PMID:3069843
| Citation |
Inoue, K, Kuramitsu, S, Aki, K, Watanabe, Y, Takagi, T, Nishigai, M, Ikai, A and Kagamiyama, H (1988) Branched-chain amino acid aminotransferase of Escherichia coli: overproduction and properties. J. Biochem. 104:777-84 |
|---|---|
| Abstract |
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 alpha-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. |
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| Keywords |
Amino Acid Sequence; Amino Acids/analysis; Aspartate Aminotransferases/analysis; Chromatography, Gel; Chromatography, Ion Exchange; Circular Dichroism; Escherichia coli/enzymology; Hydrogen-Ion Concentration; Kinetics; Microscopy, Electron; Molecular Sequence Data; Molecular Weight; Pyridoxal Phosphate/analysis; Recombinant Proteins; Schiff Bases; Spectrum Analysis; Transaminases/biosynthesis; Transaminases/isolation & purification |
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