Molecular Cloning and Characterization of Chondroitin Polymerase from Escherichia coli Strain K4

• Published in: The Journal of biological chemistry 2002-06, Vol.277 (24), p.21567-21575
• Main Authors: Ninomiya, Toshio, Sugiura, Nobuo, Tawada, Akira, Sugimoto, Kazunori, Watanabe, Hideto, Kimata, Koji
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Blotting, Southern; Blotting, Western; Cations; Chondroitin - chemistry; Chondroitinases and Chondroitin Lyases - chemistry; Chondroitinases and Chondroitin Lyases - genetics; Chromatography, Gel; Chromatography, High Pressure Liquid; Cloning, Molecular; Dermatan Sulfate - chemistry; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Escherichia coli - enzymology; Glycosyltransferases - chemistry; Glycosyltransferases - metabolism; Heparin - chemistry; Hexosyltransferases - chemistry; Hexosyltransferases - genetics; Hyaluronic Acid - chemistry; Kinetics; Models, Genetic; Molecular Sequence Data; Multigene Family; Oligosaccharides - chemistry; Polymers - chemistry; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M201719200

Escherichia coli strain K4 produces the K4 antigen, a capsule polysaccharide consisting of a chondroitin backbone (GlcUA β(1–3)-GalNAc β(1–4)) n to which β-fructose is linked at position C-3 of the GlcUA residue. We molecularly cloned region 2 of the K4 capsular gene cluster essential for biosynthesis of the polysaccharide, and we further identified a gene encoding a bifunctional glycosyltransferase that polymerizes the chondroitin backbone. The enzyme, containing two conserved glycosyltransferase sites, showed 59 and 61% identity at the amino acid level to class 2 hyaluronan synthase and chondroitin synthase from Pasteurella multocida , respectively. The soluble enzyme expressed in a bacterial expression system transferred GalNAc and GlcUA residues alternately, and polymerized the chondroitin chain up to a molecular mass of 20 kDa when chondroitin sulfate hexasaccharide was used as an acceptor. The enzyme exhibited apparent K m values for UDP-GlcUA and UDP-GalNAc of 3.44 and 31.6 μ m , respectively, and absolutely required acceptors of chondroitin sulfate polymers and oligosaccharides at least longer than a tetrasaccharide. In addition, chondroitin polymers and oligosaccharides and hyaluronan polymers and oligosaccharides served as acceptors for chondroitin polymerization, but dermatan sulfate and heparin did not. These results may lead to elucidation of the mechanism for chondroitin chain synthesis in both microorganisms and mammals.