alpha-KDOase activity in oyster and synthesis of alpha- and beta-4-methylumbelliferyl ketosides of 3-deoxy-D-manno-octulosonic acid.
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).
Li
Y T
YT
Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA. yli@tmcpop.tmc.tulane.edu
Wang
L X
LX
Pavlova
N V
NV
Li
S C
SC
Lee
Y C
YC
D000818:Animals; D006027:Glycosides / Q000138:chemical synthesis*; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D010125:Ostreidae / Q000201:enzymology*; D010179:Pancreas / Q000201:enzymology; D011522:Protons; D013400:Sugar Acids / Q000737:chemistry*; D014166:Transferases / Q000302:isolation & purification / Q000378:metabolism*
D006027:Glycosides; D011522:Protons; D013400:Sugar Acids; C002532:2-keto-3-deoxyoctonate; D014166:Transferases; C019917:3-deoxy-D-manno-octulosonate transferase