The disulfide bond in the Aeromonas hydrophila lipase/acyltransferase stabilizes the structure but is not required for secretion or activity.
Vibrio and Aeromonas spp. secrete an unusual 35-kDa lipase that shares several properties with mammalian lecithin-cholesterol acyltransferase. The Aeromonas hydrophila lipase contains two cysteine residues that form an intramolecular disulfide bridge. Here we show that changing either of the cysteines to serine does not reduce enzymatic activity, indicating that the disulfide bond is not required for correct folding. However, when either of the cysteines is replaced, the enzyme is more readily denatured by urea and more sensitive to degradation by trypsin than is the wild-type enzyme, evidence that the bridge has an important role in stabilizing the protein's structure. The two mutant proteins with serine-for-cysteine replacements were secreted by Aeromonas salmonicida containing the cloned genes, although the levels of both in the culture supernatants were lower than the level of the wild-type enzyme. When the general secretory pathway was blocked with carbonyl cyanide chlorophenylhydrazone, the cell-associated pools of the mutant enzymes appeared to be degraded, whereas the wild-type pool remained stable. We conclude that reduced extracellular levels of the mutant proteins are the result of their increased sensitivities to proteases encountered inside the cell during export.
Brumlik
M J
MJ
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
van der Goot
F G
FG
Wong
K R
KR
Buckley
J T
JT
D000217:Acyltransferases / Q000737:chemistry* / Q000378:metabolism*; D016980:Aeromonas hydrophila / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D001427:Bacterial Toxins / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability / Q000235:genetics; D006868:Hydrolysis / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D052899:Pore Forming Cytotoxic Proteins; D012694:Serine / Q000235:genetics; D013329:Structure-Activity Relationship; D014357:Trypsin
D001427:Bacterial Toxins; D004220:Disulfides; D052899:Pore Forming Cytotoxic Proteins; D012694:Serine; C032676:aerolysin; D000217:Acyltransferases; C020597:glycerophospholipid-cholesterol acyltransferase; D014357:Trypsin; D003545:Cysteine