Improving Hydrolytic Activity and Enantioselectivity of Epoxide Hydrolase from Phanerochaete chrysosporium by Directed Evolution.

Epoxide hydrolases (EHs) catalyze the conversion of epoxides into vicinal diols. The epoxide hydrolase gene from P. chrysosporium was previously cloned and subjected to site-directed mutation to study its enzyme activity, but the results were unsatisfactory. This study used error prone PCR and DNA shuffling to construct a PchEHA mutation library. We performed mutation-site combinations on PchEHA based on enzyme activity measurement results combined with directed evolution technology. More than 15,000 mutants were randomly selected for the preliminary screening of PchEHA enzyme activity alongside 38 mutant strains with increased enzyme activity or enantioselectivity. Protein expression and purification were conducted to determine the hydrolytic activity of PchEHA, and three mutants increased their activity by more than 95% compared with that of the wt. After multiple rounds of screening and site-specific mutagenesis, we found that F3 offers the best enzyme activity and enantioselectivity; furthermore, the molecular docking results confirmed this result. Overall, this study uncovered novel mutants with potential value as industrial biocatalysts.

Linoleic acid-derived diol 12,13-DiHOME enhances NLRP3 inflammasome activation in macrophages.

12,13-dihydroxy-9z-octadecenoic acid (12,13-DiHOME) is a linoleic acid diol derived from cytochrome P-450 (CYP) epoxygenase and epoxide hydrolase (EH) metabolism. 12,13-DiHOME is associated with inflammation and mitochondrial damage in the innate immune response, but how 12,13-DiHOME contributes to these effects is unclear. We hypothesized that 12,13-DiHOME enhances macrophage inflammation through effects on NOD-like receptor protein 3 (NLRP3) inflammasome activation. To test this hypothesis, we utilized human monocytic THP1 cells differentiated into macrophage-like cells with phorbol myristate acetate (PMA). 12,13-DiHOME present during lipopolysaccharide (LPS)-priming of THP1 macrophages exacerbated nigericin-induced NLRP3 inflammasome activation. Using high-resolution respirometry, we observed that priming with LPS+12,13-DiHOME altered mitochondrial respiratory function. Mitophagy, measured using mito-Keima, was also modulated by 12,13-DiHOME present during priming. These mitochondrial effects were associated with increased sensitivity to nigericin-induced mitochondrial depolarization and reactive oxygen species production in LPS+12,13-DiHOME-primed macrophages. Nigericin-induced mitochondrial damage and NLRP3 inflammasome activation in LPS+12,13-DiHOME-primed macrophages were ablated by the mitochondrial calcium uniporter (MCU) inhibitor, Ru265. 12,13-DiHOME present during LPS-priming also enhanced nigericin-induced NLRP3 inflammasome activation in primary murine bone marrow-derived macrophages. In summary, these data demonstrate a pro-inflammatory role for 12,13-DiHOME by enhancing NLRP3 inflammasome activation in macrophages.

Epoxyeicosatrienoic Acids Prevent Cardiac Dysfunction in Viral Myocarditis via Interferon Type I Signaling.

Myocarditis is a challenging inflammatory disease of the heart, and better understanding of its pathogenesis is needed to develop specific drug therapies. Epoxyeicosatrienoic acids (EETs), active molecules synthesized by CYP (cytochrome P450) enzymes from arachidonic acids and hydrolyzed to less active dihydroxyeicosatrienoic acids by sEH (soluble epoxide hydrolase), have been attributed anti-inflammatory activity. Here, we investigated whether EETs have immunomodulatory activity and exert protective effects on coxsackie B3 virus-induced myocarditis. Viral infection altered eicosanoid epoxide and diol levels in both patients with myocarditis and in the murine heart and correlated with the increased expression and activity of sEH after coxsackie B3 virus infection. Administration of a sEH inhibitor prevented coxsackie B3 virus-induced cardiac dysfunction and inflammatory infiltration. Importantly, EET/sEH inhibitor treatment attenuated viral infection or improved viral resistance by activating type I IFN (interferon) signaling. At the molecular level, EETs enhanced the interaction between GSK3ß (glycogen synthase kinase-3 beta) and TBK1 (TANK-binding kinase 1) to promote IFN-ß production. Our findings revealed that EETs and sEH inhibitors prevent the progress of coxsackie B3 virus-induced myocarditis, particularly by promoting viral resistance by increasing IFN production.

Cloning and characterization of Aedes aegypti juvenile hormone epoxide hydrolases (JHEHs).

Juvenile hormone epoxide hydrolase (JHEH) plays an important role in the metabolism of juvenile hormone III (JH III) in insects. To study the role that JHEH plays in female Aedes aegypti JHEH 1, 2, and 3 complementary DNA (cDNAs) were cloned and sequenced. Northern blot analyses show that the three transcripts are expressed in the head thorax, the gut, the ovaries, and the fat body of females. Molecular modeling shows that the enzyme is a homodimer that binds JH III acid (JH IIIA) at the catalytic groove better than JH III. The cDNA of JHEH 1 and 2 are very similar indicating close relationship. Knocking down of jheh 1, 2, and 3 in adult female and larval Ae. aegypti using double-stranded RNA (dsRNA) did not affect egg development or caused adult mortality. Larvae that were fed bacterial cells expressing dsRNA against jheh 1, 2, and 3 grew normally. Treating blood-fed female Ae. aegypti with [12-3 H](10R) JH III and analyzing the metabolites by C18 reversed phase chromatography showed that JHEH preferred substrate is not JH III but JH IIIA. Genomic analysis of jheh 1, 2, and 3 indicate that jheh 1 and 2 are transcribed from a 1.53 kb DNA whereas jheh 3 is transcribed from a 10.9 kb DNA. All three genes are found on chromosome two at distinct locations. JHEH 2 was expressed in bacterial cells and purified by Ni affinity chromatography. Sequencing of the recombinant protein by MS/MS identified JHEH 2 as the expressed recombinant protein.

Cloning and Characterization of Drosophila melanogaster Juvenile Hormone Epoxide Hydrolases (JHEH) and Their Promoters.

Juvenile hormone epoxide hydrolase (JHEH) plays an important role in the metabolism of JH III in insects. To study the control of JHEH in female Drosophila melanogaster, JHEH 1, 2 and 3 cDNAs were cloned and sequenced. Northern blot analyses showed that the three transcripts are expressed in the head thorax, the gut, the ovaries and the fat body of females. Molecular modeling shows that the enzyme is a homodimer that binds juvenile hormone III acid (JH IIIA) at the catalytic groove better than JH III. Analyses of the three JHEH promoters and expressing short promoter sequences behind a reporter gene (lacZ) in D. melanogaster cell culture identified a JHEH 3 promoter sequence (626 bp) that is 10- and 25-fold more active than the most active promoter sequences of JHEH 2 and JHEH 1, respectively. A transcription factor (TF) Sp1 that is involved in the activation of JHEH 3 promoter sequence was identified. Knocking down Sp1 using dsRNA inhibited the transcriptional activity of this promoter in transfected D. melanogaster cells and JH III and 20HE downregulated the JHEH 3 promoter. On the other hand, JH IIIA and farnesoic acid did not affect the promoter, indicating that JH IIIA is JHEH's preferred substrate. A transgenic D. melanogaster expressing a highly activated JHEH 3 promoter behind a lacZ reporter gene showed promoter transcriptional activity in many D. melanogaster tissues.

Plasma Oxylipin Profile Discriminates Ethnicities in Subjects with Non-Alcoholic Steatohepatitis: An Exploratory Analysis.

Non-alcoholic fatty liver disease (NAFLD) is a common liver pathology that includes steatosis, or non-alcoholic fatty liver (NAFL), and non-alcoholic steatohepatitis (NASH). Without a clear pathophysiological mechanism, it affects Hispanics disproportionately compared to other ethnicities. Polyunsaturated fatty acids (PUFAs) and inflammatory lipid mediators including oxylipin (OXL) and endocannabinoid (eCB) are altered in NAFLD and thought to contribute to its pathogenesis. However, the existence of ethnicity-related differences is not clear. We employed targeted lipidomic profiling for plasma PUFAs, non-esterified OXLs and eCBs in White Hispanics (HIS, n = 10) and Caucasians (CAU, n = 8) with biopsy-confirmed NAFL, compared with healthy control subjects (HC; n = 14 HIS; n = 8 CAU). NAFLD was associated with diminished long chain PUFA in HIS, independent of histological severity. Differences in plasma OXLs and eCBs characterized ethnicities in NASH, with lower arachidonic acid derived OXLs observed in HIS. The secondary analysis comparing ethnicities within NASH (n = 12 HIS; n = 17 CAU), confirms these ethnicity-related differences and suggests lower lipoxygenase(s) and higher soluble epoxide hydrolase(s) activities in HIS compared to CAU. While causes are not clear, these lipidomic differences might be with implications for NAFLD severity and are worth further investigation. We provide preliminary data indicating ethnicity-specific lipidomic signature characterizes NASH which requires further validation.

Structure-function relationship between soluble epoxide hydrolases structure and their tunnel network.

Enzymes with buried active sites maintain their catalytic function via a single tunnel or tunnel network. In this study we analyzed the functionality of soluble epoxide hydrolases (sEHs) tunnel network, by comparing the overall enzyme structure with the tunnel's shape and size. sEHs were divided into three groups based on their structure and the tunnel usage. The obtained results were compared with known substrate preferences of the studied enzymes, as well as reported in our other work evolutionary analyses data. The tunnel network architecture corresponded well with the evolutionary lineage of the source organism and large differences between enzymes were observed from long fragments insertions. This strategy can be used during protein re-engineering process for large changes introduction, whereas tunnel modification can be applied for fine-tuning of enzyme.

Oxidative stress-related genes (EPHX1 and MnSOD) polymorphism and risk of pre-eclampsia: a meta-analysis.

BACKGROUND: Previous studies have detected the association of polymorphisms in oxidative stress-related genes EPHX1 and MnSOD with pre-eclampsia (PE) risk, but the results are inconsistent among studies. Thus, a meta-analysis was performed to obtain more conclusive results. METHODS: Eligible studies were retrieved in PubMed, Web of Science, EMBASE, Scopus, and CNKI. Odds ratios (ORs) with 95% confidence intervals (CIs) were utilized to evaluate the relationship between EPHX1 rs1051740, EPHX1 rs2234922, MnSOD rs4880 polymorphisms, and PE susceptibility in the genetic models. The subgroup analysis was also performed. RESULTS: Fourteen studies with a total of 4250 participants were included, including 1784 PE patients and 2466 healthy women. There was a statistically significant association between EPHX1 rs1051740 polymorphism and PE in Caucasians within the allele, dominant, heterozygous, and homozygous models (OR = 0.79, 95% CI = 0.64-0.98; OR = 0.64, 95% CI = 0.47-0.87; OR = 0.61, 95% CI = 0.44-0.85; OR = 0.63, 95% CI = 0.42-0.97, respectively). There was a statistically significant association between EPHX1 rs2234922 polymorphism and PE in Middle Easterners within the recessive and homozygous models (OR = 3.59, 95% CI = 1.25-10.32; OR = 3.99, 95% CI = 1.38-11.49, respectively). There was no statistically significant association between MnSOD rs4880 polymorphism and PE within five genetic models. Subgroup analysis didn't reveal any association between MnSOD rs4880 polymorphism and PE in Asians, Caucasians, or Middle Easterners. CONCLUSIONS: This meta-analysis shows a significant association between the EPHX1 rs1051740 and PE risk in Caucasians. Meantime, there was a statistically significant association between EPHX1 rs2234922 polymorphism and PE in Middle Easterners.

Proteasome Inhibitors and Their Pharmacokinetics, Pharmacodynamics, and Metabolism.

The proteasome is responsible for mediating intracellular protein degradation and regulating cellular function with impact on tumor and immune effector cell biology. The proteasome is found predominantly in two forms, the constitutive proteasome and the immunoproteasome. It has been validated as a therapeutic drug target through regulatory approval with 2 distinct chemical classes of small molecular inhibitors (boronic acid derivatives and peptide epoxyketones), including 3 compounds, bortezomib (VELCADE), carfilzomib (KYPROLIS), and ixazomib (NINLARO), for use in the treatment of the plasma cell neoplasm, multiple myeloma. Additionally, a selective inhibitor of immunoproteasome (KZR-616) is being developed for the treatment of autoimmune diseases. Here, we compare and contrast the pharmacokinetics (PK), pharmacodynamics (PD), and metabolism of these 2 classes of compounds in preclinical models and clinical studies. The distinct metabolism of peptide epoxyketones, which is primarily mediated by microsomal epoxide hydrolase, is highlighted and postulated as a favorable property for the development of this class of compound in chronic conditions.

GSK2256294 Decreases sEH (Soluble Epoxide Hydrolase) Activity in Plasma, Muscle, and Adipose and Reduces F2-Isoprostanes but Does Not Alter Insulin Sensitivity in Humans.

[Figure: see text].

An epoxide hydrolase from endophytic Streptomyces shows unique structural features and wide biocatalytic activity.

The genus Streptomyces is characterized by the production of a wide variety of secondary metabolites with remarkable biological activities and broad antibiotic capabilities. The presence of an unprecedented number of genes encoding hydrolytic enzymes with industrial appeal such as epoxide hydrolases (EHs) reveals its resourceful microscopic machinery. The whole-genome sequence of Streptomyces sp. CBMAI 2042, an endophytic actinobacterium isolated from Citrus sinensis branches, was explored by genome mining, and a putative α/ß-epoxide hydrolase named B1EPH2 and encoded by 344 amino acids was selected for functional and structural studies. The crystal structure of B1EPH2 was obtained at a resolution of 2.2 Šand it was found to have a similar fold to other EHs, despite its hexameric quaternary structure, which contrasts with previously solved dimeric and monomeric EH structures. While B1EPH2 has a high sequence similarity to EHB from Mycobacterium tuberculosis, its cavity is similar to that of human EH. A group of 12 aromatic and aliphatic racemic epoxides were assayed to determine the activity of B1EPH2; remarkably, this enzyme was able to hydrolyse all the epoxides to the respective 1,2-diols, indicating a wide-range substrate scope acceptance. Moreover, the (R)- and (S)-enantiomers of styrene oxide, epichlorohydrin and 1,2-epoxybutane were used to monitor enantiopreference. Taken together, the functional and structural analyses indicate that this enzyme is an attractive biocatalyst for future biotechnological applications.

Genetic and clinical risk factors associated with phenytoin-induced cutaneous adverse drug reactions in Thai population.

OBJECTIVE: This study aimed to describe the genetic and clinical risk factors associated with phenytoin-induced cutaneous adverse drug reactions (PHT-induced cADRs) in Thai patients. METHOD: A retrospective case-control study was conducted among 88 PHT- cADRs (25 SJS/TEN, 37 DRESS/DIHS and 26 MPE) compared to 70 PHT-tolerant controls during 2008-2017. Genotyping was performed by Taqman RT-PCR (EPHX1 337 T > C, EPHX1 416A > G and CYP2C9*3), pyrosequencing (UGT1A1*28, UGT1A1*6) and polymerase chain reaction-sequence-specific oligonucleotide probe (HLA-B). Chi-squared test and binary logistic regression were used to identify factors associated with PHT-cADRs. RESULTS: Multivariate analysis showed that HLA-B*46:01 was significantly associated with all PHT-induced cADRs (OR 2.341; 95% CI, 1.078-5.084; P = .032). Age of ≥60 years showed a significant association with PHT-induced SJS/TEN (OR 3.600; 95% CI, 1.214-10.672; P = .021). CYP2C9*3 was almost reaching statistically associated with an increased risk of PHT-induced SJS/TEN (OR 4.800; 95% CI, 0.960-23.990; P = .056). While HLA-B*56:02/04 was found to have a significant association with PHT-induced DRESS/DIHS (OR 29.312; 95% CI, 1.213-707.994; P = .038). Moreover, female gender and HLA-B*40:01 were associated with an increased risk of PHT-induced MPE at OR 5.734; 95% CI, 0.910-58.351; P = .042 and OR 3.647; 95% CI, 1.193-11.147; P = .023, respectively. CONCLUSION: Both clinical (advanced age, female gender) and genetic factors (HLA-B*46:01, CYP2C9*3, HLA-B*56:02/04 and HLA-B*40:01) contributed to the risk of PHT-induced cADRs. Further studies with larger sample size may be warranted to confirm these findings and also the influence of EPHX1 gene.

Studies on the Catalytic Promiscuity of Limonene Epoxide Hydrolases in the Non-hydrolytic Ring Opening of 1,2-Epoxides.

The non-hydrolytic ring opening of 1,2-epoxides in the presence of limonene epoxide hydrolases (LEHs) and different nucleophiles has been investigated. Lyophilized, wild-type LEHs were tested in selected water-saturated organic solvents in the presence of cyclohexene oxide as substrate and different alcohols, thiols and primary amines as nucleophiles. Although the LEHs retained an appreciable catalytic activity under different reaction conditions, formation of the desired 1,2-substituted cyclohexanols was not observed. Alternatively, LEH variants incapable of performing the hydrolytic reaction were generated by site-directed mutagenesis and tested in aqueous media in the presence of different water-soluble nucleophiles and cyclohexene oxide. Under defined reaction conditions, an acceleration of up to about threefold of the spontaneous reaction rate was observed in the presence of sodium azide and potassium thiocyanate as nucleophiles.

[Study on the relationship between EPHX1 gene polymorphism and antioxidant capacity in patients with chronic obstructive pulmonary disease].

Objective: To explore the difference of mRNA, protein expression levels and the indexes of peripheral blood antioxidant capacity in peripheral blood lymphocytes of different EPHX1 genotypes in chronic obstructive pulmonary disease(COPD). Methods: A case-control study was conducted to collect peripheral blood samples of 220 stable chronic COPD patients with smoking history and 230 healthy smokers (control group) from October 2016 to February 2018 in the First Affiliated Hospital of Kunming Medical University, and the genetic testing was carried out according to the operation instructions of BigDye Terminator v1.1 DNA Sequencing Kit. Based on their EPHX1 exon 3 and exon 4 polymorphism status, the EPHX1 was classified into 4 groups, i. e., normal activity, slow activity, extremely slow activity and fast activity. Then COPD patients were allocated to either a slow activity group (slow and very slow activity) or a fast activity group (normal and fast activity) according to EPHX1 genotype and gene activity. The expression of EPHX1 mRNA and protein in peripheral blood lymphocytes were detected by qRT-PCR and Western blot, and indexes of serum antioxidant capacity was detected by corresponding kits. Results: (1)The 2(-ΔΔCt) of the control group was 1.000, and the 2(-ΔΔCt) of the COPD group was 1.052±0.023. There was no significant difference in the level of EPHX1 mRNA expression between the two groups (t=1.992 P=0.865). The level of EPHX1 mRNA expression in the slow activity group was not different significantly compared to that in the fast-active group (1.053±0.023 vs 1.048±0.021, t=1.133, P=0.260). (2)The level of EPHX1 protein expression by Western blot analysis showed that the EHPX1/GAPDH gray ratio was not different significantly between the COPD group and the control group (0.613±0.089 vs 0.602±0.075, t=0.805, P=0.422). The level of EPHX1 protein expression in the slow activity group was not significantly different compared to that in the fast activity group (0.606±0.088 vs 0.622±0.092, t=-0.786 P=0.434). (3)There were significant differences in indexes of antioxidant capacity between the control group and the COPD group (P<0.05). There were significant differences in indexes of antioxidant capacity between the slow activity group and the fast activity group of COPD patients (P<0.05). Conclusions: The different antioxidant capacity of COPD patients with different EPHX1 genotypes may be related to the polymorphism of EPHX1 gene affecting the activity of microsomal epoxidase, but not to the level of EPHX1 mRNA and protein expression.

Exploring Solanum tuberosum Epoxide Hydrolase Internal Architecture by Water Molecules Tracking.

Several different approaches are used to describe the role of protein compartments and residues in catalysis and to identify key residues suitable for the modification of the activity or selectivity of the desired enzyme. In our research, we applied a combination of molecular dynamics simulations and a water tracking approach to describe the water accessible volume of Solanum tuberosum epoxide hydrolase. Using water as a molecular probe, we were able to identify small cavities linked with the active site: (i) one made up of conserved amino acids and indispensable for the proper positioning of catalytic water and (ii) two others in which modification can potentially contribute to enzyme selectivity and activity. Additionally, we identified regions suitable for de novo tunnel design that could also modify the catalytic properties of the enzyme. The identified hot-spots extend the list of the previously targeted residues used for modification of the regioselectivity of the enzyme. Finally, we have provided an example of a simple and elegant process for the detailed description of the network of cavities and tunnels, which can be used in the planning of enzyme modifications and can be easily adapted to the study of any other protein.

Epoxide Hydrolase Conformational Heterogeneity for the Resolution of Bulky Pharmacologically Relevant Epoxide Substrates.

The conformational landscape of Bacillus megaterium epoxide hydrolase (BmEH) and how it is altered by mutations that confer the enzyme the ability to accept bulky epoxide substrates has been investigated. Extensive molecular dynamics (MD) simulations coupled to active site volume calculations have unveiled relevant features of the enzyme conformational dynamics and function. Our long-timescale MD simulations identify key conformational states not previously observed by means of X-ray crystallography and short MD simulations that present the loop containing one of the catalytic residues, Asp239, in a wide-open conformation, which is likely involved in the binding of the epoxide substrate. Introduction of mutations M145S and F128A dramatically alters the conformational landscape of the enzyme. These singly mutated variants can accept bulky epoxide substrates due to the disorder induced by mutation in the α-helix containing the catalytic Tyr144 and some parts of the lid domain. These changes impact the enzyme active site, which is substantially wider and more complementary to the bulky pharmacologically relevant epoxide substrates.

Anti-tubercular activity of a natural stilbene and its synthetic derivatives.

Objectives: Tuberculosis (TB) and multidrug- and extensively drug-resistant TB in particular are remaining a major global health challenge and efficient new drugs against TB are needed. This study evaluated the anti-tubercular activity of a natural stilbene and its synthetic derivatives against M. tuberculosis. Methods: Isopropylstilbene and its synthetic derivatives were analyzed for their anti-tubercular activity against M. tuberculosis ATCC 27294 as well as multidrug- and extensively drug-resistant M. tuberculosis clinical isolates by using MGIT 960 instrumentation and EpiCenter software equipped with TB eXiST module. Cytotoxic effects of drug candidates were determined by a MTT dye reduction assay using A549 adenocarcinomic human alveolar basal epithelial cells. Results: Growth of M. tuberculosis ATCC 27294 was suppressed by the natural isopropylstilbene HB64 as well as synthetic derivatives DB56 and DB55 at 25 µg/ml. Growth of clinical isolates MDR and XDR M. tuberculosis was suppressed by HB64 at 100 µg/ml as well as by synthetic derivatives DB56 and DB55 at 50 µg/ml and 25 µg/ml, respectively. No anti-tubercular activity was demonstrated for synthetic derivatives DB53, EB251, and RB57 at 100 µg/ml. Toxicity in terms of IC50 values of HB64, DB55 and DB56 were 7.92 µg/ml, 12.15 µg/ml and 16.01 µg/ml, respectively. Conclusions: Synthetical derivatives of stilbene might be effective candidates as anti-tubercular drugs. However, toxicity of these substances as determined by IC50 values might limit therapeutic success in vivo. Further investigations should address lowering the toxicity for parenteral administration by remodeling stilbene derivatives.

Improving the 'tool box' for robust industrial enzymes.

The speed of sequencing of microbial genomes and metagenomes is providing an ever increasing resource for the identification of new robust biocatalysts with industrial applications for many different aspects of industrial biotechnology. Using 'natures catalysts' provides a sustainable approach to chemical synthesis of fine chemicals, general chemicals such as surfactants and new consumer-based materials such as biodegradable plastics. This provides a sustainable and 'green chemistry' route to chemical synthesis which generates no toxic waste and is environmentally friendly. In addition, enzymes can play important roles in other applications such as carbon dioxide capture, breakdown of food and other waste streams to provide a route to the concept of a 'circular economy' where nothing is wasted. The use of improved bioinformatic approaches and the development of new rapid enzyme activity screening methodology can provide an endless resource for new robust industrial biocatalysts.This mini-review will discuss several recent case studies where industrial enzymes of 'high priority' have been identified and characterised. It will highlight specific hydrolase enzymes and recent case studies which have been carried out within our group in Exeter.

CYP1A1, CYP2E1 and EPHX1 polymorphisms in sporadic colorectal neoplasms.

AIM: To investigate the contribution of polymorphisms in the CYP1A1, CYP2E1 and EPHX1 genes on sporadic colorectal cancer (SCRC) risk. METHODS: Six hundred forty-one individuals (227 patients with SCRC and 400 controls) were enrolled in the study. The variables analyzed were age, gender, tobacco and alcohol consumption, and clinical and histopathological tumor parameters. The CYP1A1*2A, CYP1A1*2C CYP2E1*5B and CYP2E1*6 polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The EPHX1 Tyr113His, EPHX1 His139Arg and CYP1A1*2C polymorphisms were detected by real-time PCR. Chi-squared test and binary logistic regression were used in the statistical analysis. Haplotype analysis was conducted using the Haploview program, version 2.05. RESULTS: Age over 62 years was a risk factor for SCRC development (OR = 7.54, 95%CI: 4.94-11.50, P < 0.01). Male individuals were less susceptible to SCRC (OR = 0.55, 95%CI: 0.35-0.85, P < 0.01). The CYP2E1*5B polymorphism was associated with SCRC in the codominant (heterozygous genotype: OR = 2.66, 95%CI: 1.64-4.32, P < 0.01), dominant (OR = 2.82, 95%CI: 1.74-4.55, P < 0.01), overdominant (OR = 2.58, 95%CI: 1.59-4.19, P < 0.01), and log-additive models (OR = 2.84, 95%CI: 1.78-4.52, P < 0.01). The CYP2E1*6 polymorphism was associated with an increased SCRC risk in codominant (heterozygous genotype: OR = 2.81, 95%CI: 1.84-4.28, P < 0.01; homozygous polymorphic: OR = 7.32, 95%CI: 1.85-28.96, P < 0.01), dominant (OR = 2.97, 95%CI: 1.97-4.50, P < 0.01), recessive (OR = 5.26, 95%CI: 1.35-20.50, P = 0.016), overdominant (OR = 2.64, 95%CI: 1.74-4.01, P < 0.01), and log-additive models (OR = 2.78, 95%CI: 1.91-4.06, P < 0.01). The haplotype formed by the minor alleles of the CYP2E1*5B (C) and CYP2E1*6 (A) polymorphisms was associated with SCRC (P = 0.002). However, the CYP1A1*2A, CYP1A1*2C, EPHX1 Tyr113His and EPHX1 His139Arg polymorphisms were not associated with SCRC. CONCLUSION: In conclusion, the results demonstrated that CYP2E1*5B and CYP2E1*6 minor alleles play a role in the development of SCRC.

Comparing Different Strategies in Directed Evolution of Enzyme Stereoselectivity: Single- versus Double-Code Saturation Mutagenesis.

Saturation mutagenesis at sites lining the binding pockets of enzymes constitutes a viable protein engineering technique for enhancing or inverting stereoselectivity. Statistical analysis shows that oversampling in the screening step (the bottleneck) increases astronomically as the number of residues in the randomization site increases, which is the reason why reduced amino acid alphabets have been employed, in addition to splitting large sites into smaller ones. Limonene epoxide hydrolase (LEH) has previously served as the experimental platform in these methodological efforts, enabling comparisons between single-code saturation mutagenesis (SCSM) and triple-code saturation mutagenesis (TCSM); these employ either only one or three amino acids, respectively, as building blocks. In this study the comparative platform is extended by exploring the efficacy of double-code saturation mutagenesis (DCSM), in which the reduced amino acid alphabet consists of two members, chosen according to the principles of rational design on the basis of structural information. The hydrolytic desymmetrization of cyclohexene oxide is used as the model reaction, with formation of either (R,R)- or (S,S)-cyclohexane-1,2-diol. DCSM proves to be clearly superior to the likewise tested SCSM, affording both R,R- and S,S-selective mutants. These variants are also good catalysts in reactions of further substrates. Docking computations reveal the basis of enantioselectivity.