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Crystal structure of the hexachlorocyclohexane dehydrochlorinase (LinA-type2): mutational analysis, thermostability and enantioselectivity.

ABSTRACT: Hexachlorocyclohexane dehydrochlorinase (LinA) mediates dehydrochlorination of ?-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 ?-HCH. Contrastingly, we identified that -type2 protein prefers the (+) enantiomer of ?-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.

SUBMITTER: Macwan AS

PROVIDER: S-EPMC3507683 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Crystal structure of the hexachlorocyclohexane dehydrochlorinase (LinA-type2): mutational analysis, thermostability and enantioselectivity.

Macwan Ankit S AS Kukshal Vandna V Srivastava Nidhi N Javed Saleem S Kumar Ashwani A Ramachandran Ravishankar R

PloS one 20121127 11

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 ...[more]

PMID: 23209726

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Project description:Enterovirus D68 (EV-D68) is one of the many nonpolio enteroviruses that cause mild to severe respiratory illness. The nonstructural protein 3Dpol is an RNA-dependent RNA polymerase (RdRP) of EV-D68 which plays a critical role in the replication of the viral genome and represents a promising drug target. Here, we report the first three-dimensional crystal structure of the RdRP from EV-D68 in complex with the substrate GTP to 2.3-Å resolution. The RdRP structure is similar to structures of other viral RdRPs, where the three domains, termed the palm, fingers, and thumb, form a structure resembling a cupped right hand. Particularly, an N-terminal fragment (Gly1 to Phe30) bridges the fingers and the thumb domains, which accounts for the enhanced stability of the full-length enzyme over the truncation mutant, as assessed by our thermal shift assays and the dynamic light scattering studies. Additionally, the GTP molecule bound proximal to the active site interacts with both the palm and fingers domains to stabilize the core structure of 3Dpol Interestingly, using limited proteolysis assays, we found that different nucleoside triphosphates (NTPs) stabilize the polymerase structure by various degrees, with GTP and CTP being the most and least stabilizing nucleosides, respectively. Lastly, we derived a model of the core structure of 3Dpol stabilized by GTP, according to our proteolytic studies. The biochemical and biophysical characterizations conducted in this study help us to understand the stability of EV-D68-3Dpol, which may extend to other RdRPs as well.IMPORTANCE Enterovirus D68 (EV-D68) is an emerging viral pathogen, which caused sporadic infections around the world. In recent years, epidemiology studies have reported an increasing number of patients with respiratory diseases globally due to the EV-D68 infection. Moreover, the infection has been associated with acute flaccid paralysis and cranial nerve dysfunction in children. However, there are no vaccines and antiviral treatments specifically targeting the virus to date. In this study, we solved the crystal structure of the RNA-dependent RNA polymerase of EV-D68 and carried out systematic biophysical and biochemical characterizations on the overall and local structural stability of the wild-type (WT) enzyme and several variants, which yields a clear view on the structure-activity relationship of the EV-D68 RNA polymerase.

Dataset Information

Crystal structure of the hexachlorocyclohexane dehydrochlorinase (LinA-type2): mutational analysis, thermostability and enantioselectivity.

Publications

Crystal structure of the hexachlorocyclohexane dehydrochlorinase (LinA-type2): mutational analysis, thermostability and enantioselectivity.

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