Branched-Chain Amino Acid Aminotransferase of Escherichia coli: Overproduction and Properties1

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.

The specificity and kinetic mechanism of branched‐chain amino acid aminotransferase from Escherichia coli studied with a new improved coupled assay procedure and the enzyme's potential for biocatalysis

“…This has been possible because of the efficiency of the coupling system employing 2-hydroxyglutarate dehydrogenase to monitor production of 2-oxoglutarate [6]. The previous detailed kinetic study of the enzyme [8] was for the opposite direction of reaction.…”

“…Surprisingly, the K m value of BCAT for 2-oxovalerate (product L-norvaline) was 7.5-fold higher than that for 2-oxoisocaproate (0.6 mM versus 0.08 mM), while k cat values were similar. In the case of pyruvate, it is noteworthy that Inoue et al [8] reported that this oxoacid cannot serve as an amino acceptor for BCAT. In fact, although its K m is very high (~57 mM), the k cat value is only 5-fold lower than that for the best substrates.…”

“…However, significant product inhibition of GDH was observed at low glutamate concentration and this is inescapable since glutamate serves as both substrate for BCAT and product for GDH [6]. Further characterization was carried out in 1988 by Inoue et al [8], who determined detailed kinetic parameters in the direction of glutamate formation by the method of Velick and Vavra [9], but only for biological amino acids. They reported that E. coli BCAT shows activity towards various aliphatic amino acids, of which L-isoleucine was the best substrate, showing the highest catalytic efficiency value (k cat /K m ).…”

Characterization and Role of the Branched-Chain Aminotransferase (BcaT) Isolated from Lactococcus lactis subsp. cremoris NCDO 763

“…While 25 gene sequences are available in gene banks, only a few bacterial BcaTs have been well characterized (10,27,29,32,37,48,49). All of these enzymes belong to class IV of the pyridoxal phosphate-dependent aminotransferases (3,24).…”

Isolation of a Mutant Auxotrophic forL-Alanine and Identification of Three Major Aminotransferases That SynthesizeL-Alanine inEscherichia coli

“…[1][2][3][4] In view of L-alanine formation, AvtA is the only genetically characterized aminotransferase that aminates pyruvate to produce L-alanine using valine as the amino donor, 5,6) but an AvtA-deficient mutant has no nutritional requirement. 7) On one hand, it is known that purified AspC and IlvE do not catalyze transamination between L-alanine and -ketoglutarate, 8,9) and that TyrB uses L-alanine as the amino donor only negligibly as compared to its favored substrates, the aromatic amino acids. 10) Hence, it has been assumed that Lalanine is synthesized by at least one unidentified aminotransferase in conjunction with AvtA.…”