Influence of active site and tyrosine modification on the secretion and activity of the Aeromonas hydrophila lipase/acyltransferase.
Aeromonas sp. secrete a lipase/acyltransferase that shares several properties with the mammalian plasma enzyme lecithin:cholesterol acyltransferase. Reaction of the enzyme with tetranitromethane led to modification of 2 tyrosines and a nearly 80% decline in enzyme activity. Replacing Tyr230 with Phe altered the activity of the enzyme in the same way as did treatment with tetranitromethane. Unlike the wild type enzyme, which preferentially hydrolyzes the 2-position acyl chain of phosphatidylcholine, the Y230F mutant enzyme did not discriminate between the 1- and 2-positions of the phospholipid. Tyr230 may be necessary to correctly position phospholipid substrates at the active site. Several amino acids around the active site Ser16 of the lipase were also changed. Replacing Ser18 with Gly, bringing the enzyme's sequence into line with the "lipase consensus sequence," resulted in reduced secretion of the protein and complete loss of activity. Changing this serine to Val led to an inactive protein that was not secreted at all. Substituting Phe13 in the hydrophobic region of the consensus sequence with Ser also prevented secretion, although the mutant protein appeared to be active. The Aeromonas lipase may represent a distinct group of lipolytic enzymes which have a novel active site structure.
Robertson
D L
DL
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
Hilton
S
S
Wong
K R
KR
Koepke
A
A
Buckley
J T
JT
D000217:Acyltransferases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016980:Aeromonas hydrophila / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli; D007700:Kinetics; D008049:Lipase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008322:Mammals; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013379:Substrate Specificity; D013774:Tetranitromethane / Q000378:metabolism; D014443:Tyrosine
D011994:Recombinant Proteins; D014443:Tyrosine; D000217:Acyltransferases; C085461:Aeromonas hydrophilia lipase-acyltransferase; D008049:Lipase; D013774:Tetranitromethane