Identification of the catalytic triad of the lipase/acyltransferase from Aeromonas hydrophila.
Aeromonas hydrophila secretes a lipolytic enzyme that has several properties in common with the mammalian enzyme lecithin-cholesterol acyltransferase. We have recently shown that it is a member of a newly described group of proteins that contain five similar blocks of sequence arranged in the same order in their primary structures (C. Upton and J. T. Buckley, Trends Biochem. Sci. 233:178-179, 1995). Assuming that, like other lipases, these enzymes have a Ser-Asp-His catalytic triad, we used these blocks to predict which aspartic acid and histidine would be at the active site of the Aeromonas enzyme. Targeted residues were replaced with other amino acids by site-directed mutagenesis, and the effects on secretion and activity were assessed. Changing His-291 to asparagine completely abolished enzyme activity, although secretion by the bacteria was not affected. Only very small amounts of the D116N mutant appeared in the culture supernatant, likely because it is sensitive to periplasmic proteases it encounters en route. Assays of crude preparations containing this variant showed no detectable enzyme activity. We conclude that, together with Ser-16, which we have identified previously, Asp-116 and His-291 compose the catalytic triad of the enzyme.
Brumlik
M J
MJ
Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada.
Buckley
J T
JT
D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016980:Aeromonas hydrophila / Q000201:enzymology*; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004269:DNA, Bacterial; D006639:Histidine / Q000378:metabolism; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship
D004269:DNA, Bacterial; D001224:Aspartic Acid; D012694:Serine; D006639:Histidine; D000217:Acyltransferases; C085461:Aeromonas hydrophilia lipase-acyltransferase; D008049:Lipase