A library of Tau class GSTs (glutathione transferases) was constructed by DNA shuffling using the DNA encoding the Glycine max GSTs GmGSTU2-2, GmGSTU4-4 and GmGSTU10-10. The parental GSTs are >88% identical at the sequence level; however, their specificity varies towards different substrates. The DNA library contained chimaeric structures of alternated segments of the parental sequences and point mutations. Chimaeric GST sequences were expressed in Escherichia coli and their enzymatic activities towards CDNB (1-chloro-2,4-dinitrobenzene) and the herbicide fluorodifen (4-nitrophenyl α,α,α-trifluoro-2-nitro-p-tolyl ether) were determined. A chimaeric clone (Sh14) with enhanced CDNB- and fluorodifen-detoxifying activities, and unusual co-operative kinetics towards CDNB and fluorodifen, but not towards GSH, was identified. The structure of Sh14 was determined at 1.75 Å (1 Å=0.1 nm) resolution in complex with S-(p-nitrobenzyl)-glutathione. Analysis of the Sh14 structure showed that a W114C point mutation is responsible for the altered kinetic properties. This was confirmed by the kinetic properties of the Sh14 C114W mutant. It is suggested that the replacement of the bulky tryptophan residue by a smaller amino acid (cysteine) results in conformational changes of the active-site cavity, leading to enhanced catalytic activity of Sh14. Moreover, the structural changes allow the strengthening of the two salt bridges between Glu66 and Lys104 at the dimer interface that triggers an allosteric effect and the communication between the hydrophobic sites.
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Structure of the alpha1, beta1, gamma1 isoform of AMPK bound to a chlorinated analog of A769662 (‘Cl-A769662’). Alpha colored in purple, beta in blue, gamma in light pink. Cl-A769662 shown in yellow sticks and AMP bound to gamma shown in green sticks. Image kindly provided by Francis Rajamohan (Groton, U.S.A.). - PDF Icon PDF LinkFront Matter
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Research Article|
February 24 2016
Directed evolution of Tau class glutathione transferases reveals a site that regulates catalytic efficiency and masks co-operativity
Irine Axarli;
Irine Axarli
1
*Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece
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Abdi W. Muleta;
Abdi W. Muleta
1
†Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Biocity, Turku, FIN-20521, Finland
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Dimitrios Vlachakis;
Dimitrios Vlachakis
‡Bioinformatics and Medical Informatics Laboratory, Biomedical Research Foundation of the Academy of Athens, GR-11527 Athens, Greece
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Sophia Kossida;
Sophia Kossida
‡Bioinformatics and Medical Informatics Laboratory, Biomedical Research Foundation of the Academy of Athens, GR-11527 Athens, Greece
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Georgia Kotzia;
Georgia Kotzia
*Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece
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Anastasios Maltezos;
Anastasios Maltezos
*Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece
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Prathusha Dhavala;
Prathusha Dhavala
†Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Biocity, Turku, FIN-20521, Finland
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Anastassios C. Papageorgiou;
Anastassios C. Papageorgiou
†Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Biocity, Turku, FIN-20521, Finland
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Nikolaos E. Labrou
Nikolaos E. Labrou
2
*Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece
2To whom correspondence should be addressed (email lambrou@aua.gr).
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Publisher: Portland Press Ltd
Received:
August 24 2015
Revision Received:
November 20 2015
Accepted:
December 04 2015
Accepted Manuscript online:
December 04 2015
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2016 Authors; published by Portland Press Limited
2016
Biochem J (2016) 473 (5): 559–570.
Article history
Received:
August 24 2015
Revision Received:
November 20 2015
Accepted:
December 04 2015
Accepted Manuscript online:
December 04 2015
Citation
Irine Axarli, Abdi W. Muleta, Dimitrios Vlachakis, Sophia Kossida, Georgia Kotzia, Anastasios Maltezos, Prathusha Dhavala, Anastassios C. Papageorgiou, Nikolaos E. Labrou; Directed evolution of Tau class glutathione transferases reveals a site that regulates catalytic efficiency and masks co-operativity. Biochem J 1 March 2016; 473 (5): 559–570. doi: https://doi.org/10.1042/BJ20150930
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