A hydrolytic γ-glutamyl transpeptidase from thermo-acidophilic archaeon Picrophilus torridus: binding pocket mutagenesis and transpeptidation

Abstract

γ-Glutamyl transpeptidase of a thermo-acidophilic archaeon Picrophilus torridus was cloned and expressed using E. coli Rosetta-pET 51b(+) expression system. The enzyme was expressed at 37 °C/200 rpm with γ-GT production of 1.99 U/mg protein after 3 h of IPTG induction. It was improved nearby 10-fold corresponding to 18.92 U/mg protein in the presence of 2 % hexadecane. The enzyme was purified by Ni(2+)-NTA with a purification fold of 3.6 and recovery of 61 %. It was synthesized as a precursor heterodimeric protein of 47 kDa with two subunits of 30 kDa and 17 kDa, respectively, as revealed by SDS-PAGE and western blot. The enzyme possesses hydrolase activity with optima at pH 7.0 and 55 °C. It was thermostable with a t (1/2) of 1 h at 50 °C and 30 min at 60 °C, and retained 100 % activity at 45 °C even after 24 h. It was inhibited by azaserine and DON and PMSF. Ptγ-GT shared 37 % sequence identity and 53 % homology with an extremophile γ-GT from Thermoplasma acidophilum. Functional residues identified by in silico approaches were further validated by site-directed mutagenesis where Tyr327 mutated by Asn327 introduced significant transpeptidase activity.

Bacterial Gamma-Glutamyl Transpeptidase, an Emerging Biocatalyst: Insights Into Structure–Function Relationship and Its Biotechnological Applications

“…Substitution of three aromatic residues with threonine (W385T), tyrosine (F417Y), and alanine (W525A), present at the corresponding positions in EcGGT, resulted in enhanced transpeptidase activity for all three mutants, while a 5-14% decrease in hydrolytic activity was observed. Likewise, in another report from Picrophilus torridus GGT (PtGGT), the replacement of an aromatic residue tyrosine at position 327 by asparagine (corresponding residue in EcGGT) introduced significant transpeptidase activity in PtGGT, while the native enzyme exhibited only hydrolytic activity (Rajput et al, 2013). Comparative docking of the acceptor ligand Gly-Gly in the structural model of PtGGT and its Y327N mutant identified some residues with suggested importance in acceptor recognition and binding.…”

Development of transgenic Brassica juncea lines for reduced seed sinapine content by perturbing phenylpropanoid pathway genes

“…The in-silico structural analysis and molecular docking were performed following Rajput et al (2013) [ 16 ]. In brief, the predicted amino acid sequences of SGT and SCT enzymes were subjected to PSI-Blast search against PDB database to select the suitable template for homology modelling.…”

“Phylogenetic and evolutionary analysis of functional divergence among Gamma glutamyl transpeptidase (GGT) subfamilies”

“…However, the details of its mode of binding and inhibition are not known in detail yet. Other than its medical significance, GGT also happens to be a biotechnologically useful enzyme [ 20 , 29 – 34 ]. The three dimensional structures of GGTs from varied organisms, including human GGT1, E. coli , H. pylori, B. subtilis , B. licheniformis , B. halodurans and T. acidophilum , with and without substrate analogues, small ions or inhibitors bound to the protein, are now available (Additional file 2 ) [ 17 , 29 – 31 , 35 – 37 ].…”