Dissection of the two transferase activities of the Pasteurella multocida hyaluronan synthase: two active sites exist in one polypeptide.
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. PmHAS1-703 is a soluble, active HA synthase suggesting that the carboxyl-terminus is involved in membrane association of the native enzyme. PmHAS1-650 is inactive as a HA synthase, but retains GlcNAc-transferase activity. Within the pmHAS 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 HA 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.
Jing
W
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Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73104, USA.
DeAngelis
P L
PL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D005816:Genetic Complementation Test; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000076002:Hyaluronan Synthases; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D016979:Pasteurella multocida / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D014166:Transferases; D029867:Xenopus Proteins
D008565:Membrane Proteins; D009097:Multienzyme Complexes; D029867:Xenopus Proteins; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases