title: Crystal structure of the hexachlorocyclohexane dehydrochlorinase (LinA-type2): mutational analysis, thermostability and enantioselectivity.
abstract: Hexachlorocyclohexane dehydrochlorinase (LinA) mediates dehydrochlorination of γ-HCH to 1, 3, 4, 6-tetrachloro-1,4-cyclohexadiene that constitutes first step of the aerobic degradation pathway. We report the 3.5 Å crystal structure of a thermostable LinA-type2 protein, obtained from a soil metagenome, in the hexagonal space group P6(3)22 with unit cell parameters a = b = 162.5, c = 186.3 Å, 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 suggests that thermostability of LinA-type2 might partly arise due to presence of higher number of ionic interactions, along with 4% increase in the intersubunit buried surface area. Mutational analysis involving the differing residues between the -type1 and -type2 proteins, circular dichroism experiments and functional assays suggest that Q20 and G23 are determinants of stability for LinA-type2. It was earlier reported that LinA-type1 exhibits enantioselectivity for the (-) enantiomer of α-HCH. Contrastingly, we identified that -type2 protein prefers the (+) enantiomer of α-HCH. Structural analysis and molecular 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 specific biotechnological applications.
authors.affiliation: Environmental Biotechnology Division, CSIR-Indian Institute of Toxicology Research, Mahatma Gandhi Marg, Lucknow, India.
authors.forename: Ankit S
authors.initials: AS
authors.lastname: Macwan
authors.forename: Vandna
authors.initials: V
authors.lastname: Kukshal
authors.forename: Nidhi
authors.initials: N
authors.lastname: Srivastava
authors.forename: Saleem
authors.initials: S
authors.lastname: Javed
authors.forename: Ashwani
authors.initials: A
authors.lastname: Kumar
authors.forename: Ravishankar
authors.initials: R
authors.lastname: Ramachandran
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D002942:Circular Dichroism / Q000379:methods; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray / Q000379:methods; D004252:DNA Mutational Analysis; D001556:Hexachlorocyclohexane / Q000737:chemistry; D007306:Insecticides / Q000737:chemistry; D008190:Lyases / Q000737:chemistry*; D008956:Models, Chemical; D008968:Molecular Conformation; D009154:Mutation; D011485:Protein Binding; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012492:Salts / Q000737:chemistry; D042301:Sphingomonadaceae / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D014466:Ultraviolet Rays
chemical_list: D001426:Bacterial Proteins; D007306:Insecticides; D012492:Salts; D001556:Hexachlorocyclohexane; D008190:Lyases