|
type |
Journal Article |
authors |
Inoue, K.; Kuramitsu, S.; Aki, K.; Watanabe, Y.; Takagi, T.; Nishigai, M.; Ikai, A.; Kagamiyama, H. |
title |
Branched-chain amino acid aminotransferase of Escherichia coli: overproduction and properties |
journal |
J Biochem (Tokyo) |
Activity |
2.6.1.42 |
Family |
2.6.1.42.a |
sel |
selected |
ui |
3069843 |
year |
(1988) |
volume |
104 |
number |
5 |
pages |
777-84 |
| |
keywords |
Amino Acid Sequence |
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. |
last changed |
2017/06/29 20:44 |
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