Induction, localization, and purification of a novel sialidase, deaminoneuraminidase (KDNase), from Sphingobacterium multivorum.
Recently, we reported the discovery of a new type of sialidase, KDNase, which specifically hydrolyzes the ketosidic linkages of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), but not N-acylneuraminyl linkages. We now report that this enzyme, designated KDNase SM, is an inducible enzyme that is localized in the periplasm of Sphingobacterium multivorum. Growth of S. multivorum in the presence of KDN-containing oligosaccharide alditols, KDNalpha2-->3Galbeta1-->3GalNAc alpha1-->3[KDNalpha2--> (8KDN alpha2-->)n-->6]GalNAcol, as a sole carbon source induced KDNase SM activity 15 40-fold, compared with growth in the absence of inducer. KDN, Neu5Ac, or Neu5Ac oligomers were ineffective as inducers. The enzyme was released from the periplasm of induced cells by cold osmotic shock and purified 700-fold to homogeneity. The specific activity of the pure enzyme was 82,100 units/mg of protein. KDNase SM activity resided in a single polypeptide chain with an estimated molecular weight of approximately 47,500. Enzyme activity was maximal at near neutral pH. The availability of pure KDNase will now make it possible to study the structure and functional role of KDN-glycoconjugates and to determine the molecular mechanism whereby the enzyme can discriminate between KDN and N-acylneuraminic acid.
Nishino
S
S
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo-7, Tokyo 113, Japan.
Kuroyanagi
H
H
Terada
T
T
Inoue
S
S
Inoue
Y
Y
Troy
F A
FA
Kitajima
K
K
D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D006090:Gram-Negative Bacteria / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D009005:Monosaccharides / Q000494:pharmacology; D009844:Oligosaccharides / Q000494:pharmacology; D012988:Soil Microbiology
D009005:Monosaccharides; D009844:Oligosaccharides; D006026:Glycoside Hydrolases; C091615:deaminoneuraminate hydrolase