Alteration of the Fatty Acid Substrate Specificity of Lysophosphatidate Acyltransferase by Site-Directed Mutagenesis

Abstract

The JC201 strain ofEschericia colicontains 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 fromEschericia coliwas 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 specificityin vitrofor oleoyl-CoA and linoleoyl-CoA; whereas a change to leucine increased the substrate specificity for lignoceroyl-CoA.