title: 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.
authors.affiliation: Department of Microbiology, University of Delhi, South Campus, New Delhi, 110021, India.
authors.forename: Rinky
authors.initials: R
authors.lastname: Rajput
authors.forename: Ved Vrat
authors.initials: VV
authors.lastname: Verma
authors.forename: Vishal
authors.initials: V
authors.lastname: Chaudhary
authors.forename: Rani
authors.initials: R
authors.lastname: Gupta
mesh_terms: D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013822:Thermoplasma / Q000201:enzymology / Q000235:genetics; D019603:Thermoplasmales / Q000201:enzymology* / Q000235:genetics*; D005723:gamma-Glutamyltransferase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D011994:Recombinant Proteins; D005723:gamma-Glutamyltransferase