5AGY

CRYSTAL STRUCTURE OF A TAU CLASS GST MUTANT FROM GLYCINE

Summary for 5AGY

• Entry DOI: 10.2210/pdb5agy/pdb
• Descriptor: GLUTATHIONE S-TRANSFERASE, 4-NITROPHENYL METHANETHIOL, S-(P-NITROBENZYL)GLUTATHIONE, ... (5 entities in total)
• Functional Keywords: transferase, binding site, catalytic domain, enzyme, induction, detoxification, herbicides, kinetics, site-directed mutagenesis, soy beans, glutathione transferase, protein stability, catalytic mechanism, xenobiotic binding, allosterism
• Biological source: GLYCINE MAX (SOYBEAN)
• Total number of polymer chains: 2
• Total formula weight: 52696.64

Authors

Axarli, I.,Muleta, A.W.,Vlachakis, D.,Kossida, S.,Kotzia, G.,Dhavala, P.,Papageorgiou, A.C.,Labrou, N.E. (deposition date: 2015-02-04, release date: 2015-12-16, Last modification date: 2024-01-10)

Primary citation

Axarli, I.,Muleta, A.W.,Vlachakis, D.,Kossida, S.,Kotzia, G.,Maltezos, A.,Dhavala, P.,Papageorgiou, A.C.,Labrou, N.E.
Directed Evolution of Tau Class Glutathione Transferases Reveals a Site that Regulates Catalytic Efficiency and Masks Cooperativity.
Biochem.J., 473:559-, 2016

PubMed 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.

PubMed: 26637269
DOI: 10.1042/BJ20150930

Experimental method

X-RAY DIFFRACTION (1.75 Å)