, Volume 17, Issue 1, pp 29–41 | Cite as

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

  • Rinky Rajput
  • Ved Vrat Verma
  • Vishal Chaudhary
  • Rani GuptaEmail author
Original Paper


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.


Picrophilus 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.


  1. Abe K, Ito Y, Ohmachi T, Asada Y (1995) Purification and properties of two isozymes of gamma glutamyltranspeptidase from Bacillus subtilis TAM-4. Biosci Biotechnol Biochem 61:1621–1625CrossRefGoogle Scholar
  2. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  3. Angelov A, Futterer O, Valerius O, Braus GH, Liebl W (2005) Properties of the recombinant glucose/galactose dehydrogenase from the extreme thermoacidophile, Picrophilus torridus. FEBS J 27:1054–1062CrossRefGoogle Scholar
  4. Angelov A, Putyrski M, Liebl W (2006) Molecular and biochemical characterization of α-glucosidase and α-mannosidase and their clustered genes from the thermoacidophilic archaeon Picrophilus torridus. J Bacteriol 188:7123–7131PubMedCrossRefGoogle Scholar
  5. Boanca G, Sand A, Barycki JJ (2006) Uncoupling the enzymatic and autoprocessing activities of Helicobacter pylori gamma-glutamyltranspeptidase. J Biol Chem 281:9029–19037CrossRefGoogle Scholar
  6. Boanca G, Sand A, Okada T, Suzuki H, Kumagai H, Fukuyama K, Barycki JJ (2007) Autoprocessing of Helicobacter pylori gamma-glutamyltranspeptidase leads to the formation of a threonine–threonine catalytic dyad. J Biol Chem 282:534–541PubMedCrossRefGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  8. Burg BVD (2003) Extremophiles as a source for novel enzymes. Curr Opin Microbiol 6:213–218PubMedCrossRefGoogle Scholar
  9. Canutescu AA, Shelenkov AA, Dunbrack RL (2003) A graph theory algorithm for protein side-chain prediction. Protein Sci 12:2001–2014PubMedCrossRefGoogle Scholar
  10. 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
  11. Castellano I, Merlino A, Rossi M, La Cara F (2010) Biochemical and structural properties of gamma-glutamyl transpeptidase from Geobacillus thermodenitrificans: an enzyme specialized in hydrolase activity. Biochimie 92:464–474PubMedCrossRefGoogle Scholar
  12. 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
  13. Cavicchioli R (2007) Archaea: molecular and cellular biology. ASM Press, Washington DCGoogle Scholar
  14. De Vendittis E, Castellano I, Cotugno R, Ruocco MR, Raimo G, Masullo M (2008) Adaptation of model proteins from cold to hot environments involves continuous and small adjustments of average parameters related to amino acid composition. J Theor Biol 250:156–171PubMedCrossRefGoogle Scholar
  15. Duhovny DS, Inbar Y, Nussinov R, Wolfson HJ (2005) PatchDock and SymmDock: servers for rigid and symmetric docking. Nucleic Acids Res 33:363–367CrossRefGoogle Scholar
  16. Dundas J, Ouyang Z, Tseng J, Binkowski A, Turpaz Y, Liang J (2006) CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. Nucleic Acids Res 34:116–118CrossRefGoogle Scholar
  17. 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
  18. Futterer O, Angelov A, Liesegang H, Gottschalk G, Schleper C, Schepers B, Dock C, Antranikian G, Liebl W (2004) Genome sequence of Picrophilus torridus and its implications for life around pH 0. Proc Natl Acad Sci USA 101:9091–9096PubMedCrossRefGoogle Scholar
  19. 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
  20. Giridhar R, Srivastava AK (2000) Productivity enhancement in l-sorbose fermentation using oxygen vector. Enzyme Microb Technol 27:537–541PubMedCrossRefGoogle Scholar
  21. Haney PJ, Badger JH, Buldak GL, Reich CI, Woese CR, Olsen GJ (1999) Thermal adaptation analyzed by comparison of protein sequences from mesophilic and extremely thermophilic Methanococcus species. Proc Natl Acad Sci USA 96:3578–3583PubMedCrossRefGoogle Scholar
  22. Hooft RW, Vriend R, Sander C, Abola EE (1996) Errors in protein structures. Nature 381:272PubMedCrossRefGoogle Scholar
  23. 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
  24. Kinlough CL, Poland PA, Bruns JB, Hughey RP (2005) Gamma-glutamyltranspeptidase: disulfide bridges, propeptide cleavage, and activation in the endoplasmic reticulum. Methods Enzymol 401:426–449PubMedCrossRefGoogle Scholar
  25. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948PubMedCrossRefGoogle Scholar
  26. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereo-chemical quality of protein structure. J Appl Crystallogr 26:283–291CrossRefGoogle Scholar
  27. Lee DH, Blomhoff R, Jacobs DR (2004) Is serum gamma-glutamyltransferase a marker of oxidative stress? Free Radic Res 38:535–539PubMedCrossRefGoogle Scholar
  28. Lin LL, Chou PR, Hua YW, Hsu WH (2006) Overexpression, one-step purification, and biochemical characterization of a recombinant gamma-glutamyltranspeptidase from Bacillus licheniformis. Appl Microbiol Biotechnol 73:103–112PubMedCrossRefGoogle Scholar
  29. McGuffin LJ, Bryson K, Jones DT (2000) The PSIPRED protein structure prediction server. Bioinformatics Appl Note 16:405–406Google Scholar
  30. Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, Olson AJ (1998) Automated docking using a Lamarckian genetic algorithm and and empirical binding free energy function. J Comput Chem 19:1639–1662CrossRefGoogle Scholar
  31. Murty NAR, Tiwary E, Sharma R, Nair N, Gupta R (2011) Gamma-glutamyl transpeptidase from Bacillus pumilus KS 12: decoupling autoprocessing from catalysis and molecular characterization of N-terminal region. Enzyme Microb Technol 50:159–164PubMedCrossRefGoogle Scholar
  32. Ohlsson BG, Westrom BR, Karlsson BW (1986) Enzymoblotting: a method for localizing proteinases and their zymogens using para-nitroanilide substrates after agarose gel electrophoresis and transfer to nitrocellulose. Anal Biochem 152:239–244PubMedCrossRefGoogle Scholar
  33. Okada T, Suzuki H, Wada K, Kumagai H, Fukuyama K (2006) Crystal structures of gamma-glutamyltranspeptidase from Escherichia coli, a key enzyme in glutathione metabolism, and its reaction intermediate. Proc Natl Acad Sci USA 103:6471–6476PubMedCrossRefGoogle Scholar
  34. Pompella A, Corti A, Paolicchi A, Giommarelli C, Zunino F (2007) Gamma-glutamyltransferase, redox regulation and cancer drug resistance. Curr Opin Pharmacol 7:360–366PubMedCrossRefGoogle Scholar
  35. Reher M, Fuhrer T, Bott M, Schonheit P (2010) The non-phosphorylative Entner-Doudoroff Pathway in the thermoacidophilic euryarchaeon Picrophilus torridus involves a novel 2-keto-3-deoxygluconate-specific aldolase. J Bacteriol 192:964–974PubMedCrossRefGoogle Scholar
  36. Ruepp A, Graml W, Santos-Martinez MLS, Koretke KK, Volker C, Mewes HW, Frishman D, Stocker S, Lupas AN, Baumeister W (2000) The genome sequence of the thermo-acidophilic scavenger Thermoplasma acidophilum. Nature 407:508PubMedCrossRefGoogle Scholar
  37. Ruppert J, Welch W, Jain AN (1997) Automatic identification and representation of protein binding sites for molecular docking. Protein Sci 6:524–533PubMedCrossRefGoogle Scholar
  38. Schepers B, Theimann V, Antranikian G (2006) Characterization of a novel glucoamylase from the thermoacidophilic archaeon Picrophilus torridus heterologously expressed in E. coli. Eng Life Sci 6:311–317CrossRefGoogle Scholar
  39. Seeliger D, De Groot BL (2010) Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J Comput Aided Mol Des 24:417–422PubMedCrossRefGoogle Scholar
  40. Shuai Y, Zhang T, Mu W, Jiang B (2011) Purification and characterization of gamma-glutamyltranspeptidase from Bacillus subtilis SK11.004. J Agric Food Chem 59:6233–6238PubMedCrossRefGoogle Scholar
  41. Suzuki H, Kumagai H (2002) Autocatalytic processing of gamma-glutamyltranspeptidase. J Biol Chem 277:43536–43543PubMedCrossRefGoogle Scholar
  42. Suzuki H, Kumagai H, Tochikura T (1986) Gamma-glutamyltranspeptidase from Escherichia coli K-12: purification and properties. J Bacteriol 168:1325–1331PubMedGoogle Scholar
  43. Suzuki H, Yamada C, Kato K (2007) Gamma-glutamyl compounds and their enzymatic production using bacterial gamma-glutamyltranspeptidase. Amino Acids 32:333–340PubMedCrossRefGoogle Scholar
  44. Szilagyi A, Zavodsky P (2000) Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey. Struct Fold Des 8:493–504CrossRefGoogle Scholar
  45. Takagi M, Tamaki H, Miyamoto Y, Leonardi R, Hanada S, Jackowski S, Chohnan S (2010) Pantothenate kinase from the thermoacidophilic archaeon Picrophilus torridus. J Bacteriol 192:233–241PubMedCrossRefGoogle Scholar
  46. 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
  47. Tate SS, Meister A (1981) Gamma-glutamyl transpeptidase: catalytic, structural and functional aspects. Mol Cell Biochem 39:357–368PubMedCrossRefGoogle Scholar
  48. Tate SS, Meister A (1985) Gamma-Glutamyl transpeptidase from kidney. Methods Enzymol 113:400–419PubMedCrossRefGoogle Scholar
  49. 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
  50. Wang Q, Yao Z, Xun Z, Xu X, Xu H, Wei P (2008) Properties and catalytic mechanism of γ-glutamyltranspeptidase from Bacillus subtilis NX-2. Frontiers of Chem Engineer in China 2:456–461CrossRefGoogle Scholar
  51. Willard L, Ranjan A, Zhang H, Monzavi H, Boyko RF, Sykes BD, Wishart DS (2003) VADAR: a web server for quantitative evaluation of protein structure quality. Nucleic Acids Res 31:3316–3319PubMedCrossRefGoogle Scholar

Copyright information

© Springer Japan 2012

Authors and Affiliations

  • Rinky Rajput
    • 1
  • Ved Vrat Verma
    • 1
  • Vishal Chaudhary
    • 1
  • Rani Gupta
    • 1
    Email author
  1. 1.Department of MicrobiologyUniversity of DelhiNew DelhiIndia

Personalised recommendations