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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To cite this article: Lindahl TL, Macwan AS, Ramstr€ om S. Protease-activated receptor 4 is more important than protease-activated receptor 1 for the thrombin-induced procoagulant effect on platelets. J Thromb Haemost 2016; 14:1639-41.See also French SL, Arthur JF, Lee H, Nesbitt WS, Andrews RK, Gardiner EE, Hamilton JR. Inhibition of protease-activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood. This issue, pp 1642-54.Thrombin is the most important enzyme in coagulation and also a potent activator of platelets. It activates platelets via N-terminal cleavage of protease-activated receptors (PARs) 1 and 4. Although structurally similar, both PARs have distinct and complementary roles; for example, PAR1 is more sensitive than PAR4 to low concentrations of thrombin [1], whereas PAR4 gives rise to a more prolonged calcium mobilization after activation compared with a transient 'spike' by PAR1 [2]. Moreover, PAR4 is more important for clot lysis resistance [3]. After activation, a subpopulation of the platelets become procoagulant by allowing the formation of coagulation factor complexes on their surface. They do not participate in aggregation but facilitate generation of even more thrombin and, consequently, amplification of the response [4]. Experimental data suggest that collagen exposed during vessel damage in combination with thrombin is the most potent trigger for formation of procoagulant platelets [5], but the signaling processes regulating the formation of the aggregatory and procoagulant platelet subpopulations are still not known in detail.The article by Hamilton and co-workers in this issue of JTH [6] shows for the first time that activation of PAR4 is crucial for the thrombin-induced activation of platelets in an in vitro thrombus model; that is, platelets adhered on a collagen surface in a flow chamber system. Two tools were essential for this study, a rabbit polyclonal antibody directed towards the cleavage site for thrombin at the N-terminal of PAR4 and a fluorescence resonance energy transfer-based thrombin activity sensor linked to an anti-CD41a antibody, to prevent washing out by the flowing blood, which was invented by Welsh et al. [7]. The anti-PAR4 antibody only attenuated the peak of the thrombin-induced cytosolic calcium but abolished the sustained elevation of cytosolic calcium; thus PAR4 was responsible for the sustained elevation, in accordance with previous studies [8][9][10][11][12]. Presumably this sustained elevation is essential for the creation of the negatively charged surface and the procoagulant activity, which is supported by the known very strong effect in this direction by the calcium ionophore A23187 (see Fig. 4G in [6] and [13]).The relative importance of PAR1 and PAR4 for different aspects of platelet activation is far from clear; published studies have shown various, even contradicting, results. To be able to answer questions regarding the relative importance of PAR1 and PAR4 in the platelet procoagulant response, we need to st...

As platelet activation is an irreversible and potentially harmful event, platelet stimulatory signaling must be tightly regulated to ensure the filtering-out of inconsequential fluctuations of agonist concentrations in the vascular milieu. Herein, we show that platelet activation via G protein-coupled receptors is gradient-dependent, i.e., determined not only by agonist concentrations per se but also by how rapidly concentrations change over time. We demonstrate that gradient-dependent inhibition is a common feature of all major platelet stimulatory G protein-coupled receptors, while platelet activation via the non-G protein-coupled receptor glycoprotein VI is strictly concentration-dependent. By systematically characterizing the effects of variations in temporal agonist concentration gradients on different aspects of platelet activation, we demonstrate that gradient-dependent inhibition of protease-activated receptors exhibits different kinetics, with platelet activation occurring at lower agonist gradients for protease-activated receptor 4 than for protease-activated receptor 1, but shares a characteristic bimodal effect distribution, as gradient-dependent inhibition increases over a narrow range of gradients, below which aggregation and granule secretion is effectively shut off. In contrast, the effects of gradient-dependent inhibition on platelet activation via adenosine diphosphate and thromboxane receptors increase incrementally over a large range of gradients. Furthermore, depending on the affected activation pathway, gradient-dependent inhibition results in different degrees of refractoriness to subsequent autologous agonist stimulation. Mechanistically, our study identifies an important role for the cyclic adenosine monophosphate-dependent pathway in gradient-dependent inhibition. Together, our findings suggest that gradient-dependent inhibition may represent a new general mechanism for hemostatic regulation in platelets.