Thermal Adaptation Of Cytosolic Malate Dehydrogenases Indexed By Evolutional Strategies And Enzymatic Characteristics From Marine Molluscs

A remarkable research model combining the in vitro measurements of enzymatic characteristic,in silico molecular dynamics simulation(MDS)and in vitro protein expression of site-directed mutagenesis(SDM)of the cytosolic malate dehydrogenases(cMDHs)was developed.This research model is shown to provide a powerful tool for examining thermal adaptation of orthologous enzymes by providing a quantitative index of protein structural changes in marine molluscs.To explore the relationship between structure-function and to characterize how amino acid substitutions alter stability of different regions of the enzyme,the thermal adaptation of orthologous cMDHs from two differently thermal adapted snails that are among the most heat-tolerant animals were studied.Then,the temperature effects on cMDH orthologs from differently thermally adapted congeners of 12 marine molluscs whose field body temperatures span a range of 60? were examined.It's help to develop a relationship analysis between the thermal adaptation of structure-function and the biogeographic distributions of the species.Based on the above results,further comparisons on thermal adaptation mechanisms of cMDH orthologs from 26 marine molluscs including Antarctic scallop and tropical snail were carried on.These results shown how amino acid usage in different regions of the enzyme varies with the adaptation temperature.(1)Snails of the genus Echinolittorina are among the most heat-tolerant animals with a thermal limit up to>550C.We demonstrate that heat stability of function(indexed by KMNADH)and structure(indexed by residual activity)of cMDHs of two congeners(E.malaccana and E.radiata)exceeds values previously found for orthologs of this protein from less thermophilic species.The ortholog of E.malaccana is more heat stable than that of E.radiata,in keeping with the congeners' thermal environments.Only two inter-congener differences in amino acid sequence were identified.In both cases(residues 48#and 114#),a glycine(Gly)in the E.malaccana ortholog is replaced by a serine(Ser)in the E.radiata ortholog.MDS method was applied to explore the relationship between structure and function and to characterize how amino acid substitutions alter stability of different regions of the enzyme.These computational methods allow determination of thermal effects on fine-scale movements of protein components,by estimating the root mean square deviation(RMSD)in atom position over time and the root mean square fluctuation(RMSF)for individual residues.The minor changes of E.radiata cMDH in amino acid sequence favor temperature-adaptive change in flexibility of regions in and around the active sites.Interspecific differences in effects of temperature on fine-scale protein movements are consistent with the differences in thermal effects on binding and rates of heat denaturation.(2)Orthologous proteins of species adapted to different temperatures exhibit differences in stability and function that are interpreted to reflect adaptive variation in structural flexibility.However,quantifying flexibility and comparing flexibility across proteins has remained a challenge.To address this issue,we examined temperature effects on cMDH orthologs from differently thermally adapted congeners of five genera of marine molluscs whose field body temperatures span a range of 60?.We describe consistent patterns of convergent evolution in adaptation of function(KMNADH)and structural stability(residual activity).To determine how these differences depend on flexibilities of overall structure and of regions known to be important in binding and catalysis,we performed MDS analyses.The results revealed a significant negative correlation between adaptation temperature and heat-induced increase of backbone atom movements(RMSD).RMSFs of movement by individual amino acid residues varied across the sequence in a qualitatively similar pattern among orthologs.Regions of sequence involved in ligand binding and catalysis(termed mobile regions 1 and 2,MR1 and MR2,respectively)showed the largest values for RMSF.Heat-induced changes in RMSF values across the sequence and,importantly,in MR1 and MR2 were greatest in cold-adapted species.MDS methods are shown to provide powerful tools for examining adaptation of enzymes by providing a quantitative index of protein flexibility and identifying sequence regions where adaptive change in flexibility occurs.(3)Comparative studies of orthologous proteins of species evolved at different temperatures have revealed consistent patterns of temperature-related variation in thermal stabilities of structure and function.However,the precise mechanisms by which interspecific variations in sequence foster these adaptive changes remain largely unknown.Here,we compare orthologs of cMDH from marine molluscs adapted to temperatures ranging from-1.9?(Antarctica)to?55?(South China coast)and show how amino acid usage in different regions of the enzyme(surface,intermediate depth,and protein core)varies with adaptation temperature.To link the effects of specific amino acid substitutions with adaptive variations in enzyme thermal stability,we combined in vitro SDM and in vitro protein experimentation with in silico mutagenesis using MDS techniques.SDM and MDS methods generally but not invariably yielded common effects on protein stability.MDS analysis is shown to provide insights into how specific amino acid substitutions affect the conformational flexibilities of MRs of the enzyme that are essential for binding and catalysis.Whereas these substitutions invariably lie outside of the MRs,they effectively transmit their flexibility-modulating effects to the MRs through linked interactions among surface residues.This discovery illustrates that regions of the protein surface lying outside of the site of catalysis can help establish an enzyme's thermal responses and foster evolutionary adaptation of function.In the present study,the thermal adaptation of the key metabolism enzymes from marine molluscs ranging from Antarctica to tropics indexed by the enzymatic characteristics and evolutional strategy were studied.These results can provide a further insight into the thermal adaptation mechanism of proteins from marine species,provide a new research pattern in the field.It's curcial for understanding the impacts of global warming on biogeographic distribution of marine molluscs.