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 P6322 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.
LinA-type1 and LinA-type2 are two well-characterized variants of the enzyme ‘hexachlorocyclohexane (HCH)-dehydrochlorinase’. They differ from each other at ten amino acid positions and exhibit differing enantioselectivity for the transformation of the (–) and (+) enantiomers of α-HCH. Amino acids responsible for this enantioselectivity, however, are not known. An in silico docking analysis identified four amino acids (K20, L96, A131, and T133) in LinA-type1 that could be involved in selective binding of the substrates. Experimental studies with constructed mutant enzymes revealed that a combined presence of three amino acid changes in LinA-type1, i.e. K20Q, L96C, and A131G, caused a reversal in its preference from the (–) to the (+) enantiomer of α-HCH. This preference was enhanced by the additional amino acid change T133 M. Presence of these four changes also caused the reversal of enantioselectivity of LinA-type1 for δ-HCH, and β-, γ-, and δ-pentachlorocyclohexens. Thus, the residues K20, L96, A131, and T133 in LinA-type1 and the residues Q20, C96, G131, and M133 in LinA-type 2 appear to be important determinants for the enantioselectivity of LinA enzymes.Electronic supplementary materialThe online version of this article (doi:10.1007/s10532-017-9786-9) contains supplementary material, which is available to authorized users.
Background Although flow cytometry is often brought forward as a preferable method in the setting of thrombocytopenia, the relative effects of low sample counts on results from flow cytometry‐based platelet function testing ( FC ‐ PFT ) in comparison with light transmission aggregometry ( LTA ) and multiple electrode aggregometry ( MEA ) has not been reported. Objectives To compare the effects of different sample platelet counts (10, 50, 100, and 200 × 10 9 L −1 ) on platelet activation measured with FC ‐ PFT , LTA , and MEA using the same anticoagulant and agonist concentrations as for the commercial MEA test. Methods Platelets were stimulated with two commonly used platelet agonists ( ADP [6.5 μmol L −1 ] and PAR 1‐ AP [ TRAP , 32 μmol L −1 ]). The specified sample platelet counts were obtained by combining platelet‐rich and platelet poor hirudinized plasma in different proportions with or without red blood cells. Results For FC , P‐selectin exposure and PAC ‐1 binding was reduced at 10 × 10 9 L −1 after stimulation with PAR 1‐ AP (by approximately 20% and 50%, respectively), but remained relatively unchanged when ADP was used as agonist (n = 9). The platelet count‐dependent effects observed with PAR 1‐ AP were eliminated when samples were pre‐incubated with apyrase, implying that reduced purinergic signaling was the main underlying factor (n = 5). Both aggregometry‐based PFT s showed a 50% reduction at 50 × 10 9 L −1 and more than 80% reduction at 10 × 10 9 L −1 , irrespective of agonist used (n = 7). Conclusions Although FC ‐ PFT is generally preferable to aggregometry‐based PFT s in situations with low sample platelet counts, a careful optimization of experimental parameters is still required in order to eliminate platelet count‐related effects.
Hexachlorocyclohexane dehydrochlorinase (LinA) mediates first step of aerobic degradation of a chlorinated insecticide γ-hexachlorocyclohexane (γ-HCH). In this study, we describe characterization of a novel variant (LinA-type2) that is distinct from reported LinAs and is substantially more thermostable than archetypal LinA-UT26. LinA-type2 remains active even after 8 h of incubation at 45 °C, when nearly 50% activity of LinA-UT26 is lost after incubation for 60 min at the same temperature. Circular dichroism analysis revealed that secondary structures of LinA-UT26 and LinA-type2 are similar, but their Tm was 45 and 65 °C, respectively. Thermostability of LinA-type2 makes it suitable for bioreactors where allowance for higher temperatures can be of advantage.
The chlorinated insecticide gamma-hexachlorocyclohexane (gamma-HCH) is sequentially metabolized by the products of linA, linB, linC, linD, linE, and linF genes to beta-ketoadipate, which is subsequently mineralized. Two or more copies of these genes are present in the bacterium Pseudomonas aeruginosa ITRC-5 that was isolated earlier by selective enrichment on technical-HCH. At least one copy of linA, linB, linC, linD, and possibly linE is lost from ITRC-5 upon its growth on gamma-HCH. All the lin genes, however, are lost when the bacterium was grown in Luria-Bertani (LB) medium. The loss of lin genes is accompanied with the loss/rearrangement of insertion sequence IS6100 genes. Concomitant to the loss of lin genes, the degradation of HCH-isomers by "gamma-HCH grown cells" is slower, when compared with "technical-HCH grown cells", and is completely lost by "LB-grown cells". The selective loss of lin genes during different growth conditions has not been reported before and is expected to help in understanding the dynamism of degradative genes.
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