Alteration of the fatty acid substrate specificity of lysophosphatidate acyltransferase by site-directed mutagenesis.
The JC201 strain of Eschericia coli contains a temperature-sensitive lesion in lysophosphatidate acyltransferase (LPAT) activity. The LPAT gene from JC201 was isolated by PCR and a single mutant nucleotide, adenine-440, was identified by DNA sequence analysis. Site-directed mutagenesis converted the mutant adenine-440 back to the native guanine-440 nucleotide. The restored LPAT gene rescued JC201 cells at the non-permissive temperature. The fatty acid substrate specificity of LPAT from Eschericia coli was altered by site-directed mutagenesis of a single amino acid in the restored LPAT gene. Threonine-122 of LPAT was changed to alanine or leucine. A change from threonine-122 to alanine increased the substrate specificity in vitro for oleoyl-CoA and linoleoyl-CoA; whereas a change to leucine increased the substrate specificity for lignoceroyl-CoA.
Morand
L Z
LZ
Department of Food Science and Technology, University of California, Davis 95616, USA. lzmorand@ucdavis.edu
Patil
S
S
Quasney
M
M
German
J B
JB
D051103:1-Acylglycerol-3-Phosphate O-Acyltransferase; D000217:Acyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000225:Adenine; D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001667:Binding, Competitive / Q000235:genetics; D004398:Dyneins; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D005227:Fatty Acids / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics; D006147:Guanine; D007930:Leucine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity / Q000235:genetics; D013696:Temperature; D013912:Threonine / Q000235:genetics; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005227:Fatty Acids; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D006147:Guanine; D000217:Acyltransferases; D051103:1-Acylglycerol-3-Phosphate O-Acyltransferase; C495091:plsC protein, E coli; D004398:Dyneins; C083745:SLC1 protein, S cerevisiae; D007930:Leucine; D000225:Adenine; D000409:Alanine