Crystal Structure of the Hexachlorocyclohexane Dehydrochlorinase (LinA-Type2) Mutational Analysis, Thermostability and Enantioselectivity 英文参考文献.docVIP

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Crystal Structure of the Hexachlorocyclohexane Dehydrochlorinase (LinA-Type2) Mutational Analysis, Thermostability and Enantioselectivity 英文参考文献.doc

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CrystalStructureoftheHexachlorocyclohexane Dehydrochlorinase(LinA-Type2):MutationalAnalysis, ThermostabilityandEnantioselectivity AnkitS.Macwan1,VandnaKukshal3,NidhiSrivastava1,SaleemJaved2,AshwaniKumar1*, RavishankarRamachandran3* 1EnvironmentalBiotechnologyDivision,CSIR-IndianInstituteofToxicologyResearch,MahatmaGandhiMarg,Lucknow,India,2DepartmentofBiochemistry,Hamdard University,NewDelhi,India,3MolecularandStructuralBiologyDivision,CSIR-CentralDrugResearchInstitute,ChattarManzil,MahatmaGandhiMarg,Lucknow,India Abstract Hexachlorocyclohexane dehydrochlorinase (LinA) mediates dehydrochlorination of c-HCH to 1, 3, 4, 6-tetrachloro-1,4- cyclohexadienethatconstitutesfirststep oftheaerobic degradation pathway.Wereport the3.5A? crystalstructureofa thermostable LinA-type2 protein, obtained from a soil metagenome, in the hexagonal space group P6322 with unit cell ? parameters a=b=162.5, c=186.3A, respectively. The structure was solved by molecular replacement using the co- ordinates of LinA-type1 that exhibits mesophile-like properties. Structural comparison of LinA-type2 and -type1 proteins suggeststhatthermostabilityofLinA-type2mightpartlyariseduetopresenceofhighernumberofionicinteractions,along with4%increaseintheintersubunitburiedsurfacearea.Mutationalanalysisinvolvingthedifferingresiduesbetweenthe- type1and-type2proteins,circulardichroismexperimentsandfunctionalassayssuggestthatQ20andG23aredeterminants ofstabilityforLinA-type2.ItwasearlierreportedthatLinA-type1exhibitsenantioselectivityforthe(2)enantiomerofa- HCH.Contrastingly,weidentifiedthat-type2proteinprefersthe(+)enantiomerofa-HCH.Structuralanalysisandmolecular docking experiments suggest that changed residues K20Q, L96C and A131G, vicinal to the active site are probably responsible for the altered enantioselectivity of LinA-type2. Overall the study has identified features responsible for the thermostability and enantioselectivity of LinA-type2 that can be exploited for the design of variants for specif