Branched-chain amino acid aminotransferase of Escherichia coli: overproduction and properties.
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.
Inoue
K
K
Department of Medical Chemistry, Osaka Medical College.
Kuramitsu
S
S
Aki
K
K
Watanabe
Y
Y
Takagi
T
T
Nishigai
M
M
Ikai
A
A
Kagamiyama
H
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001219:Aspartate Aminotransferases / Q000032:analysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011732:Pyridoxal Phosphate / Q000032:analysis; D011994:Recombinant Proteins; D012545:Schiff Bases; D013057:Spectrum Analysis; D000637:Transaminases / Q000096:biosynthesis* / Q000302:isolation & purification
D000596:Amino Acids; D011994:Recombinant Proteins; D012545:Schiff Bases; D011732:Pyridoxal Phosphate; D000637:Transaminases; D001219:Aspartate Aminotransferases; C019798:branched-chain-amino-acid transaminase