Although α- and β-linked 3-deoxy-D-manno-octulosonic acid (KDO) is found in lipopolysaccharides (LPSs) of Gram-negative bacteria, capsular polysaccharides of microorganisms, and plants, very little is known about its degradation. Using both thin-layer chromatography and the periodate-thiobarbituric acid reaction, we found that the hepatopancreas of oyster (Crassostrea virginica) contained an enzyme (α-Kdoase) capable of releasing α-linked KDO from LPSs. To facilitate the studies of α-Kdoase, we have carried out the synthesis of 4-methylumbelliferyl-α-Kdo (α-Kdo-MU) by conjugating the glycosyl chloride of the per-O-acetylated methylester of KDO with methylumbelliferone by the SN2 type reaction and the catalyzed phase-transfer. In both cases, the β-anomer was obtained as the major product with a yield of about 80%, whereas the yield of α-anomer was only about 7%. Attempts to increase the yield of α-anomer were not successful. α-Kdo-MU was used as substrate to follow the purification of α-Kdoase from oyster hepatopancreas. The pH optimum for oyster α-Kdoase was determined to be 4.5 using Re-LPS as substrate and 3.0 using α-KDO-MU as substrate. The enzyme was found to be stable in the pH range of 3-8. This enzyme released KDO from different LPSs, including Re-LPS from Escherichia coli and Salmonella minnesota, Rd-LPS from S. minnesota, and de-O-acyl-Re-LPS (Kiang, J., Szu, S. C., Wang, L.X., Tang, M., and Lee, Y. C. (1997) Anal. Biochem. 245, 97-101).
biosynthesis, synthesis, acid, Kdo, activity, 3-deoxy-D-manno-octulosonic acid, glycoside, oyster, synthase
Journal NLM ID: 2985121RPublisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Institutions: Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112, Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218
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