Identification and molecular cloning of a chondroitin synthase from Pasteurella multocida type F.
Pasteurella multocida Type F, the minor fowl cholera pathogen, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported that the capsule was removed by treating microbes with chondroitin AC lyase. We found by acid hydrolysis that the polysaccharide contained galactosamine and glucuronic acid. We molecularly cloned a Type F polysaccharide synthase and characterized its enzymatic activity. The 965-residue enzyme, called P. multocida chondroitin synthase (pmCS), is 87% identical at the nucleotide and the amino acid level to the hyaluronan synthase, pmHAS, from P. multocida Type A. A recombinant Escherichia coli-derived truncated, soluble version of pmCS (residues 1-704) was shown to catalyze the repetitive addition of sugars from UDP-GalNAc and UDP-GlcUA to chondroitin oligosaccharide acceptors in vitro. Other structurally related sugar nucleotide precursors did not substitute in the elongation reaction. Polymer molecules composed of approximately 10(3) sugar residues were produced, as measured by gel filtration chromatography. The polysaccharide synthesized in vitro was sensitive to the action of chondroitin AC lyase but resistant to the action of hyaluronan lyase. This is the first report identifying a glycosyltransferase that forms a polysaccharide composed of chondroitin disaccharide repeats, [beta(1,4)GlcUA-beta(1,3)GalNAc](n). In analogy to known hyaluronan synthases, a single polypeptide species, pmCS, possesses both transferase activities.
DeAngelis
P L
PL
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Padgett-McCue
A J
AJ
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D014453:Glucuronosyltransferase / Q000235:genetics; D016695:Glycosyltransferases / Q000235:genetics*; D000076002:Hyaluronan Synthases; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases; D016979:Pasteurella multocida / Q000201:enzymology / Q000235:genetics*; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014166:Transferases; D029867:Xenopus Proteins
D008565:Membrane Proteins; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D014166:Transferases; D016695:Glycosyltransferases; D017350:N-Acetylgalactosaminyltransferases; D014453:Glucuronosyltransferase; C412725:chondroitin synthase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases