cDNA cloning, recombinant expression, and site-directed mutagenesis of bovine liver carnitine octanoyltransferase--Arg505 binds the carboxylate group of carnitine.
The cDNA for bovine liver carnitine octanoyltransferase (COT) has been cloned by a combination of lambda gt11 library screening and 3' rapid amplification of cDNA ends (3'-RACE). The cDNA comprises 338 bases of 5' non-coding sequence, a reading frame of 1839 bases including the stop codon, and 820 bases of 3' non-coding DNA. The deduced amino acid sequence of 612 residues predicts a protein with a calculated mass of 70263 Da and pI 6.28. The enzyme was expressed in recombinant soluble form in Escherichia coli and was purified by a two-step procedure to near-homogeneity with a yield of purified protein of 2-3 mg/l culture. Recombinant COT had similar kinetic properties to those of the enzyme isolated directly from beef liver. Arg505 in COT, conserved in all reported carnitine acyltransferase sequences but replaced by asparagine or isoleucine in the choline acetyltransferases, was converted to asparagine by site-directed mutagenesis. This single mutation resulted in a greater than 1650-fold increase in the Km value for COT towards carnitine, but had little effect on the value of k(cat) or the Km value for the acyl-CoA substrate. In addition, although choline was an extremely poor substrate for COT, the k(cat)/Km ratio towards this substrate was increased fourfold as a result of the mutation. These data support the notion that Arg505 in COT, and other carnitine acyltransferases, contributes to substrate binding by forming a salt bridge with the carboxylate moiety of carnitine.
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Department of Veterans Affairs Medical Center, and Department of Biochemistry and Biophysics, University of California, San Francisco 94121, USA. ciaran@itsa.ucsf.edu
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002264:Carboxylic Acids / Q000378:metabolism*; D002331:Carnitine / Q000737:chemistry / Q000378:metabolism*; D002333:Carnitine Acyltransferases / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D002264:Carboxylic Acids; D018076:DNA, Complementary; D011994:Recombinant Proteins; D001120:Arginine; D002333:Carnitine Acyltransferases; C030162:carnitine octanoyltransferase; D002331:Carnitine