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-galactonononic 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, KDN␣233Gal133GalNAc␣133[KDN␣23 (8KDN␣23) n 36]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.The natural occurrence of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), 1 deaminated neuraminic acid, was first discovered in 1986 by Nadano et al. (1). Subsequently, an increasing number of other KDN-glycoconjugates have been reported (2-14). Substitution of the aminoacyl group at C-5 on Neu5Ac or Neu5Gc by a hydroxyl group blocks almost completely the action of bacterial exosialidases that are commonly used for the identification and structure-function studies of sialoglycoconjugates (1,8,15).Recently, we isolated and identified a Gram-negative soil bacterium, Sphingobacterium multivorum, that produced a new sialidase that selectively catalyzed the hydrolysis of different types of KDN-ketosidic linkages (16). The partial purification of this enzyme, designated KDNase SM, and its substrate specificity were also described previously (16). The most important feature of KDNase SM was the complete absence of sialidase activity that could release Neu5Ac and Neu5Gc residues from a variety of naturally occurring Neu5Ac-and Neu5Gc-containing glycoconjugates. This unique specificity was in distinct contrast with other eukaryotic KDN-sialidases that hydrolyzed both KDN and N-acylneuraminic acid ␣-ketosidic linkages (17, 18). Since the first report of KDN residues in fish egg polysialoglycoproteins (1), it has been presumed that KDN residues would not likely be restricted in occurrence to only lower vertebrates but rather would also be expressed on mammalian cells. With the recent findings that KDN-glycoconjugates are implicated in fertilization in fish egg polysialoglycoproteins (4,7,8), it became important to determine the extent of the natural occurrence of KDN-glycoconjugates in other eukaryotic organisms. While anti-KDN antibodies have been used to show that K...