Cloning and expression of 3-deoxy-d-manno-oct-2-ulosonic acid ?-ketoside hydrolase from oyster hepatopancreas†.

ABSTRACT: We have previously reported that oyster hepatopancreas contained three unusual ?-ketoside hydrolases: (i) a 3-deoxy-d-manno-oct-2-ulosonic acid ?-ketoside hydrolase (?-Kdo-ase), (ii) a 3-deoxy-D-glycero-D-galacto-non-2-ulosonic acid ?-ketoside hydrolase and (iii) a bifunctional ketoside hydrolase capable of cleaving both the ?-ketosides of Kdn and Neu5Ac (Kdn-sialidase). After completing the purification of Kdn-sialidase, we proceeded to clone the gene encoding this enzyme. Unexpectedly, we found that instead of expressing Kdn-sialidase, our cloned gene expressed ?-Kdo-ase activity. The full-length gene, consisting of 1176-bp (392 amino acids, Mr 44,604), expressed an active recombinant ?-Kdo-ase (R-?-Kdo-ase) in yeast and CHO-S cells, but not in various Escherichia coli strains. The deduced amino acid sequence contains two Asp boxes (S(277)PDDGKTW and S(328)TDQGKTW) commonly found in sialidases, but is devoid of the signature FRIP-motif of sialidase. The R-?-Kdo-ase effectively hydrolyzed the Kdo in the core-oligosaccharide of the structurally defined lipopolysaccharide (LPS), Re-LPS (Kdo(2)-Lipid A) from Salmonella minnesota R595 and E. coli D31m4. However, Rd-LPS from S. minnesota R7 that contained an extra outer core phosphorylated heptose was only slowly hydrolyzed. The complex type LPS from Neisseria meningitides A1 and M992 that contained extra 5-6 sugar units at the outer core were refractory to R-?-Kdo-ase. This R-?-Kdo-ase should become useful for studying the structure and function of Kdo-containing glycans.

SUBMITTER: Nakagawa T 

PROVIDER: S-EPMC4634316 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature