Applied Microbiology and Biotechnology

, Volume 103, Issue 1, pp 251–263 | Cite as

Enhancement in the catalytic activity of Sulfolobus solfataricus P2 (+)-γ-lactamase by semi-rational design with the aid of a newly established high-throughput screening method

  • Shuaihua Gao
  • Yingxiu Lu
  • Yuanyuan Li
  • Rong Huang
  • Guojun ZhengEmail author
Biotechnologically relevant enzymes and proteins


(−)-γ-Lactam ((−)-2-azabicyclo[2.2.1]hept-5-en-3-one) has attracted increasing attention as the chiral intermediate of carbocyclic nucleosides most of which serve as pharmaceutical agents such as anti-HIV/HBV drugs abacavir and carbovir. So far, developing in vitro (+)-γ-lactamase-mediated biotransformation has been one of the most efficient approaches for the production of (−)-γ-lactam. In this study, the catalytic activity of the (+)-γ-lactamase from Sulfolobus solfataricus P2 was engineered by semi-rational design. Molecular docking and molecular dynamics simulation were carried out to target the key positions relevant to catalytic activity. Nine amino acid residues were selected for site saturation mutagenesis. To expedite the screening process, a sensitive colorimetric high-throughput screening method was established based on the Rimini test which was originally applied to distinguish primary amines from secondary amines. The screening process resulted in the achievement of several efficient mutants: V203N, V203Q, I336H, I336R, and Y388H. Synergy effects led to four final mutants (V203N/I336R, V203N/Y388H, I336R/Y388H, and V203N/I336R/Y388H) with enhanced enzyme activity after the combination of positive single mutants. The best mutant V203N/Y388H/I336R displayed a 21-fold higher enzyme efficiency (kcat/KM) compared to the wild-type enzyme. The result demonstrated that the biotransformation using the triple mutant as the catalyst reached > 49% conversion and > 99% enantiomeric excess at 80 °C after 2 h, which made it a good catalyst candidate to produce (−)-γ-lactam. The possible mechanism responsible for the improvement in the catalytic activity was explicated by analyzing the protein-ligand binding modes and interaction between the protein and the ligand.


High-throughput screening γ-Lactam (+)-γ-lactamase Molecular docking Site-saturation mutagenesis Semi-rational design 


Funding information

This work was supported by the National Natural Science Foundation of China (NSFC, Grant No. 21706005), National Great Science and Technology Projects (2018ZX09721001), and the Fundamental Research Funds for the Central Universities (No. ZY1713).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9428_MOESM1_ESM.pdf (497 kb)
ESM 1 (PDF 497 kb)


  1. Alzate-Morales JH, Contreras R, Soriano A, Tunon I, Silla E (2007) A computational study of the protein-ligand interactions in CDK2 inhibitors: using quantum mechanics/molecular mechanics interaction energy as a predictor of the biological activity. Biophys J 92(2):430–439. CrossRefPubMedGoogle Scholar
  2. Assaf Z, Eger E, Vitnik Z, Fabian WMF, Ribitsch D, Guebitz GM, Faber K, Hall M (2014) Identification and application of enantiocomplementary lactamases for vince lactam derivatives. Chemcatchem 6:2517–2521CrossRefGoogle Scholar
  3. Assaf Z, Faber K, Hall M (2016) Scope, limitations and classification of lactamases. J Biotechnol 235:11–23. CrossRefPubMedGoogle Scholar
  4. Baumgarten RL (1977) Sodium nitroprusside reagent. A classification test for aliphatic and aryl amines. J Chem Educ 54(3):189CrossRefGoogle Scholar
  5. Behrens GA, Hummel A, Padhi SK, Schätzle S, Bornscheuer UT (2011) Discovery and protein engineering of biocatalysts for organic synthesis. Adv Synth Catal 353(13):2191–2215CrossRefGoogle Scholar
  6. Berenguer J, Perez-Elias MJ, Bellon JM, Knobel H, Rivas-Gonzalez P, Gatell JM, Miguelez M, Hernandez-Quero J, Flores J, Soriano V, Santos I, Podzamczer D, Sala M, Camba M, Resino S, Spanish Abacavir L, Zidovudine Cohort Study G (2006) Effectiveness and safety of abacavir, lamivudine, and zidovudine in antiretroviral therapy-naive HIV-infected patients: results from a large multicenter observational cohort. J Acquir Immune Defic Syndr 41(2):154–159CrossRefGoogle Scholar
  7. Bommarius AS, Blum JK, Abrahamson MJ (2011) Status of protein engineering for biocatalysts: how to design an industrially useful biocatalyst. Curr Opin Chem Biol 15(2):194–200CrossRefGoogle Scholar
  8. Bornscheuer UT, Huisman GW, Kazlauskas RJ, Lutz S, Moore JC, Robins K (2012) Engineering the third wave of biocatalysis. Nature 485(7397):185–194CrossRefGoogle Scholar
  9. Boutureira O, Matheu MI, Diaz Y, Castillon S (2013) Advances in the enantioselective synthesis of carbocyclic nucleosides. Chem Soc Rev 42(12):5056–5072. CrossRefPubMedGoogle Scholar
  10. Chebrou H, Bigey F, Arnaud A, Galzy P (1996) Study of the amidase signature group. Biochim Biophys Acta 1298(2):285–293CrossRefGoogle Scholar
  11. Cilia E, Fabbri A, Uriani M, Scialdone GG, Ammendola S (2005) The signature amidase from Sulfolobus solfataricus belongs to the CX3C subgroup of enzymes cleaving both amides and nitriles. Ser195 and Cys145 are predicted to be the active site nucleophiles. FEBS J 272(18):4716–4724. CrossRefPubMedGoogle Scholar
  12. Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: a sequence logo generator. Genome Res 14:1188–1190CrossRefGoogle Scholar
  13. De Camp WH (1993) Chiral drugs: the FDA perspective on manufacturing and control. J Pharm Biomed Anal 11(11–12):1167–1172CrossRefGoogle Scholar
  14. Dessailly BH, Lensink MF, Wodak SJ (2007) Relating destabilizing regions to known functional sites in proteins. BMC Bioinformatics 8:141. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Devane WA, Breuer A, Sheskin T, Järbe TU, Eisen MS, Mechoulam R (1992) A novel probe for the cannabinoid receptor. J Med Chem 35(11):2065–2069CrossRefGoogle Scholar
  16. Elisa C, Sergio A (2010) Identification of the amino acid residues affecting the catalytic pocket of the Sulfolobus solfataricus signature amidase. Protein Pept Lett 17(2):146–150CrossRefGoogle Scholar
  17. Gao SH, Zhu SZ, Huang R, Lu YX, Zheng GJ (2015) Efficient synthesis of the intermediate of abacavir and carbovir using a novel (+)-gamma-lactamase as a catalyst. Bioorg Med Chem Lett 25(18):3878–3881. CrossRefPubMedGoogle Scholar
  18. Gao S, Huang R, Zhu S, Li H, Zheng G (2016) Identification and characterization of a novel (+)-gamma-lactamase from Microbacterium hydrocarbonoxydans. Appl Microbiol Biotechnol 100(22):9543–9553. CrossRefPubMedGoogle Scholar
  19. Gao S, Zhou Y, Zhang W, Wang W, Yu Y, Mu Y, Wang H, Gong X, Zheng G, Feng Y (2017a) Structural insights into the γ-lactamase activity and substrate enantioselectivity of an isochorismatase-like hydrolase from Microbacterium hydrocarbonoxydans. Sci Rep 7:44542CrossRefGoogle Scholar
  20. Gao S, Zhu S, Huang R, Li H, Wang H, Zheng G (2017b) Engineering the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans for the kinetic resolution of Vince lactam. Appl Environ Microbiol 12(10):e0186654Google Scholar
  21. Giordano C, Ammendola S (2008) Characterization of mutants of Sulfolobus solfataricus signature amidase able to hydrolyse R-ketoprofen amide. Protein Pept Lett 15(6):617–623CrossRefGoogle Scholar
  22. Griffiths GJ, Previdoli FE (1993) Diels-Alder reaction of methanesulfonyl cyanide with cyclopentadiene. Industrial synthesis of 2-azabicyclo[2.2.1]hept-5-en-3-one. J Org Chem 58(22):6129–6131CrossRefGoogle Scholar
  23. He YC, Ma CL, Xu JH, Zhou L (2011) A high-throughput screening strategy for nitrile-hydrolyzing enzymes based on ferric hydroxamate spectrophotometry. Appl Microbiol Biotechnol 89(3):817–823. CrossRefPubMedGoogle Scholar
  24. Hogrefe HH, Cline J, Youngblood GL, Allen RM (2002) Creating randomized amino acid libraries with the QuikChange multi site-directed mutagenesis kit. Biotechniques 33(5):1164–1165CrossRefGoogle Scholar
  25. Holt-Tiffin KE (2009) (+)- and (−)-2-Azabicyclo [2.2.1]hept-5-en-3-one extremely useful synthons. Chim Oggi 27:23–25Google Scholar
  26. Homeyer N, Gohlke H (2012) Free energy calculations by the molecular mechanics Poisson-Boltzmann surface area method. Mol Inf 31(2):114–122. CrossRefGoogle Scholar
  27. Kagan HB, Phat DT (1972) Asymmetric catalytic reduction with transition metal complexes. I. Catalytic system of rhodium(I) with (−)-2,3–0-isopropylidene-2,3-dihydroxy-1,4-bis(diphenylphosphino)butane, a new chiral diphosphine. Jamchemsoc (18):6429–6433Google Scholar
  28. Ku HK, Lim HM, Oh KH, Yang HJ, Jeong JS, Kim SK (2013) Interpretation of protein quantitation using the Bradford assay: comparison with two calculation models. Anal Biochem 434(1):178–180. CrossRefPubMedGoogle Scholar
  29. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. CrossRefGoogle Scholar
  30. Laidler KJ (1984) The development of the Arrhenius equation. J Chem Educ 61(6):494CrossRefGoogle Scholar
  31. Lutz S (2010) Beyond directed evolution—semi-rational protein engineering and design. Curr Opin Biotechnol 21(6):734–743CrossRefGoogle Scholar
  32. Mayerhöfer TG, Mutschke H, Popp J (2016) Employing theories far beyond their limits—the case of the (Boguer-) Beer–Lambert law. ChemPhysChem 17(13):1948–1955CrossRefGoogle Scholar
  33. Nei M, Kumar S (2000) Molecular evolution and phylogenetics. Oxford University PressGoogle Scholar
  34. Ohtaki A, Murata K, Sato Y, Noguchi K, Miyatake H, Dohmae N, Yamada K, Yohda M, Odaka M (2010) Structure and characterization of amidase from Rhodococcus sp. N-771: insight into the molecular mechanism of substrate recognition. Biochim Biophys Acta 1804(1):184–192. CrossRefPubMedGoogle Scholar
  35. Patricelli MP, Cravatt BF (2000) Clarifying the catalytic roles of conserved residues in the amidase signature family. J Biol Chem 275(25):19177–19184. CrossRefPubMedGoogle Scholar
  36. Patricelli MP, Lovato MA, Cravatt BF (1999) Chemical and mutagenic investigations of fatty acid amide hydrolase: evidence for a family of serine hydrolases with distinct catalytic properties. Biochemistry 38(31):9804–9812. CrossRefPubMedGoogle Scholar
  37. Reetz MT (2011) Laboratory evolution of stereoselective enzymes: a prolific source of catalysts for asymmetric reactions. Angew Chem Int Ed Engl 50(1):138–174. CrossRefPubMedGoogle Scholar
  38. Reetz MT, Bocola M, Carballeira JD, Zha D, Vogel A (2005) Expanding the range of substrate acceptance of enzymes: combinatorial active-site saturation test. Angew Chem Int Ed Engl 44(27):4192–4196. CrossRefPubMedGoogle Scholar
  39. Reetz MT, Carballeira JD, Vogel A (2006) Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability. Angew Chem Int Edi 45(46):7745–7751CrossRefGoogle Scholar
  40. Reetz MT, Bocola M, Wang LW, Sanchis J, Cronin A, Arand M, Zou J, Archelas A, Bottalla AL, Naworyta A (2009) Directed evolution of an enantioselective epoxide hydrolase: uncovering the source of enantioselectivity at each evolutionary stage. J Am Chem Soc 131(21):7334–7343CrossRefGoogle Scholar
  41. Ren L, Zhu S, Shi Y, Gao S, Zheng G (2015) Enantioselective resolution of gamma-lactam by a novel thermostable type II (+)-gamma-lactamase from the hyperthermophilic archaeon Aeropyrum pernix. Appl Biochem Biotechnol 176(1):170–184. CrossRefPubMedGoogle Scholar
  42. Singh R, Vince R (2012) 2-Azabicyclo[2.2.1]hept-5-en-3-one: chemical profile of a versatile synthetic building block and its impact on the development of therapeutics. Chem Rev 112(8):4642–4686. CrossRefPubMedGoogle Scholar
  43. Taylor SJC, Sutherland AG, Lee CWR, Thomas S, Roberts SM, Evans C (1990) Chemoenzymatic synthesis of (−)-carbovir utilizing a whole cell catalyzed resolution of 2-azabicyclo[2.2.1]hept-5-en-3-one. J Chem Soc Chem Commun 16:1120–1121CrossRefGoogle Scholar
  44. Taylor SJC, Mccague R, Wisdom R, Lee C, Dickson K, Ruecroft G, Obrien F, Littlechild J, Bevan J, Roberts SM, Evans CT (1993) Development of the biocatalytic resolution of 2-azabicyclo[2.2.1]hept-5-en-3-one as an entry to single-enantiomer carbocyclic nucleosides. Tetrahedron-Asymmetry 4:1117–1128CrossRefGoogle Scholar
  45. Taylor SJ, Brown RC, Keene PA, Taylor IN (1999) Novel screening methods—the key to cloning commercially successful biocatalysts. Bioorg Med Chem 7(10):2163–2168CrossRefGoogle Scholar
  46. Toogood HS, Brown RC, Line K, Keene PA, Taylor SJC, McCague R, Littlechild JA (2004) The use of a thermostable signature amidase in the resolution of the bicyclic synthon (rac)-gamma-lactam. Tetrahedron 60(3):711–716. CrossRefGoogle Scholar
  47. Wang J, Zhu Y, Zhao G, Zhu J, Wu S (2015a) Characterization of a recombinant (+)-gamma-lactamase from Microbacterium hydrocarbonoxydans which provides evidence that two enantiocomplementary gamma-lactamases are in the strain. Appl Microbiol Biotechnol 99(7):3069–3080. CrossRefPubMedGoogle Scholar
  48. Wang JJ, Zhu JG, Wu S (2015b) Immobilization on macroporous resin makes E-coli RutB a robust catalyst for production of (−) Vince lactam. Appl Microbiol Biotechnol 99(11):4691–4700. CrossRefPubMedGoogle Scholar
  49. Wu X, Tian Z, Jiang X, Zhang Q, Wang L (2018) Enhancement in catalytic activity of Aspergillus niger XynB by selective site-directed mutagenesis of active site amino acids. Appl Microbiol Biotechnol 102(1):249–260. CrossRefPubMedGoogle Scholar
  50. Xie Y, An J, Yang G, Wu G, Zhang Y, Cui L, Feng Y (2014) Enhanced enzyme kinetic stability by increasing rigidity within the active site. J Biol Chem 289(11):7994–8006CrossRefGoogle Scholar
  51. Xue TY, Xu GC, Han RZ, Ni Y (2015) Soluble expression of (+)-gamma-lactamase in Bacillus subtilis for the enantioselective preparation of abacavir precursor. Appl Biochem Biotechnol 176(6):1687–1699. CrossRefPubMedGoogle Scholar
  52. Yuen GJ, Weller S, Pakes GE (2008) A review of the pharmacokinetics of abacavir. Clin Pharmacokinet 47(6):351–371. CrossRefPubMedGoogle Scholar
  53. Zhu S, Gong C, Song D, Gao S, Zheng G (2012) Discovery of a novel (+)-gamma-lactamase from Bradyrhizobium japonicum USDA 6 by rational genome mining. Appl Environ Microbiol 78(20):7492–7495. CrossRefPubMedPubMedCentralGoogle Scholar
  54. Zhu S, Huang R, Gao S, Li X, Zheng G (2016) Discovery and characterization of a second extremely thermostable (+)-gamma-lactamase from Sulfolobus solfataricus P2. J Biosci Bioeng 121(5):484–490. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Chemical Resources EngineeringBeijing University of Chemical TechnologyBeijingPeople’s Republic of China
  2. 2.Department of ChemistryUniversity of CaliforniaBerkeleyUSA
  3. 3.California Institute for Quantitative BiosciencesUniversity of CaliforniaBerkeleyUSA

Personalised recommendations