Journal
GLYCOBIOLOGY
Volume 10, Issue 9, Pages 883-889Publisher
OXFORD UNIV PRESS INC
DOI: 10.1093/glycob/10.9.883
Keywords
capsule; glycosyltransferase; hyaluronan; polysaccharide; synthase
Categories
Funding
- NIGMS NIH HHS [R01-GM56497] Funding Source: Medline
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM056497] Funding Source: NIH RePORTER
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Type A Pasteurella multocida, an animal pathogen, employs a hyaluronan [HA] capsule to avoid host defenses. PmHAS, the 972-residue membrane-associated hyaluronan synthase, catalyzes the transfer of both GlcNAc and GlcUA to form the HA polymer. To define the catalytic and membrane-associated domains, pmHAS mutants were analyzed. PmHAS(1-703) is a soluble, active HA synthase suggesting that the carboxyl-terminus is involved in membrane association of the native enzyme, PmHAS(1-650) is inactive as a HA synthase, but retains GlcNAc-transferase activity. Within the pmNAS sequence, there is a duplicated domain containing a short motif, Asp-Gly-Ser, that is conserved among many beta-glycosyltransferases. Changing this aspartate in either domain to asparagine, glutamate, or lysine reduced the WA synthase activity to low levels. The mutants substituted at residue 196 possessed GlcUA-transferase activity while those substituted at residue 477 possessed GlcNAc-transferase activity. The Michaelis constants of the functional transferase activity of the various mutants, a measure of the apparent affinity of the enzymes for the precursors, were similar to wild-type values. Furthermore, mixing D196N and D477K mutant proteins in the same reaction allowed HA polymerization at levels similar to the wild-type enzyme, These results provide the first direct evidence that the synthase polypeptide utilizes two separate glycosyltransferase sites.
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