Volume 272, Number 42,
Issue of October 17, 1997
pp. 26419-26424
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
-KDOase Activity in Oyster and Synthesis of - and
-4-Methylumbelliferyl Ketosides of
3-Deoxy-D-manno-octulosonic Acid (KDO)
(Received for publication, May 16, 1997)
Yu-Teh
Li
,
Lai-Xi
Wang
¶
,
Nadejda V.
Pavlova
,
Su-Chen
Li
and
Yuan C.
Lee
¶
From the 
Department of Biochemistry, Tulane
University School of Medicine, New Orleans, Louisiana 70112 and the
¶ Department of Biology, The Johns Hopkins University, Baltimore,
Maryland 21218
Although 
- and 
-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 (-KDOase) capable of releasing -linked KDO from LPSs. To facilitate the studies of -KDOase, we have carried out the synthesis of 4-methylumbelliferyl--KDO (-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 -anomer was
obtained as the major product with a yield of about 80%, whereas the
yield of -anomer was only about 7%. Attempts to increase the yield
of -anomer were not successful. -KDO-MU was used as substrate to follow the purification of -KDOase from oyster hepatopancreas. The
pH optimum for oyster -KDOase was determined to be 4.5 using Re-LPS
as substrate and 3.0 using -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).
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