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

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.

Expanding the Plant GSTome Through Directed Evolution: DNA Shuffling for the Generation of New Synthetic Enzymes With Engineered Catalytic and Binding Properties

“…Thus, RNA from stressed tissues (leaf, root, and shoot treated for 48 h) was reverse transcribed and the GST genes were amplified using PCRs and degenerate primers. The PCR amplicons of putative GST genes were cloned and the resulted recombinant plasmids were isolated, mixed, and used for in vitro recombination by DNA shuffling (Zhao and Arnold, 1997 ; Axarli et al, 2016 , 2017 ). Following in vitro recombination, a single PCR product was cloned into the pEXP5-CT/TOPO®TA plasmid.…”

“…Previous studies have established that in several tau class GSTs, although the H-site of neighboring subunits is remote, a reasonable communication between them exists. For example, in the case of a mutant form of Gm GST4-4 (Axarli et al, 2016 ) structural examination revealed that Lys104, which is located at the dimer interface, plays a key role in inter-subunit communication as well as in the cooperative allosteric kinetics observed with this enzyme.…”

“…Screening of the library and expression of the recombinant enzymes were carried out as described by Axarli et al ( 2016 ). Enzyme purification was carried out using affinity chromatography on S-hexyl-GSH-Agarose as previously described (Axarli et al, 2009a ).…”

Structure and Dynamics of FosA-Mediated Fosfomycin Resistance in Klebsiella pneumoniae and Escherichia coli

“…Although supernatant was combined with 250 nl of mother liquor (7% [vol/vol] ethylene glycol, 7% [wt/vol] polyethylene glycol 6000 [PEG 6000], 0.1 M HEPES [pH 6.95]) in sitting drops. For fosfomycin-bound FosA KP , protein was concentrated to 9 mg/ml, combined with 6 mM fosfomycin disodium salt (Sigma-Aldrich; purity as determined by thin-layer chromatography [TLC], Ͼ98%) and 6 mM MnCl 2 , and centrifuged (19,150 ϫ g for 5 min). One microliter of supernatant was combined in hanging drops with 1 l of mother liquor (0.22 M KBr, 20% [wt/vol] PEG 2000 monomethyl ether).…”

Differential Roles for VviGST1, VviGST3, and VviGST4 in Proanthocyanidin and Anthocyanin Transport in Vitis vinífera

“…SWISS-MODEL ( Biasini et al, 2014 ) was used to select 3D models crystallized with the closest sequence homology and also to construct comparative model structures. The model with the best homology to GST3, GST4, and TT19 was the crystalline structure of a Phi class GST from Populus tremula × P. tremuloide PttGSTF1 (PDB code 4RI6; Pégeot et al, 2014 ) and for GST1 was the structure of a Tau class GST from G. max GmGSTU10-10 (PDB code 5AGY; Axarli et al, 2016 ). The best models for GST proteins obtained by SWISS-MODEL were improved by molecular dynamic simulation and equilibration methods using Nano Molecular Dynamics (NAMD v. 2.10; Phillips et al, 2005 ), the Chemistry of Harvard Molecular Modeling (CHARMM27) force field ( Schlenkrich et al, 1996 ) and the TIP3P model for water ( Jorgensen et al, 1983 ).…”