title: 2-Keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN)- and N-acetylneuraminic acid-cleaving sialidase (KDN-sialidase) and KDN-cleaving hydrolase (KDNase) from the hepatopancreas of oyster, Crassostrea virginica.
abstract: KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid), a sialic acid analog, has been found to be widely distributed in nature. Despite the structural similarity between KDN and Neu5Ac, alpha-ketosides of KDN are refractory to conventional sialidases. We found that the hepatopancreas of the oyster, Crassostrea virginica, contains two KDN-cleaving sialidases but is devoid of conventional sialidase. The major sialidase, KDN-sialidase, effectively cleaves alpha-ketosidically linked KDN and also slowly cleaves the alpha-ketosides of Neu5Ac. The minor sialidase, KDNase, is specific for alpha-ketosides of KDN. We were able to separate these two KDN-cleaving enzymes using hydrophobic interaction and cation-exchange chromatographies. The rate of hydrolysis of 4-methylumbelliferyl-alpha-KDN (MU-KDN) by KDN-sialidase is 30 times faster than that of MU-Neu5Ac in the presence of 0.2 M NaCl, whereas in the absence of NaCl this ratio is only 8. KDNase hydrolyzes MU-KDN over 500 times faster than MU-Neu5Ac and is not affected by NaCl. KDN-sialidase purified to electrophoretically homogeneous form was found to have a molecular mass of 25 kDa and an isoelectric point of 8.4. One of the three tryptic peptides derived from KDN-sialidase contains the consensus motif, SXDXGXTW, that has been found in all conventional sialidases. Kinetic analysis of the inhibition of the hydrolysis of MU-KDN and MU-Neu5Ac by 2, 3-dehydro-2-deoxy-KDN (KDN2-en) and 2,3-dehydro-2-deoxy-(Neu5Ac2-en) suggests that KDN-sialidase contains two separate active sites for the hydrolysis of KDN and Neu5Ac. Both KDN-sialidase and KDNase effectively hydrolyze KDN-G(M3), KDNalpha2-->3Gal beta1-->4Glc, KDNalpha2-->6Galbeta1-->4Glc, KDNalpha2-->6-N-acetylgalactosaminitol, KDNalpha2-->6(KDNalpha2-->3)N-acetylgalactosaminitol, and KDNalpha2-->6(GlcNAcbeta1-->3)N-acetylgalactosaminitol. However, only KDN-sialidase also slowly hydrolyzes G(M3), Neu5Acalpha2-->3Galbeta1-->4Glc, and Neu5Acalpha2-->6Galbeta1-->4Glc. These two KDN-cleaving sialidases should be useful for studying the structure and function of KDN-containing glycoconjugates.
authors.affiliation: Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA.
authors.forename: N V
authors.initials: NV
authors.lastname: Pavlova
authors.forename: J A
authors.initials: JA
authors.lastname: Yuziuk
authors.forename: H
authors.initials: H
authors.lastname: Nakagawa
authors.forename: M
authors.initials: M
authors.lastname: Kiso
authors.forename: S C
authors.initials: SC
authors.lastname: Li
authors.forename: Y T
authors.initials: YT
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004064:Digestive System / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006868:Hydrolysis; D007525:Isoelectric Focusing; D007700:Kinetics; D008628:Mercury / Q000494:pharmacology; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000378:metabolism*; D009994:Osmolar Concentration; D010125:Ostreidae / Q000201:enzymology*; D013400:Sugar Acids / Q000378:metabolism*
chemical_list: D013400:Sugar Acids; C050008:3-deoxyglycero-galacto-nonulosonic acid; D006026:Glycoside Hydrolases; C091615:deaminoneuraminate hydrolase; D009439:Neuraminidase; D008628:Mercury