Molecular cloning of cDNA species for rat and mouse liver alpha-methylacyl-CoA racemases

cDNA species coding for alpha-methylacyl-CoA racemase were cloned from rat and mouse liver cDNA libraries and characterized. The rat liver lambda gt11 cDNA expression library was screened with anti-racemase IgG [Schmitz, Albers, Fingerhut and Conzelmann(1995) fur. J. Biochem. 231, 815-822]. Several full-length clones were obtained that contained an open reading frame of 1083 bp, coding for a protein of 361 amino acid residues with a predicted molecular mass of 39 679 Da. The sequences of three peptides that were isolated by HPLC from a tryptic digest of purified rat liver racemase fully matched the cDNA-derived amino acid sequence. The cDNA coding for mouse racemase was cloned from a mouse liver lambda ZAP cDNA expression library and sequenced. The coding region of 1080 bp codes for a 360-residue protein (molecular mass 39 558 Dal that shares 89.7 % similarity with the rat protein. Expression of the rat racemase as a recombinant protein in Escherichia coli with the pTrcHisB-expression vector yielded enzymically active protein. The amino acid sequences of alpha-methylacyl-CoA racemases do not resemble any known sequence of beta-oxidation or auxiliary enzymes, supporting the view of a highly diverse evolutionary origin of enzymes acting on fatty acyl-CoA S-esters.