Cloning and expression of the mammalian cytosolic branched chain aminotransferase isoenzyme.
The cDNA for the rat cytosolic branched chain aminotransferase (BCATc) has been cloned. The BCATc cDNA encodes a polypeptide of 410 amino acids with a calculated molecular mass of 46.0 kDa. By Northern blot analysis, BCATc 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 found in rat brain cytosol. Comparison of the rat BCATc sequence with available data bases identified the Escherichia coli (and Salmonella typhimurium) branched chain aminotransferase (BCAT) and revealed a Haemophilus influenzae BCAT, a yeast BCAT, which is hypothesized to be a mitochondrial form of the enzyme, and the murine BCATc (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 BCATc sequence was 84% identical with murine BCATc, 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-amino 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 BCATc with D-AAT suggests that the folding pattern of the overlapping mammalian BCATc sequence is similar to that of D-AAT and indicates that orientation of the pyridoxal phosphate cofactor in the active site of the eukaryotic BCAT is the same as in D-AAT. Thus, BCAT are the only eukaryotic aminotransferases to abstract and replace the proton on the re face of the pyridoxal phosphate cofactor. Finally, requirements for recognition of substrate L-amino acid and alpha-carboxylate binding are discussed.
Hutson
S M
SM
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157, USA.
Bledsoe
R K
RK
Hall
T R
TR
Dawson
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D003001:Cloning, Molecular; D016384:Consensus Sequence; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression; D006193:Haemophilus influenzae / Q000201:enzymology; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012486:Salmonella typhimurium / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D000637:Transaminases / Q000096:biosynthesis* / Q000737:chemistry*; D014162:Transfection
D017931:DNA Primers; D007527:Isoenzymes; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D000637:Transaminases; C019798:branched-chain-amino-acid transaminase