A Molecular Dynamics Simulation Study On The Temperature-Adaptation Mechanisms Of The Extremophilic Malate Dehydrogenases

Enzymes with adaptability to the extreme temperatures play a key role in maintaining the normal physiological and metabolic activities of extremophiles and,therefore,understanding the temperature-adaptation mechanisms of enzymes is of great significance for not only the basic research of enzymology,but also for the enzyme engineering and industrial applications(including applications to industry,agriculture,food ? medicine,and biotechnology).Due to the complexity of the topological organizations and dynamic behaviors of the protein 3D structure,different enzyme families could adopt different strategies to maintain the balanced relationships among the structural stability,conformational flexibility,and catalytic activity at the extreme temperatures,and in particular,studies related to the temperature-adaptation mechanisms of the multimeric enzymes are rarely reported.In the current study,the psychrophilic malate dehydrogenase(MDH)from an Arctic bacterium Aquaspirillum arcticum(Aa-MDH)and its thermophilic counterpart,Tf-MDH from a hot spring bacterium Thermus flavus,were taken as the comparative objects and subjected to multiple-replica molecular dynamics(MD)simulations to explore the strategies and mechanisms of the temperature adaptation of the dimeric MDH.The results of comparative analyses of the concatenated equilibrium trajectories in terms of the dynamics-related properties indicate that,when compared to Tf-MDH,Aa-MDH exhibited greater structural fluctuations,a higher inter-subunit positional variability,and higher global and local flexibilities;the results of the essential dynamics(ED)analysis indicate that Aa-MDH has greater freedoms of molecular motions than Tf-MDH and the most significant motion modes lead to the opposite consequences of the conformational changes in the two MDHs,i.e.,the approach/departure of the two subunits and the shrinkage/enlargement of the coenzyme-substrate-binding cavity in Tf-MDH/Aa-MDH.The comparison between the constructed free energy landscapes(FELs)of the two enzymes revealed that Tf-MDH has a lower conformational entropy and higher thermostability than Aa-MDH.The comparison between the calculated structural/geometrical parameters indicates that Tf-MDH has more intra-molecular and inter-subunit noncovalent interactions and a more compact structural packing than Aa-MDH.The calculated values of the binding free energy between the two subunits show that Tf-MDH has a higher inter-subunit binding affinity than Aa-MDH,and this is due to stronger inter-subunit electrostatic interaction forces in Tf-MDH than in Aa-MDH.Synthesizing our calculation results,we consider that the variations in the strength of inter-subunit electrostatic interaction could not only affect the conformational flexibility of the inter-subunit interfaces and the relative positional variability of the two subunits,but also regulate the overall structural stability,global conformational flexibility,or even the local flexibility of the structural regions responsible for substrate binding and catalytic activity.Therefore,strengthening and weakening the electrostatic interaction strength between the two subunits are very likely to be the temperature-adaptation strategies adopted by Aa-MDH and Tf-MDH,respectively.Several screened “key residues”,which are considered to be determinants for the inter-monomer binding affinity,are also likely to be the “key residues” in determining the temperature adaptability of MDH.It can be concluded that Aa-MDH has adapted to the cold environment through the “global-flexibility” mechanism rather than the currently generally accepted “local rigidity/flexibility” mechanism used to explain the cold adaptation of enzymes.Nevertheless,the question of which specific regions with significantly increased local flexibility lead to the decrease in the activation free energy of Aa-MDH still remains open to study.The results presented in this study not only reveal the temperature-adaptation strategies adopted by malate dehydrogenase,but also contributed to an in-depth understanding of the structure-dynamics-function relationships and temperature-adaptation mechanism of MDH;last but not least,this study provides candidate amino acid residue sites for the enzyme-directed modification to improve the catalytic activity and thermal stability of MDH.