, 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


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


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



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.


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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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