The present study describes the isolation of a protein from Escherichia coli possessing kynurenine aminotransferase (KAT) activity and its identification as aspartate aminotransferase (AspAT). KAT catalyses the transamination of kynurenine and 3-hydroxykynurenine to kynurenic acid and xanthurenic acid respectively, and the enzyme activity can be easily detected in E. coli cells. Separation of the E. coli protein possessing KAT activity through various chromatographic steps led to the isolation of the enzyme. N-terminal sequencing of the purified protein determined its first 10 N-terminal amino acid residues, which were identical with those of the E. coli AspAT. Recombinant AspAT (R-AspAT), homologously expressed in an E. coli/pET22b expression system, was capable of catalysing the transamination of both l-kynurenine (Km = 3mM; Vmax = 7.9μmol·min−1·mg−1) and 3-hydroxy-dl-kynurenine (Km = 3.7mM; Vmax = 1.25μmol·min−1·mg−1) in the presence of pyruvate as an amino acceptor, and exhibited its maximum activity at temperatures between 50–60°C and at a pH of approx. 7.0. Like mammalian KATs, R-AspAT also displayed high glutamine transaminase K activity when l-phenylalanine was used as an amino donor (Km = 8mM; Vmax = 20.6μmol·min−1·mg−1). The exact match of the first ten N-terminal amino acid residues of the KAT-active protein with that of AspAT, in conjunction with the high KAT activity of R-AspAT, provides convincing evidence that the identity of the E. coli protein is AspAT.
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December 2001
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Research Article|
December 10 2001
Kynurenine aminotransferase and glutamine transaminase K of Escherichia coli: identity with aspartate aminotransferase
Qian HAN;
Qian HAN
Department of Pathobiology, University of Illinois at Champaign-Urbana, 2001 S. Lincoln Avenue, Urbana, IL 61802, U.S.A.
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Jianmin FANG;
Jianmin FANG
Department of Pathobiology, University of Illinois at Champaign-Urbana, 2001 S. Lincoln Avenue, Urbana, IL 61802, U.S.A.
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Jianyong LI
Jianyong LI
1
Department of Pathobiology, University of Illinois at Champaign-Urbana, 2001 S. Lincoln Avenue, Urbana, IL 61802, U.S.A.
1To whom correspondence should be addressed (e-mail jli2@uiuc.edu).
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Publisher: Portland Press Ltd
Received:
July 30 2001
Revision Received:
September 10 2001
Accepted:
October 16 2001
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2001
2001
Biochem J (2001) 360 (3): 617–623.
Article history
Received:
July 30 2001
Revision Received:
September 10 2001
Accepted:
October 16 2001
Citation
Qian HAN, Jianmin FANG, Jianyong LI; Kynurenine aminotransferase and glutamine transaminase K of Escherichia coli: identity with aspartate aminotransferase. Biochem J 15 December 2001; 360 (3): 617–623. doi: https://doi.org/10.1042/bj3600617
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