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Glycobiology Advance Access published online on June 10, 2003
Glycobiology, doi:10.1093/glycob/cwg085
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Submitted on May 9, 2002
Revised on May 9, 2003
Accepted on May 22, 2003

© 2003 Oxford University Press

ORIGINAL ARTICLES

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

Wei Jing 1 and Paul L. DeAngelis 1*

1 Dept. of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, Univ. of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd, Oklahoma City, OK 73104

* To whom correspondence should be addressed. E-mail: paul-deangelis{at}ouhsc.edu.

Abstract

Type A Pasteurella multocida produce a hyaluronan [HA] capsule to enhance infection. The 972-residue hyaluronan synthase, pmHAS, polymerizes the linear HA polysaccharide composed of alternating {beta}3N-acetylglucosamine [GlcNAc]-{beta}4glucuronic 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 that is essential for HA synthase activity. D247N or D249N mutants possessed only GlcUA-transferase activity while 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 E396 or D370 possessed only GlcUA-transferase activity.

Type F P. multocida synthesizes an unsulfated chondroitin ({beta}3N-acetylgalactosamine [GalNAc]-{beta}4GlcUA) 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.


capsule, chondroitin, glycosaminoglycan, glycosyltransferase, hyaluronan
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