Analysis of the two active sites of the hyaluronan synthase and the chondroitin synthase of Pasteurella multocida

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Abstract
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 β3N-acetylglucosamine (GlcNAc)-β4glucuronic 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 (β3GalNAc-β4GlcUA) 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.

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