Induction, Localization, and Purification of a Novel Sialidase, Deaminoneuraminidase (KDNase), from Sphingobacterium multivorum

Satoru Nishino,Hidehito Kuroyanagi,Takaho Terada,Sadako Inoue,Yasuo Inoue,Frederic A Troy,Ken Kitajima

doi:10.1074/jbc.271.6.2909

Abstract

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.

Highlights

We reported the discovery of a new type of sialidase, KDNase, which hydrolyzes the ketosidic linkages of 2-keto-3-deoxy-D-glycero-D-galactonononic acid (KDN), but not N-acylneuraminyl linkages
We report that this enzyme, designated KDNase SM, is an inducible enzyme that is localized in the periplasm of Sphingobacterium multivorum
1 The abbreviations used are: KDN, 3-deoxy-D-glycero-D-galacto-2nonulosonic acid; Sia, sialic acid; KDN-glycoconjugates, KDN-containing glycoconjugates; KDNase, an enzyme which catalyzes the hydrolysis of ketosidic linkages of KDN; KDN-oligosaccharide alditols (KDN-OS), KDN␣2–3Gal␤1– 3GalNAc␣1–3[KDN␣2-(8KDN␣2-)n-6]GalNAcol with n ϭ 5; 4-MUKDN, 4-methylumbelliferyl KDN; KDNase SM, KDNase isolated from Sphingobacterium multivorum; BSA, bovine serum albumin

Summary

EXPERIMENTAL PROCEDURES

Bacteria—Isolation and growth of a strain of Sphingobacterium multivorum that could grow on KDN-oligosaccharide alditols as the sole carbon source were carried out as described previously [16]. Release of KDNase SM by Osmotic Shock from S. multivorum— Washed cells (1.2 ϫ 1013), grown at 25 °C in 160 ml of M9 medium supplemented with 1% (w/v) casamino acids and glucose, as described above, were inoculated into 500 ml of M9 medium containing 0.1% (w/v) enriched KDN-OS as the sole carbon source, and incubated at 25 °C for 43 h. Cells (8 g, wet weight) were harvested and washed in cold 0.03 M NaCl-0.01 M Tris-HCl buffer (pH 7.1) by centrifugation at 13,000 ϫ g for 20 min. The effect of pH on the KDNase SM-catalyzed hydrolysis of 4-MUKDN was studied by incubating the enzyme (2 ␮l; 7.0 milliunits) with 4-MU-KDN (1.6 ␮l; 1.6 nmol) in 20 ␮l of 0.1 M NaCl-containing 0.1% (w/v) BSA and 0.1 M Tris acetate buffer that was adjusted to pH 4.5– 8.0. Thermal stability of the enzyme was estimated by measuring the activity after preincubation of the reaction mixture at 4, 25, and 37 °C for 0, 15, 30, and 60 min

RESULTS

KDNase activitya Percent activity

Specific activity

DISCUSSION