Branched-chain amino acid aminotransferase of Escherichia coli: overproduction and properties.

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Citation

Inoue K, Kuramitsu S, Aki K, Watanabe Y, Takagi T, Nishigai M, Ikai A, Kagamiyama H

Branched-chain amino acid aminotransferase of Escherichia coli: overproduction and properties.

J Biochem. 1988 Nov;104(5):777-84.

PubMed ID
3069843 [ View in PubMed
]
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.

DrugBank Data that Cites this Article

Polypeptides
NameUniProt ID
Branched-chain-amino-acid aminotransferaseP0AB80Details