Analysis of the two active sites of the hyaluronan synthase and the chondroitin synthase of Pasteurella multocida.
Type A Pasteurella multocida produces a hyaluronan (HA) capsule to enhance infection. The 972-residue HA synthase, pmHAS, polymerizes the linear HA polysaccharide composed of alternating beta3N-acetylglucosamine (GlcNAc)-beta4glucuronic acid (GlcUA). We demonstrated previously that pmHAS possesses two independent glycosyltransferase sites. Here we further define the sites and putative motifs. Deletion of residues 1-117 does not affect HA polymerizing activity. The carboxyl-terminal boundary of the GlcUA-transferase resides within residues 686-703. Both transferase sites contain a DXD motif essential for HA synthase activity. D247N or D249N mutants possessed only GlcUA-transferase activity, whereas D527N or D529N mutants possessed only GlcNAc-transferase activity, further confirming our assignment of the two active sites within the synthase polypeptide. A potential role of the DXD motif in substrate binding was supported by experiments utilizing high UDP-sugar concentrations that partially rescued the activity of certain mutants. The WGGED sequence motif is involved in GlcNAc-transferase activity because mutants with substitutions at E369 or D370 possessed only GlcUA-transferase activity. Type F P. multocida synthesizes an unsulfated chondroitin (beta3GalNAc-beta4GlcUA) capsule. A chimeric enzyme consisting of residues 1-427 of pmHAS and residues 421-704 of pmCS, the homologous chondroitin synthase, was an active HA synthase. The converse chimeric enzyme consisting of residues 1-420 of pmCS and residues 428-703 of pmHAS was a functional chondroitin synthase. Analyses of a panel of pmHAS/pmCS chimeric enzymes identified a 44-residue region, corresponding to pmHAS residues 225-265, involved in UDP-hexosamine selectivity. Overall, these findings further support the model of two independent transferase sites within a single polypeptide.
Jing
Wei
W
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., Oklahoma City, OK 73104, USA.
DeAngelis
Paul L
PL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D014453:Glucuronosyltransferase; D000076002:Hyaluronan Synthases; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016979:Pasteurella multocida / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013379:Substrate Specificity; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014530:Uridine Diphosphate / Q000378:metabolism
D008670:Metals; D014530:Uridine Diphosphate; D014166:Transferases; D017350:N-Acetylgalactosaminyltransferases; D014453:Glucuronosyltransferase; C412725:chondroitin synthase; D000076002:Hyaluronan Synthases