α-KDOase Activity in Oyster and Synthesis of α- and β-4-Methylumbelliferyl Ketosides of 3-Deoxy-d-manno-octulosonic Acid (KDO)

Abstract

Although alpha- and beta-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 (alpha-KDOase) capable of releasing alpha-linked KDO from LPSs. To facilitate the studies of alpha-KDOase, we have carried out the synthesis of 4-methylumbelliferyl-alpha-KDO (alpha-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 beta-anomer was obtained as the major product with a yield of about 80%, whereas the yield of alpha-anomer was only about 7%. Attempts to increase the yield of alpha-anomer were not successful. alpha-KDO-MU was used as substrate to follow the purification of alpha-KDOase from oyster hepatopancreas. The pH optimum for oyster alpha-KDOase was determined to be 4.5 using Re-LPS as substrate and 3.0 using alpha-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).