title: Cloning and expression of 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase from oyster hepatopancreas†.
abstract: We have previously reported that oyster hepatopancreas contained three unusual α-ketoside hydrolases: (i) a 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase (α-Kdo-ase), (ii) a 3-deoxy-D-glycero-D-galacto-non-2-ulosonic acid α-ketoside hydrolase and (iii) a bifunctional ketoside hydrolase capable of cleaving both the α-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 α-Kdo-ase activity. The full-length gene, consisting of 1176-bp (392 amino acids, Mr 44,604), expressed an active recombinant α-Kdo-ase (R-α-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-α-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-α-Kdo-ase. This R-α-Kdo-ase should become useful for studying the structure and function of Kdo-containing glycans.
authors.affiliation: Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA 70112, USA.
authors.forename: Tetsuto
authors.initials: T
authors.lastname: Nakagawa
authors.affiliation: Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA 70112, USA.
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Shimada
authors.affiliation: Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA 70112, USA.
authors.forename: Nadejda V
authors.initials: NV
authors.lastname: Pavlova
authors.affiliation: Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA 70112, USA.
authors.forename: Su-Chen
authors.initials: SC
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA 70112, USA yli1@tulane.edu ytlscl@gmail.com.
authors.forename: Yu-Teh
authors.initials: YT
authors.lastname: Li
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D010125:Ostreidae / Q000201:enzymology* / Q000235:genetics; D010179:Pancreas / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013400:Sugar Acids / Q000378:metabolism*
chemical_list: D013400:Sugar Acids; D006026:Glycoside Hydrolases; C120356:3-deoxy-manno-oct-2-ulopyranosonic acid
keywords: glycosidase; lipid A; recombinant enzyme; sialidase; α-Kdo-ase