CLONING AND EXPRESSION OF THE MAMMALIAN CYTOSOLIC BRANCHED-CHAIN AMINOTRANSFERASE ISOENZYME

The cDNA for the rat cytosolic branched chain aminotransferase (BCAT(c )) has been cloned, The BCAT(c) cDNA encodes a polypeptide of 410 amin o acids with a calculated molecular mass of 46.0 kDa. By Northern blot analysis, BCAT(c) message of approximately 2.7 kilobases was readily detected in rat brain, but was absent from liver, a rat hepatoma cell line, kidney, and skeletal muscle. When expressed in COS-1 cells, the enzyme is immunologically indistinguishable from the native enzyme fou nd in rat brain cytosol, Comparison of the rat BCAT(c) sequence with a vailable data bases identified the Escherichia coli (and Salmonella ty phimurium) branched chain aminotransferase (BCAT) and revealed a Haemo philus influenzae BCAT, a yeast BCAT, which is hypothesized to be a mi tochondrial form of the enzyme, and the murine BCAT(c) (protein ECA39) , Calculated molecular masses for the complete proteins are 33.9 kDa, 37.9 kDa, 42.9 kDa, and 43.6 kDa, respectively. The rat BCAT(c) sequen ce was 84% identical with murine BCAT(c), 45% identical with yeast, 33 % identical with H. influenzae, 27% identical with the E. coli and S. typhimurium BCAT, and 22% identical with the evolutionary related D-am ino acid aminotransferase (D-AAT) (Tanizawa, K., Asano, S., Masu, Y., Kuramitsu, S., Kagamiyama, H., Tanaka, H., and Soda, K. (1989) J. Biol . Chem. 264, 2450-2454). Amino acid sequence alignment of BCAT(c) with D-AAT suggests that the folding pattern of the overlapping mammalian BCAT(c) sequence is similar to that of D-AAT and indicates that orient ation of the pyridoxal. phosphate cofactor in the active site of the e ukaryotic BCAT is the same as in D-AAT. Thus, BCAT are the only eukary otic aminotransferases to abstract and replace the proton on the re fa ce of the pyridoxal phosphate cofactor. Finally, requirements for reco gnition of substrate L-amino acid and alpha-carboxylate binding are di scussed.