J. Biochem, 1988, Vol. 104, No. 5 777-784
© 1988 Japanese Biochemical Society
research-article |
Branched-Chain Amino Acid Aminotransferase of Escherichia coli: Overproduction and Properties1
*Department of Medical Chemistry, Osaka Medical College Takatsuki Osaka 569
**Division of Enzyme Regulation, Institute for Enzyme Research, Tokushima University Tokushima, Tokushima 770
***Institute for Protein Research, Osaka University Suita, Osaka 565
****Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo Bunkyo-ku, Tokyo 113
2 To whom correspondence should be addressed
ilvE gene of Escherichia coli was inserted into the region downstream of the tac promotor. As a result, the branched-chain amino acid aminotransferase was overproduced by about a hundred-fold in E. coli W3110. The overproduced aminotransferase was purified from cell extracts about 40-fold to homogeneity. Chemical and physicochemical analyses confirmed that it was a product of the ilvE gene. The enzyme existed in a hexamer with a subunit molecular weight of 34,000; the double trimer model of the enzyme presumed by the previous chemical cross-linking experiments (Lee-Peng, F.-C. et al. (1979) J. Bacteriol. 139, 339–345) was supported by electron micrographs. The circular dichroic (CD) spectrum of branch-chain amino acid aminotransferase had double negative maxima at 210 and 220 nm. The 
-helical content was estimated to be about 40% from the CD spectrum in the region of 200 to 250 nm. The absorption spectrum of the enzyme showed two peaks at 330 and 410 nm. There was no pH-dependent spectral shift. The CD spectrum of the coenzyme, pyridoxal 5'-phosphate, had negative peaks at 330 and 410 nm. These spectral properties of branched-chain amino acid aminotransferase were quite different from those of E. coli aspartate aminotransferase. Each subunit bound approximately 1 mol of pyridoxal 5' phosphate. A lysyl residue, which forms a Schiff base with the aldehyde group of the pyridoxal 5'-phosphate, was identified in the primary structure of the enzyme.
1 This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
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