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Biochem J. 2016 Mar 1;473(5):559-70. doi: 10.1042/BJ20150930. Epub 2015 Dec 4.

Directed evolution of Tau class glutathione transferases reveals a site that regulates catalytic efficiency and masks co-operativity.

Author information

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.
2
Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Biocity, Turku, FIN-20521, Finland.
3
Bioinformatics and Medical Informatics Laboratory, Biomedical Research Foundation of the Academy of Athens, GR-11527 Athens, Greece.
4
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 lambrou@aua.gr.

Abstract

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 Glu(66) and Lys(104) at the dimer interface that triggers an allosteric effect and the communication between the hydrophobic sites.

KEYWORDS:

allosteric regulation; enzyme catalysis; enzyme kinetics; enzyme structure; glutathione transferase; xenobiotic

PMID:
26637269
DOI:
10.1042/BJ20150930
[Indexed for MEDLINE]

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