Identification and molecular cloning of a heparosan synthase from Pasteurella multocida type D.
Pasteurella multocida Type D, a causative agent of atrophic rhinitis in swine and pasteurellosis in other domestic animals, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported previously that the capsule was removed by treating microbes with heparin lyase III. We molecularly cloned a 617-residue enzyme, pmHS, which is a heparosan (nonsulfated, unepimerized heparin) synthase. Recombinant Escherichia coli-derived pmHS catalyzes the polymerization of the monosaccharides from UDP-GlcNAc and UDP-GlcUA. Other structurally related sugar nucleotides did not substitute. Synthase activity was stimulated about 7-25-fold by the addition of an exogenous polymer acceptor. Molecules composed of approximately 500-3,000 sugar residues were produced in vitro. The polysaccharide was sensitive to the action of heparin lyase III but resistant to hyaluronan lyase. The sequence of the pmHS enzyme is not very similar to the vertebrate heparin/heparan sulfate glycosyltransferases, EXT1 and 2, or to other Pasteurella glycosaminoglycan synthases that produce hyaluronan or chondroitin. The pmHS enzyme is the first microbial dual-action glycosyltransferase to be described that forms a polysaccharide composed of beta4GlcUA-alpha4GlcNAc disaccharide repeats. In contrast, heparosan biosynthesis in E. coli K5 requires at least two separate polypeptides, KfiA and KfiC, to catalyze the same polymerization reaction.
DeAngelis
Paul L
PL
Department of Biochemistry and Molecular Biology, Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., Oklahoma City, OK 73104, USA. paul-deangelis@ouhsc.edu
White
Carissa L
CL
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004187:Disaccharides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D016695:Glycosyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D006493:Heparin / Q000378:metabolism*; D008025:Ligases / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry; D016979:Pasteurella multocida / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D004187:Disaccharides; D029968:Escherichia coli Proteins; D010455:Peptides; D011108:Polymers; D011994:Recombinant Proteins; D006493:Heparin; D016695:Glycosyltransferases; C413756:KfiA protein, E coli; C097521:KfiC protein, E coli; D017351:N-Acetylglucosaminyltransferases; C450833:heparosan synthase; D008025:Ligases