Molecular directionality of polysaccharide polymerization by the Pasteurella multocida hyaluronan synthase.
Hyaluronan (HA), a long linear polymer composed of alternating glucuronic acid and N-acetylglucosamine residues, is an essential polysaccharide in vertebrates and a putative virulence factor in certain microbes. All known HA synthases utilize UDP-sugar precursors. Previous reports describing the HA synthase enzymes from Streptococcus bacteria and mammals, however, did not agree on the molecular directionality of polymer elongation. We show here that a HA synthase, PmHAS, from Gram-negative P. multocida bacteria polymerizes the HA chain by the addition of sugar units to the nonreducing terminus. Recombinant PmHAS will elongate exogenous HA oligosaccharide acceptors to form long polymers in vitro; thus far no other HA synthase has displayed this capability. The directionality of synthesis was established definitively by testing the ability of PmHAS to elongate defined oligosaccharide derivatives. Analysis of the initial stages of synthesis demonstrated that PmHAS added single monosaccharide units sequentially. Apparently the fidelity of the individual sugar transfer reactions is sufficient to generate the authentic repeating structure of HA. Therefore, simultaneous addition of disaccharide block units is not required as hypothesized in some recent models of polysaccharide biosynthesis. PmHAS appears distinct from other known HA synthases based on differences in sequence, topology in the membrane, and putative reaction mechanism.
DeAngelis
P L
PL
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA. paul-deangelis@OUHSC.edu
D001704:Biopolymers; D002240:Carbohydrate Sequence; D014453:Glucuronosyltransferase / Q000378:metabolism*; D016695:Glycosyltransferases; D000076002:Hyaluronan Synthases; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016979:Pasteurella multocida / Q000201:enzymology*; D011134:Polysaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014166:Transferases; D029867:Xenopus Proteins
D001704:Biopolymers; D008565:Membrane Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases