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Characterization and mechanism insight of accelerated catalytic promiscuity of Sulfolobus tokodaii (ST0779) peptidase for aldol addition reaction

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Abstract

A novel peptidase from thermophilic archaea Sulfolobus tokodaii (ST0779) is examined for its catalytic promiscuity of aldol addition, which shows comparable activity as porcine pancreatic lipase (PPL, one of the best enzymes identified for biocatalytic aldol addition) at 30 °C but much accelerated activity at elevated temperature. The molecular catalytic efficiency kcat/Km (M−1 s−1) of this thermostable enzyme at 55 °C adds up to 140 times higher than that of PPL at its optimum temperature 37 °C. The fluorescence quenching analysis depicts that the binding constants of PPL are significantly higher than those of ST0779, and their numbers of binding sites show opposite temperature dependency. Thermodynamic parameters estimated by fluorescence quenching analysis unveil distinctly different substrate-binding modes between PPL and ST0779: the governing binding interaction between PPL and substrates is hydrophobic force, while the dominating substrate-binding forces for ST0779 are van der Waals and H-bonds interactions. A reasonable mechanism for ST0779-catalyzed aldol reaction is proposed based on kinetic study, spectroscopic analysis, and molecular stereostructure simulation. This work represents a successful example to identify a new enzyme for catalytic promiscuity, which demonstrates a huge potential to discover and exploit novel biocatalyst from thermophile microorganism sources.

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References

  • Bartlam M, Wang G, Yang H, Gao RJ, Zhao X, Xie GQ, Cao SG, Feng Y, Rao Z (2004) Crystal structure of an acylpeptide hydrolase/esterase from Aeropyrum pernix K1. Structure 12(8):1481–1488

    Article  CAS  PubMed  Google Scholar 

  • Branneby C, Carlqvist P, Hult K, Brinck T, Berglund P (2004) Aldol Additions with Mutant Lipase: Analysis by Experiments and Theoretical Calculations. J Mol Catal B Enzym 31(4):123–128

    Article  CAS  Google Scholar 

  • Busto E, Gotor-Fernández V, Gotor V (2010) Hydrolases: catalytically promiscuous enzymes for non-conventional reactions in organic synthesis.Chem. Soc Rev 39(11):4504–4523

    Article  CAS  Google Scholar 

  • Daudé D, Champion E, Morel S, Guieysse D, Remaud-Siméon M, André I (2013) Probing substrate promiscuity of amylosucrase from Neisseria polysaccharea. ChemCatChem 5(8):2288–2295

    Article  Google Scholar 

  • Dean SM, Greenberg WA, Wong CH (2007) Recent advances in aldolase-catalyzed asymmetric synthesis. Adv Synth Catal 349(8–9):1308–1320

    Article  CAS  Google Scholar 

  • Dowd JE, Riggs DS (1965) A comparison of estimates of Michaelis-Menten kinetic constants from various linear transformations. J Biol Chem 240(2):863–869

    CAS  PubMed  Google Scholar 

  • Eftink MR, Ghiron CA (1976) Fluorescence quenching studies with proteins. Biochemistry 15(3):672–680

    Article  CAS  PubMed  Google Scholar 

  • Egorova K, Antranikian G (2005) Industrial relevance of thermophilic Archaea. Curr Opin Microbiol 8(6):649–655

    Article  CAS  PubMed  Google Scholar 

  • Giver L, Gershenson A, Freskgard PO, Arnold FH (1998) Directed evolution of a thermostable esterase. Proc Natl Acad Sci 95(22):12809–12813

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gotor-Fernández V, Busto E, Gotor V (2006) Candida antarctica lipase B: an ideal biocatalyst for the preparation of nitrogenated organic compounds. Adv Synth Catal 348(7–8):797–812

    Article  Google Scholar 

  • Hermoso J, Pignol D, Kerfelec B, Crenon I, Chapus C, Fontecilla-Camps JC (1996) Lipase activation by nonionic detergents, the crystal structure of the porcine lipase–colipase–tetraethylene glycol monooctyl ether complex. J Biol Chem 271(30):18007–18016

    Article  CAS  PubMed  Google Scholar 

  • Hjorth A, Carriere F, Cudrey C, Woldike H, Boel E, Lawson DM, Ferrato F, Cambillau C, Dodson GG (1993) A structural domain (the lid) found in pancreatic lipases is absent in the guinea pig (phospho) lipase. Biochemistry 32(18):4702–4707

    Article  CAS  PubMed  Google Scholar 

  • Hollfelder F, Kirby AJ, Tawfik DS (1996) Off-the-shelf proteins that rival tailor-made antibodies as catalysts. Nature 383(6595):60–63

    Article  CAS  PubMed  Google Scholar 

  • Hu W, Guan Z, Deng X, He YH (2012) Enzyme catalytic promiscuity: the papain-catalyzed Knoevenagel reaction. Biochimie 94(3):656–661

    Article  CAS  PubMed  Google Scholar 

  • Hult K, Berglund P (2007) Enzyme promiscuity: mechanism and applications. Trends Biotechnol 25(5):231–238

    Article  CAS  PubMed  Google Scholar 

  • Humble MS, Berglund P (2011) Biocatalytic promiscuity. Eur J Org Chem 2011(19):3391–3401

    Article  CAS  Google Scholar 

  • Kapoor M, Gupta MN (2012) Lipase promiscuity and its biochemical applications. Process Biochem 47(4):555–569

    Article  CAS  Google Scholar 

  • Kazlauskas RJ (2005) Enhancing catalytic promiscuity for biocatalysis. Curr Opin Chem Biol 9(2):195–201

    Article  CAS  PubMed  Google Scholar 

  • Kettling U, Koltermann A, Schwille P, Eigen M (1998) Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy. Proc Natl Acad Sci U S A 95(4):1416–1420

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Leckband D (2000) Measuring the forces that control protein interaction. Ann Rev Bioph Biom 29(1):1–26

    Article  CAS  Google Scholar 

  • Li C, Feng XW, Wang N, Zhou YJ, Yu XQ (2008) Biocatalytic promiscuity: the first lipase-catalysed asymmetric aldol reaction. Green Chem 10(6):616–618

    Article  CAS  Google Scholar 

  • Li R, Zhang F, Cao SG, Xie G, Gao RJ (2012) Expression and characterization of a thermostable acyl-peptide releasing enzyme ST0779 from Sulfolobus tokodaii. Chem Res Chinese U 28(5):851–855

    CAS  Google Scholar 

  • López-Iglesias M, Busto E, Gotor V, Gotor-Fernández V (2011) Use of protease from Bacillus licheniformis as promiscuous catalyst for organic synthesis: applications in C-C and C-N bond formation reactions. Adv Synth Catal 353(13):2345–2353

    Article  Google Scholar 

  • Lou FW, Liu BK, Wu Q, Lv DS, Lin XF (2008) Candida antarctica Lipase B (CAL-B)-catalyzed carbon-sulfur bond addition and controllable selectivity in organic media. Adv Synth Catal 350(13):1959–1962

    Article  CAS  Google Scholar 

  • Michaelis L, Menten ML (1931) Die kinetik der invertinwirkung. Biochem Z 49(333–369):333–369

    Google Scholar 

  • Nardini M, Dijkstra BW (1999) α/β hydrolase fold enzymes: the family keeps growing. Curr Opin Chem Biol 9(6):732–737

    CAS  Google Scholar 

  • O'Brien PJ, Herschlag D (1999) Catalytic promiscuity and the evolution of new enzymatic activities. Chem Biol 6(4):R91–R105

    Article  PubMed  Google Scholar 

  • Pazmiño DET, Snajdrova R, Rial DV, Mihovilovic MD, Fraaije MW (2007) Altering the substrate specificity and enantioselectivity of phenylacetone monooxygenase by structure-inspired enzyme redesign. Adv Synth Catal 349(8–9):1361–1368

    Article  Google Scholar 

  • Ross PD, Subramanian S (1981) Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry 20(11):3096–3102

    Article  CAS  PubMed  Google Scholar 

  • Schmid FX (1997) Optical spectroscopy to characterize protein conformation and conformational changes. In: Protein Structure: A Practical Approach. IRL Press, Oxford, pp 261–297

    Google Scholar 

  • Segel IH (1992) Enzyme Kinetics. Weiley. pp 18–98

  • Seidel CA, Schulz A, Sauer MH (1996) Nucleobase-specific quenching of fluorescent dyes. 1. nucleobase one-electron redox potentials and their correlation with static and dynamic quenching efficiencies. J Phys Chem 100(13):5541–5553

    Article  CAS  Google Scholar 

  • Sharma N, Sharma UK, Kumar R, Katoch N, Kumar R, Sinha AK (2011) Cinnamic acids and coumarins in ionic liquid: an insight into the role of protein impurities in porcine pancreas lipase for olefinic bond formation. Adv Synth Catal 353(6):871–878

    Article  CAS  Google Scholar 

  • Szklarz GD, Paulsen MD (2002) Molecular modeling of cytochrome P450 1A1: enzyme-substrate interactions and substrate binding affinities. J Biomol Struct Dyn 20(2):155–162

    Article  CAS  PubMed  Google Scholar 

  • Takai K, Horikoshi K (1999) Genetic diversity of archaea in deep-sea hydrothermal vent environments. Genetics 152(4):1285–1297

    CAS  PubMed  PubMed Central  Google Scholar 

  • Thomas A, Allouche M, Basyn F, Brasseur R, Kerfelec B (2005) Role of the lid hydrophobicity pattern in pancreatic lipase activity. J Biol Chem 280(48):40074–40083

    Article  CAS  PubMed  Google Scholar 

  • Thorn SN, Daniels RG, Auditor MTM, Hilvert D (1995) Large rate accelerations in antibody catalysis by strategic use of haptenic charge. Nature 373:228–230

    Article  CAS  PubMed  Google Scholar 

  • Tokuriki N, Tawfik DS (2009) Protein dynamism and evolvability. Science 324(5924):203–207

    Article  CAS  PubMed  Google Scholar 

  • Wang HR, Wang Z, Zhang H, Chen G, Yue H, Wang L (2014) Enzyme catalytic promiscuity: asymmetric aldol addition reaction catalyzed by a novel thermophilic esterase in organic solvent. Green Chem Lett Rev 7(2):145–149

    Article  Google Scholar 

  • Ward LD (1985) Measurement of ligand binding to proteins by fluorescence spectroscopy. Methods Enzymol 117:400–414

    Article  CAS  PubMed  Google Scholar 

  • Wu WB, Xu JM, Wu Q, Lv DS, Lin XF (2006) Promiscuous acylases-catalyzed Markovnikov addition of N-heterocycles to vinyl esters in organic media. Adv Synth Catal 348(4–5):487–492

    Article  CAS  Google Scholar 

  • Wu Q, Liu BK, Lin XF (2010) Enzymatic promiscuity for organic synthesis and cascade process. Curr Org Chem 14(17):1966–1988

    Article  CAS  Google Scholar 

  • Xie ZB, Wang N, Zhou LH, Wan F, He T, Le ZG, Yu XQ (2013) Lipase-catalyzed stereoselective cross-aldol reaction promoted by water. ChemCatChem 5(7):1935–1940

    Article  CAS  Google Scholar 

  • Yang G, Bai A, Feng Y (2010) Molecular Redesign and Construction of New Biocatalysts. Curr Org Chem 14(14):1407–1423

    Article  CAS  Google Scholar 

  • Yang F, Wang Z, Wang H, Zhang H, Yue H, Wang L (2014) Enzyme catalytic promiscuity: lipase catalyzed synthesis of substituted 2 H-chromenes by a three-component reaction. RSC Adv 4(49):25633–25636

    Article  CAS  Google Scholar 

  • Yuryev R, Briechle S, Gruber M, Khadjawi H, Griengl, Liese A (2010) Asymmetric retro-Henry reaction catalyzed by hydroxynitrile lyase from Hevea brasiliensis. ChemCatChem 2(8):981–986

  • Zhang WW, Wang N, Feng XW, Zhang Y, Yu XQ (2014a) Biocatalytic synthesis of optically active hydroxyesters via lipase-catalyzed decarboxylative aldol reaction and kinetic resolution. Appl Biochem Biotech 173(2):535–543

    Article  CAS  Google Scholar 

  • Zhang Y, Vongvilai P, Sakulsombat M, Fischer A, Ramström O (2014b) Asymmetric synthesis of substituted thiolanes through domino thia-Michael–Henry dynamic covalent systemic resolution using lipase catalysis. Adv Synth Catal 356:987–992

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

Support from National Natural Science Foundation of China (No. 20772046) is gratefully acknowledged.

Conflict of interest

The authors declare no financial and commercial conflict of interest.

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Authors and Affiliations

  1. Key Laboratory for Molecular Enzymology and Engineering, The Ministry of Education, Jilin University, Changchun, 130021, China

    Rong Li, Hui Jian & Renjun Gao

  2. Department of Engineering, Faculty of Science and Technology, Aarhus University, Gustav Wied Vej 10, Aarhus, 8000, Denmark

    Rong Li, Bianca Perez & Zheng Guo

  3. Interdisciplinary Nanoscience Center, Aarhus University, Gustav Wieds Vej 14, 8000, Aarhus C, Denmark

    Mingdong Dong

  4. Department of Chemistry and iNANO, Aarhus University, Langelandsgade 140, 8000, Aarhus C, Denmark

    Mads Mørk Jensen & Marianne Glasius

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  1. Rong Li
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Correspondence to Renjun Gao or Zheng Guo.

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Li, R., Perez, B., Jian, H. et al. Characterization and mechanism insight of accelerated catalytic promiscuity of Sulfolobus tokodaii (ST0779) peptidase for aldol addition reaction. Appl Microbiol Biotechnol 99, 9625–9634 (2015). https://doi.org/10.1007/s00253-015-6758-z

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