Dynamic Perturbation of the Active Site Determines Reversible

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Dynamic Perturbation of the Active Site Determines Reversible Thermal Inactivation in Glycoside Hydrolase Family 12 Xukai Jiang, Wen Li, Guanjun Chen, and Lushan Wang J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.6b00692 • Publication Date (Web): 01 Feb 2017 Downloaded from http://pubs.acs.org on February 3, 2017

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Journal of Chemical Information and Modeling

Dynamic Perturbation of the Active Site Determines Reversible Thermal Inactivation in Glycoside Hydrolase Family 12

Xukai Jiang, Wen Li, Guanjun Chen and Lushan Wang* State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China

Corresponding authors: Lushan Wang, email: [email protected] Room 416 State Key Laboratory of Microbial Technology, Jinan, 250100, China Phone 86 531 88366202

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ABSTRACT The temperature dependence of enzyme catalysis is highly debated. Specifically, how high temperatures induce enzyme inactivation has broad implications for both fundamental and applied science. Here, we explored the mechanism of the reversible thermal inactivation in glycoside hydrolase family 12 (GH12) using comparative molecular dynamics simulations. First, we investigated the distribution of structural flexibility over the enzyme and found that the active site was the general thermal-sensitive region in GH12 cellulases. The dynamic perturbation of the active site before enzyme denaturation was explored through principal-component analysis, which indicated that variations in the collective motion and conformational ensemble of the active site may precisely correspond to enzyme transition from its active form to the inactive form. Furthermore, the degree of dynamic perturbation of the active site was found to be negatively correlated with the melting temperatures of GH12 enzymes, further proving the importance of the dynamic stability of the active site. Additionally, analysis of the residue-interaction network revealed that the active site in thermophilic enzyme was capable of forming additional contacts with other amino acids than that observed in the mesophilic enzyme. These interactions are likely the key mechanisms underlying the differences in rigidity of the active site. These findings provide further biophysical insights into the reversible thermal inactivation of enzymes and potential applications in future protein engineering.


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INTRODUCTION The temperature dependence of enzyme catalysis remains a hot topic in enzymology research.1 Specifically, thermal inactivation of enzymes is the most common mode of inactivation in bio-catalytic reactors, where it is necessary for the biocatalyst to work at elevated temperatures. This is due to acceleration of the reaction rate upon heating, which also reduces bacterial contamination and, in most cases, increases substrate solubility.2, 3 In practice, low enzyme thermostability limits their applications and increases industry costs.

Consequently,

a

mechanistic

investigation

of

enzyme

thermal

inactivation is of basic scientific interest and also has implications for the applied sciences. The temperature dependence of enzyme catalysis is described by the Classical Model,4, 5 which describes cooperative transition between a folded, enzymatically active state (Eact) and an unfolded, completely inactive one (Xinact). In a reactor at high temperature, efficient enzyme concentration is diluted due to the continuous transition from Eact to Xinact, and enzyme thermal inactivation was thought to be a result of irreversible enzyme denaturation.4 However, increasing evidence suggests that enzyme thermal inactivation is not completely dependent upon irreversible protein denaturation. This assumption was partly proven by observation that enzyme activity at high temperatures was lower than would be expected based on its observed thermostability, with some losses of enzyme activity being reversible.6, 7 Additionally, engineering studies showed unexpected decreases in enzyme activity in the presence of high temperatures, even after enhancing their thermostability through introduction of new non-covalent interactions into the enzyme structures.8-10 These results indicated that enzyme thermal inactivation is not entirely the result of enzyme denaturation, and that thermostability is only a necessary, but not a sufficient, factor for thermoactivity (the ability of an enzyme to perform catalysis at high temperatures).



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To account for these results, Daniel et al formulated an alternative theory called the Equilibrium Model (EM),11, 12 wherein the Eact is in equilibrium with an inactive form (Einact), and it is the inactive form that undergoes irreversible thermal inactivation to the Xinact. Compared with the Classical Model, the EM introduces an additional temperature-dependent factor associated with enzyme activity, namely the effect on the equilibrium position between the active and inactive forms of the enzyme.13 The shift in Eact/Einact equilibrium will alter the efficient concentration of the active enzyme and consequently exert a detrimental effect on the catalytic rate. It should be noted that the increase in Einact caused by high temperatures merely results in reversible inactivation of the enzyme, while the increase of Xinact (enzyme denaturation) will lead to irreversible inactivation. Therefore, Eact/Einact reversible equilibration likely provides a “thermal buffer” that protects the enzyme from thermal inactivation.13 Based on the EM, factors that determine whether the enzyme can perform catalysis at high temperatures should also include the kinetic stability associated with Eact/Einact equilibrium, except for structural stability. Despite many conceptual and kinetic studies concerning the EM, the molecular basis of Eact/Einact equilibrium and how the equilibrium shifts at high temperatures remains unclear.12, 13 In this study, we selected glycoside hydrolase family 12 (GH12) as a model due to the frequent use of cellulases for biomass conversion into biofuels and chemicals in the biorefinery industry.14 Also, GH12 enzymes are the smallest cellulases and lack carbohydrate-binding modules.15 These properties make its members ideal candidates for studying the specific effects of high temperature on the cellulase catalytic domain. Here, we investigated the molecular mechanism of reversible thermal inactivation in GH12 through comparative molecular dynamics (MD) simulations. The structural flexibility, collective motion, and conformational ensemble were characterized to illustrate the dynamic perturbation of the active site prior to protein unfolding. Additionally, the mechanism underlying the differences in thermoactivity between mesophilic

and

thermophilic

enzymes

was

explored


by

analyzing

the

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residue-interaction network. Our study provided additional biophysical insight into the reversible thermal inactivation of enzymes and potential applications for future protein engineering.

SYSTEM AND METHODS System Preparation. For this study, we constructed a dataset containing homologous mesophilic and thermophilic GH12 cellulases. Five cellulases were selected, including the mesophilic enzymes HsCel12A (PDB: 1OA3) from Hypocrea schweinitzii and TrCel12A (PDB: 1H8V) from Trichoderma reesei, and the thermophilic enzymes SsCel12A (PDB: 1OA4) from Streptomyces sp. 11AG8, HgCel12A (PDB: 1OLR) from Humicola grisea, and RmCel12A (PDB: 1H0B) from Rhodothermus marinus. These enzymes have experimentally determined crystal structures that show folding into a similar β-sandwich architecture. More importantly, their biochemical thermostabilities have been characterized by measurement of their melting temperatures which range from 49 to 95 oC. Their evolutionary locations in the phylogenetic tree of GH12 was displayed in previous study.16 These properties of the above five GH12 cellulases make them ideal candidates for studying the effect of high temperature on enzyme catalysis. All protein structures were obtained from the Protein Data Bank (www.rcsb.org). Only protein chain-A of the crystal structure was used for MD simulations. As suggested by previous study, the temperature elevation can shift the Eact/Einact equilibrium.13 Consequently, two paralleled simulations at 300 and 350 K were performed for each studied GH12 cellulase, which simulates the natural and thermal conditions of enzymes. Totally, ten simulations were constructed in present study. These simulations make it feasible to investigate the molecular basis of Eact/Einact equilibrium through exploring the structural and conformational fluctuations during the simulations. Molecular Dynamics Simulations. In present study, the protein was solvated using the SPC model.17 This model has been successfully applied into



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a variety of thermal unfolding studies.18-20 A cubic box was constructed to perform MD simulations. Water molecules that overlapped with the protein heavy atoms were removed. The total numbers of atoms in different solvent systems were greater than 30,000. To produce a neutral system with 0.1 mol/L ionic concentration, appropriate amounts of Na+ and Cl− were added by randomly replacing water molecules with ions. All MD simulations were performed under periodic boundary conditions using the GROMACS 4.5.5.21 CHARMM27 force field was used to describe the protein.22 A previous study have offered qualitative validation that the force field are also effective at highly elevated temperatures.23 To eliminate steric interference, the steepest descent method was used to perform energy minimization providing the maximum force

Dynamic Perturbation of the Active Site Determines Reversible

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