Molecular cloning and characterization of chondroitin polymerase from Escherichia coli strain K4.
Escherichia coli strain K4 produces the K4 antigen, a capsule polysaccharide consisting of a chondroitin backbone (GlcUA beta(1-3)-GalNAc beta(1-4))(n) to which beta-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 microm, 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.
Ninomiya
Toshio
T
Institute for Molecular Science of Medicine, Aichi Medical University, Yazako, Nagakute, Aichi 480-1195, Japan.
Sugiura
Nobuo
N
Tawada
Akira
A
Sugimoto
Kazunori
K
Watanabe
Hideto
H
Kimata
Koji
K
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002412:Cations; D002807:Chondroitin / Q000737:chemistry; D002810:Chondroitinases and Chondroitin Lyases / Q000737:chemistry* / Q000235:genetics; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003871:Dermatan Sulfate / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D016695:Glycosyltransferases / Q000737:chemistry / Q000378:metabolism; D006493:Heparin / Q000737:chemistry; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics*; D006820:Hyaluronic Acid / Q000737:chemistry; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D005810:Multigene Family; D009844:Oligosaccharides / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
D002412:Cations; D009844:Oligosaccharides; D011108:Polymers; D011994:Recombinant Proteins; D003871:Dermatan Sulfate; D006820:Hyaluronic Acid; D006493:Heparin; D002807:Chondroitin; D016695:Glycosyltransferases; D006602:Hexosyltransferases; C460368:chondroitin polymerase, E coli; D002810:Chondroitinases and Chondroitin Lyases