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Cellulose

, Volume 21, Issue 2, pp 937–949 | Cite as

Catalytic mechanism and origin of high activity of cellulase TmCel12A at high temperature: a quantum mechanical/molecular mechanical study

  • Peng Lian
  • Hao-Bo Guo
  • Jeremy C. Smith
  • Dong-Qing Wei
  • Hong Guo
Original Paper

Abstract

Understanding the factors that determine the catalytic efficiency of cellulases is of considerable importance in cellulosic ethanol production, especially at high temperature. The cellulase 12A from the hyperthermophile Thermotoga maritima (TmCel12A) is a possible candidate for accelerating the rate of hydrolysis via temperature elevation up to as high as 95 °C. However, the details of the catalytic mechanism and origin of the activity of TmCel12A at high temperature have not been well studied. Here, the enzyme-catalyzed reaction is explored using free energy simulations (potential of mean force) with umbrella sampling and quantum mechanical/molecular mechanical (SCC-DFTB/MM) potential at both relatively low (37 °C) and high (85 °C) temperatures. The free energy barriers for glycosylation and deglycosylation are calculated to be 22.5 ± 0.4 and 24.5 ± 0.7 kcal · mol−1 at 85 °C, respectively. The barrier for deglycosylation is found to decrease with increasing temperature or as a result of the Y61 → G mutation, consistent with experimental observations. The transition state for glycosylation and deglycosylation obtained from the simulations is in an oxocarbonium state with the −1 glucose ring having an E3 envelop (or 4H3 half-chair) conformation. A unique characteristic of the TmCel12A structure seems to be the existence of a stable moiety that may play a role in “holding” cellulose at the binding site with the correct orientation for the reaction even at 85 °C. This stable moiety (comprising hydrogen-bonded E116, E134, E227 and an active-site water molecule) may be one of the important factors for the relatively high activity of TmCel12A at high temperature.

Graphical Abstract

Keywords

Cellulase Catalytic mechanism Quantum mechanical/molecular mechanical (QM/MM) Molecular dynamics (MD) 

Notes

Acknowledgments

This work is supported in part by the National Science Foundation Award (Grant No. 0817940 to H.G.) and in part by grants from the National High-Tech R&D Program (863 Program Contract No. 2012AA020307 to D.Q.W.), the National Basic Research Program of China (973 Program) (Contract No. 2012CB721000 to D.Q.W.), and the Key Project of Shanghai Science and Technology Commission (Contract No. 11JC1406400 to D.Q.W.). P.L. is supported by a fellowship from Shanghai Jiao Tong University. JCS acknowledges support from the Bioenergy Science Center, funded by Biological and Environmental Research in the Office of Science of the U.S. Department of Energy.

References

  1. Anbar M, Gul O, Lamed R, Sezerman UO, Bayer EA (2012) Improved thermostability of Clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis. Appl Environ Microb 78(9):3458–3464CrossRefGoogle Scholar
  2. Argyros DA, Tripathi SA, Barrett TF, Rogers SR, Feinberg LF, Olson DG, Foden JM, Miller BB, Lynd LR, Hogsett DA, Caiazza NC (2011) High ethanol titers from cellulose by using metabolically engineered thermophilic, anaerobic microbes. Appl Environ Microb 77(23):8288–8294CrossRefGoogle Scholar
  3. Banerjee A, Yang W, Karplus M, Verdine GL (2005) Structure of a repair enzyme interrogating undamaged DNA elucidates recognition of damaged DNA. Nature 434(7033):612–618CrossRefGoogle Scholar
  4. Barnett CB, Naidoo KJ (2010) Ring puckering: a metric for evaluating the accuracy of AM1, PM3, PM3CARB-1, and SCC-DFTB carbohydrate QM/MM simulations. J Phys Chem B 114(51):17142–17154. doi: 10.1021/jp107620h CrossRefGoogle Scholar
  5. Barnett CB, Wilkinson KA, Naidoo KJ (2010) Pyranose ring transition state is derived from cellobiohydrolase I induced conformational stability and glycosidic bond polarization. J Am Chem Soc 132(37):12800–12803. doi: 10.1021/ja103766w CrossRefGoogle Scholar
  6. Barnett CB, Wilkinson KA, Naidoo KJ (2011) Molecular details from computational reaction dynamics for the cellobiohydrolase I glycosylation reaction. J Am Chem Soc 133(48):19474–19482CrossRefGoogle Scholar
  7. Biarnes X, Ardevol A, Planas A, Rovira C, Laio A, Parrinello M (2007) The conformational free energy landscape of beta-D-glucopyranose. Implications for substrate preactivation in beta-glucoside hydrolases. J Am Chem Soc 129(35):10686–10693. doi: 10.1021/ja068411o CrossRefGoogle Scholar
  8. Bondar AN, Elstner M, Suhai S, Smith JC, Fischer S (2004) Mechanism of primary proton transfer in bacteriorhodopsin. Structure 12(7):1281–1288CrossRefGoogle Scholar
  9. Bronnenmeier K, Kern A, Liebl W, Staudenbauer WL (1995) Purification of Thermotoga maritima enzymes for the degradation of cellulosic materials. Appl Environ Microb 61(4):1399–1407Google Scholar
  10. Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, Karplus M (1983) CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J Comput Chem 4(2):187–217CrossRefGoogle Scholar
  11. Brooks CL, Brunger A, Karplus M (1985) Active-site dynamics in protein molecules: a stochastic boundary molecular-dynamics approach. Biopolymers 24(5):843–865CrossRefGoogle Scholar
  12. Brooks BR, Brooks CL, Mackerell AD, Nilsson L, Petrella RJ, Roux B, Won Y, Archontis G, Bartels C, Boresch S, Caflisch A, Caves L, Cui Q, Dinner AR, Feig M, Fischer S, Gao J, Hodoscek M, Im W, Kuczera K, Lazaridis T, Ma J, Ovchinnikov V, Paci E, Pastor RW, Post CB, Pu JZ, Schaefer M, Tidor B, Venable RM, Woodcock HL, Wu X, Yang W, York DM, Karplus M (2009) CHARMM: the biomolecular simulation program. J Comput Chem 30(10):1545–1614CrossRefGoogle Scholar
  13. Cheng YS, Ko TP, Huang JW, Wu TH, Lin CY, Luo WH, Li Q, Ma YH, Huang CH, Wang AHJ, Liu JR, Guo RT (2012) Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop. Appl Microbiol Biotechnol 95(3):661–669CrossRefGoogle Scholar
  14. Choi TH, Liang R, Maupin CM, Voth GA (2013) Application of the SCC-DFTB method to hydroxide water clusters and aqueous hydroxide solutions. J Phys Chem B 117(17):5165–5179. doi: 10.1021/jp400953a CrossRefGoogle Scholar
  15. Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: a sequence logo generator. Genome Res 14(6):1188–1190CrossRefGoogle Scholar
  16. Davies GJ, Ducros VMA, Varrot A, Zechel DL (2003) Mapping the conformational itinerary of beta-glycosidases by X-ray crystallography. Biochem Soc Trans 31:523–527CrossRefGoogle Scholar
  17. Demain AL, Newcomb M, Wu JHD (2005) Cellulase, clostridia, and ethanol. Microbiol Mol Biol Rev 69(1):124–154CrossRefGoogle Scholar
  18. Elstner M, Porezag D, Jungnickel G, Elsner J, Haugk M, Frauenheim T, Suhai S, Seifert G (1998) Self-consistent-charge density-functional tight-binding method for simulations of complex materials properties. Phys Rev B 58(11):7260–7268CrossRefGoogle Scholar
  19. Elstner M, Frauenheim T, Suhai S (2003) An approximate DFT method for QM/MM simulations of biological structures and processes. J Mol Struct THEOCHEM 632:29–41CrossRefGoogle Scholar
  20. Gaus M, Cui QA, Elstner M (2011) DFTB3: extension of the self-consistent-charge density-functional tight-binding method (SCC-DFTB). J Chem Theory Comput 7(4):931–948CrossRefGoogle Scholar
  21. Guo H, Cui Q, Lipscomb WN, Karplus M (2001) Substrate conformational transitions in the active site of chorismate mutase: their role in the catalytic mechanism. Proc Natl Acad Sci USA 98(16):9032–9037CrossRefGoogle Scholar
  22. Guo HB, Rao N, Xu Q, Guo H (2005a) Origin of tight binding of a near-perfect transition-state analogue by cytidine deaminase: implications for enzyme catalysis. J Am Chem Soc 127(9):3191–3197CrossRefGoogle Scholar
  23. Guo HB, Wlodawer A, Guo H (2005b) A general acid–base mechanism for the stabilization of a tetrahedral adduct in a serine-carboxyl peptidase: a computational study. J Am Chem Soc 127(45):15662–15663. doi: 10.1021/ja0520565 CrossRefGoogle Scholar
  24. Guo HB, Wlodawer A, Nakayama T, Xu Q, Guo H (2006) Catalytic role of proton transfers in the formation of a tetrahedral adduct in a serine carboxyl peptidase. Biochemistry 45(30):9129–9137CrossRefGoogle Scholar
  25. Haki GD, Rakshit SK (2003) Developments in industrially important thermostable enzymes: a review. Bioresour Technol 89(1):17–34CrossRefGoogle Scholar
  26. Himmel ME, Ruth MF, Wyman CE (1999) Cellulase for commodity products from cellulosic biomass. Curr Opin Biotechnol 10(4):358–364CrossRefGoogle Scholar
  27. Himmel ME, Ding SY, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD (2007) Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315(5813):804–807CrossRefGoogle Scholar
  28. Ionescu AR, Berces A, Zgierski MZ, Whitfield DM, Nukada T (2005) Conformational pathways of saturated six-membered rings. A static and dynamical density functional study. J Phys Chem A 109(36):8096–8105CrossRefGoogle Scholar
  29. Islam SM, Roy P-N (2012) Performance of the SCC-DFTB model for description of five-membered ring carbohydrate conformations: comparison to force fields, high-level electronic structure methods, and experiment. J Chem Theory Comput 8(7):2412–2423CrossRefGoogle Scholar
  30. Javed MM, Ikram-ul-Haq, Mariyam I (2011) Multistep Mutagenesis for the over-expression of cellulase in Humicola insolens. Pak J Bot 43(1):669–677Google Scholar
  31. Jeffrey GA, Yates JH (1979) Stereographic representation of the Cremer-Pople ring-puckering parameters for pyranoid rings. Carbohyd Res 74:319–322CrossRefGoogle Scholar
  32. Jorgensen WL (1981) Quantum and statistical mechanical studies of liquids. 10. Transferable intermolecular potential functions for water, alcohols, and ethers: application to liquid water. J Am Chem Soc 103(2):335–340CrossRefGoogle Scholar
  33. Kang HJ, Uegaki K, Fukada H, Ishikawa K (2007) Improvement of the enzymatic activity of the hyperthermophilic cellulase from Pyrococcus horikoshii. Extremophiles 11(2):251–256CrossRefGoogle Scholar
  34. Kumar S, Rosenberg JM, Bouzida D, Swendsen RH, Kollman PA (1992) The weighted histogram analysis method for free-energy calculations on biomolecules. I. The method. J Comput Chem 13(8):1011–1021CrossRefGoogle Scholar
  35. 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(21):2947–2948CrossRefGoogle Scholar
  36. Lee CY, Yu KO, Kim SW, Han SO (2010) Enhancement of the thermostability and activity of mesophilic Clostridium cellulovorans EngD by in vitro DNA recombination with Clostridium thermocellum CelE. J Biosci Bioeng 109(4):331–336CrossRefGoogle Scholar
  37. Li GH, Cui Q (2003) What is so special about Arg 55 in the catalysis of cyclophilin A? Insights from hybrid QM/MM simulations. J Am Chem Soc 125(49):15028–15038CrossRefGoogle Scholar
  38. Liang CN, Fioroni M, Rodriguez-Ropero F, Xue YF, Schwaneberg U, Ma YH (2011a) Directed evolution of a thermophilic endoglucanase (Cel5A) into highly active Cel5A variants with an expanded temperature profile. J Biotechnol 154(1):46–53CrossRefGoogle Scholar
  39. Liang CN, Xue YF, Fioroni M, Rodriguez-Ropero F, Zhou C, Schwaneberg U, Ma YH (2011b) Cloning and characterization of a thermostable and halo-tolerant endoglucanase from Thermoanaerobacter tengcongensis MB4. Appl Microbiol Biotechnol 89(2):315–326CrossRefGoogle Scholar
  40. Liebl W, Ruile P, Bronnenmeier K, Riedel K, Lottspeich F, Greif I (1996) Analysis of a Thermotoga maritima DNA fragment encoding two similar thermostable cellulases, CelA and CelB, and characterization of the recombinant enzymes. Microbiology 142:2533–2542CrossRefGoogle Scholar
  41. Liu JL, Wang XM, Xu DG (2010) QM/MM study on the catalytic mechanism of cellulose hydrolysis catalyzed by cellulase Cel5A from Acidothermus cellulolyticus. J Phys Chem B 114(3):1462–1470. doi: 10.1021/jp909177e CrossRefGoogle Scholar
  42. Liu JR, Cheng YS, Ko TP, Wu TH, Ma YH, Huang CH, Lai HL, Wang AHJ, Guo RT (2011) Crystal structure and substrate-binding mode of cellulase 12A from Thermotoga maritima. Proteins 79(4):1193–1204CrossRefGoogle Scholar
  43. Mackerell AD, Feig M, Brooks CL (2004) Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. J Comput Chem 25(11):1400–1415CrossRefGoogle Scholar
  44. Maupin CM, Aradi B, Voth GA (2010) The self-consistent charge density functional tight binding method applied to liquid water and the hydrated excess proton: benchmark simulations. J Phys Chem B 114(20):6922–6931. doi: 10.1021/jp1010555 CrossRefGoogle Scholar
  45. Murashima K, Kosugi A, Doi RH (2002) Thermostabilization of cellulosomal endoglucanase EngB from Clostridium cellulovorans by in vitro DNA recombination with non-cellulosomal endoglucanase EngD. Mol Microbiol 45(3):617–626CrossRefGoogle Scholar
  46. Nakazawa H, Okada K, Onodera T, Ogasawara W, Okada H, Morikawa Y (2009) Directed evolution of endoglucanase III (Cel12A) from Trichoderma reesei. Appl Microbiol Biotechnol 83(4):649–657CrossRefGoogle Scholar
  47. Nelson KE, Clayton RA, Gill SR, Gwinn ML, Dodson RJ, Haft DH, Hickey EK, Peterson LD, Nelson WC, Ketchum KA, McDonald L, Utterback TR, Malek JA, Linher KD, Garrett MM, Stewart AM, Cotton MD, Pratt MS, Phillips CA, Richardson D, Heidelberg J, Sutton GG, Fleischmann RD, Eisen JA, White O, Salzberg SL, Smith HO, Venter JC, Fraser CM (1999) Evidence for lateral gene transfer between Archaea and Bacteria from genome sequence of Thermotoga maritima. Nature 399(6734):323–329CrossRefGoogle Scholar
  48. Niehaus TA, Elstner M, Frauenheim T, Suhai S (2001) Application of an approximate density-functional method to sulfur containing compounds. J Mol Struct (Thoechem) 541(1):185–194CrossRefGoogle Scholar
  49. Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, Pang N, Forslund K, Ceric G, Clements J, Heger A, Holm L, Sonnhammer ELL, Eddy SR, Bateman A, Finn RD (2012) The Pfam protein families database. Nucleic Acids Res 40(D1):D290–D301CrossRefGoogle Scholar
  50. Riccardi D, Konig P, Guo H, Cui Q (2008) Proton transfer in carbonic anhydrase is controlled by electrostatics rather than the orientation of the acceptor. Biochemistry 47(8):2369–2378CrossRefGoogle Scholar
  51. Saharay M, Guo H, Smith JC (2010) Catalytic mechanism of cellulose degradation by a cellobiohydrolase, CelS. PLoS ONE 5(10):e12947. doi: 10.1371/journal.pone.0012947
  52. Sattelle BM, Almond A (2010) Less is more when simulating unsulfated glycosaminoglycan 3D-structure: comparison of GLYCAM06/TIP3P, PM3-CARB1/TIP3P, and SCC-DFTB-D/TIP3P predictions with experiment. J Comput Chem 31(16):2932–2947. doi: 10.1002/jcc.21589 Google Scholar
  53. Sattelmeyer KW, Tirado-Rives J, Jorgensen WL (2006) Comparison of SCC-DFTB and NDDO-based semiempirical molecular orbital methods for organic molecules. J Phys Chem A 110(50):13551–13559CrossRefGoogle Scholar
  54. Schou C, Rasmussen G, Kaltoft MB, Henrissat B, Schulein M (1993) Stereochemistry, specificity and kinetics of the hydrolysis of reduced cellodextrins by 9 cellulases. Eur J Biochem 217(3):947–953CrossRefGoogle Scholar
  55. Shaw AJ, Podkaminer KK, Desai SG, Bardsley JS, Rogers SR, Thorne PG, Hogsett DA, Lynd LR (2008) Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield. Proc Natl Acad Sci USA 105(37):13769–13774CrossRefGoogle Scholar
  56. Srikrishnan S, Randall A, Baldi P, Da Silva NA (2012) Rationally selected single-site mutants of the Thermoascus aurantiacus endoglucanase increase hydrolytic activity on cellulosic substrates. Biotechnol Bioeng 109(6):1595–1599CrossRefGoogle Scholar
  57. Torrie GM, Valleau JP (1974) Monte-Carlo free-energy estimates using non-Boltzmann sampling: application to subcritical Lennard-Jones fluid. Chem Phys Lett 28(4):578–581CrossRefGoogle Scholar
  58. Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65(1):1–43CrossRefGoogle Scholar
  59. Viikari L, Alapuranen M, Puranen T, Vehmaanpera J, Siika-Aho M (2007) Thermostable enzymes in lignocellulose hydrolysis. Adv Biochem Eng Biotechnol 108:121–145Google Scholar
  60. Volfova O, Suchardova O, Panos J, Krumphanzl V (1985) Ethanol formation from cellulose by thermophilic bacteria. Appl Microbiol Biotechnol 22(4):246–248CrossRefGoogle Scholar
  61. Xu Q, Guo H, Wlodawer A, Guo H (2006) The importance of dynamics in substrate-assisted catalysis and specificity. J Am Chem Soc 128(18):5994–5995CrossRefGoogle Scholar
  62. Xue DS, Chen HY, Ren YR, Yao SJ (2012) Enhancing the activity and thermostability of thermostable beta-glucosidase from a marine Aspergillus niger at high salinity. Process Biochem 47(4):606–611CrossRefGoogle Scholar
  63. Yang B, Wyman CE (2004) Effect of xylan and lignin removal by batch and flowthrough pretreatment on the enzymatic digestibility of corn stover cellulose. Biotechnol Bioeng 86(1):88–95CrossRefGoogle Scholar
  64. Yang Y, Yu H, York D, Elstner M, Cui Q (2008) Description of phosphate hydrolysis reactions with the self-consistent-charge density-functional-tight-binding (SCC-DFTB) theory. 1. Parameterization. J Chem Theory Comput 4(12):2067–2084. doi: 10.1021/ct800330d CrossRefGoogle Scholar
  65. Yi ZL, Pei XQ, Wu ZL (2011) Introduction of glycine and proline residues onto protein surface increases the thermostability of endoglucanase CelA from Clostridium thermocellum. Bioresour Technol 102(3):3636–3638CrossRefGoogle Scholar
  66. Zhang YHP, Himmel ME, Mielenz JR (2006) Outlook for cellulase improvement: screening and selection strategies. Biotechnol Adv 24(5):452–481CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  1. 1.The State Key Laboratory of Microbial Metabolism, College of Life Sciences and BiotechnologyShanghai Jiao Tong UniversityShanghaiChina
  2. 2.Department of Biochemistry and Cellular and Molecular BiologyUniversity of TennesseeKnoxvilleUSA
  3. 3.Oak Ridge National LaboratoryUT/ORNL Center for Molecular BiophysicsOak RidgeUSA

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