. We studied the streptococcal metabolic pathway of unsaturated uronic acids and the structure/function relationship of its relevant isomerase and dehydrogenase. 4-Deoxy-L-threo-5-hexosulose-uronate (Dhu) nonenzymatically generated from unsaturated uronic acids was converted to 2-keto-3-deoxy-D-gluconate via 3deoxy-D-glycero-2,5-hexodiulosonate through successive reactions of gbs1892 isomerase (DhuI) and gbs1891 NADH-dependent reductase/dehydrogenase (DhuD). 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. Based on the sugar composition, mode of glycosidic bond, and sulfation level, glycosaminoglycans are classified as hyaluronan, chon-
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