Identification and molecular cloning of a unique hyaluronan synthase from Pasteurella multocida.
Type A Pasteurella multocida, a prevalent animal pathogen, employs a hyaluronan [HA] polysaccharide capsule to avoid host defenses. We utilized transposon insertional mutagenesis to identify the P. multocida HA synthase, the enzyme that polymerizes HA. A DNA fragment from a wild-type genomic library could direct HA production in vivo in Escherichia coli, a bacterium that normally does not produce HA. Analysis of truncated plasmids derived from the original clone indicated that an open reading frame encoding a 972-residue protein was responsible for HA polymerization. This identification was confirmed by expression cloning in E. coli; we observed HA capsule formation in vivo and detected activity in membrane preparations in vitro. The polypeptide size was verified by photoaffinity labeling of the native P. multocida HA synthase with azido-UDP sugar analogs. Overall, the P. multocida sequence is not very similar to the other known HA synthases from streptococci, PBCV-1 virus, or vertebrates. Instead, a portion of the central region of the new enzyme is more homologous to the amino termini of other bacterial glycosyltransferases that produce different capsular polysaccharides or lipopolysaccharides. In summary, we have discovered a unique HA synthase that differs in sequence and predicted topology from the other known enzymes.
DeAngelis
P L
PL
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA. paul-deangelis@OUHSC.edu
Jing
W
W
Drake
R R
RR
Achyuthan
A M
AM
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D014453:Glucuronosyltransferase / Q000235:genetics*; D016695:Glycosyltransferases; D000076002:Hyaluronan Synthases; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016979:Pasteurella multocida / Q000235:genetics*; D016415:Sequence Alignment; D014166:Transferases; D029867:Xenopus Proteins
D008565:Membrane Proteins; D029867:Xenopus Proteins; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases