A hydrolytic γ-glutamyl transpeptidase from thermo-acidophilic archaeon Picrophilus torridus: binding pocket mutagenesis and transpeptidation
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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 Ni2+-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.
KeywordsPicrophilus torridus Thermo-acidophile γ-Glutamyl transpeptidase Expression PET 51b(+) Site-directed mutagenesis
Financial assistance from DU/DST-PURSE grant and Misc. R & D Grant, University of Delhi is acknowledged.
- Castellano I, Merlino A (2012) γ-glutamyltranspeptidases: sequence, structure, biochemical properties,and biotechnological applications. Cell Mol Life Sci. doi: 10.1007/s00018-012-0988-3
- Castellano I, Salle AD, Merlino A, Rossi M, La Cara F (2011) Gene cloning and protein expression of γ-glutamyltranspeptidases from Thermus thermophilus and Deinococcus radiodurans: comparison of molecular and structural properties with mesophilic counterparts. Extremophiles 15:259–270PubMedCrossRefGoogle Scholar
- Cavicchioli R (2007) Archaea: molecular and cellular biology. ASM Press, Washington DCGoogle Scholar
- Eswar N, Webb B, Marti-Renom MA, Madhusudhan MS, Eramian D, Shen MY, Pieper U, Sali A (2006) Comparative protein structure modelling with MODELLER. Current protocols in bioinformatics. Wiley 15:561–5630Google Scholar
- Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins RM, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy Server. The proteomics protocols handbook, pp 571–607Google Scholar
- Kim SM, Park JT, Kim YW, Lee HS, Nyawira R, Shin HS, Park CS, Yoo SH, Kim YR, Moon TW, Park KH (2004) Properties of a novel thermostable glucomylase from the hyperthermophilic, archaeon Sulfolobus solfataricus in relation to starch processing. Appl Environ Microbiol 70:3933–3940PubMedCrossRefGoogle Scholar
- McGuffin LJ, Bryson K, Jones DT (2000) The PSIPRED protein structure prediction server. Bioinformatics Appl Note 16:405–406Google Scholar
- Takami H, Nakasone K, Takaki Y, Maeno G, Sasaki R, Masui N, Fuji F, Hirama C, Nakamura Y, Ogasawara N, Kuhara S, Horikoshi K (2000) Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acids Res 28:4317–4331PubMedCrossRefGoogle Scholar
- van der Spoel D, van Druner R, Berendsen HJC (1994) GROningen MAchine for Chemical Simulation. Department of Biophysical Chemistry, BIOSON Research Institute, Groningen Google Scholar