Biochemical Journal

Research article

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

Werner SCHMITZ, Heli M. HELANDER, J. Kalervo HILTUNEN, Ernst CONZELMANN

Abstract

cDNA species coding for α-methylacyl-CoA racemase were cloned from rat and mouse liver cDNA libraries and characterized. The rat liver λgt11 cDNA expression library was screened with anti-racemase IgG [Schmitz, Albers, Fingerhut and Conzelmann (1995) Eur. 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 39679 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 λZAP cDNA expression library and sequenced. The coding region of 1080 bp codes for a 360-residue protein (molecular mass 39558 Da) 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 α-methylacyl-CoA racemases do not resemble any known sequence of β-oxidation or auxiliary enzymes, supporting the view of a highly diverse evolutionary origin of enzymes acting on fatty acyl-CoA S-esters.