Branched-Chain Amino Acid Aminotransferase of Escherichia coli: Overproduction and Properties

• Published in: Journal of biochemistry (Tokyo) 1988, Vol.104 (5), p.777-784
• Main Authors: Inoue, Katsura, Kuramitsu, Seiki, Aki, Kenji, Watanabe, Yasushi, Takagi, Toshio, Nishigai, Masaaki, Ikai, Atsushi, Kagamiyama, Hiroyuki
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acids - analysis; Aspartate Aminotransferases - analysis; Biological and medical sciences; Biotechnology; Chromatography, Gel; Chromatography, Ion Exchange; Circular Dichroism; Escherichia coli; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; Hydrogen-Ion Concentration; Kinetics; Methods. Procedures. Technologies; Microscopy, Electron; Molecular Sequence Data; Molecular Weight; Pyridoxal Phosphate - analysis; Recombinant Proteins; Schiff Bases; Spectrum Analysis; Transaminases - biosynthesis; Transaminases - isolation & purification
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a122549

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 α-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.