Sequence and structure of epoxide hydrolases: a systematic analysis

Epoxide hydrolases (EC 3.3.2.3) are ubiquitous enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. More than 100 epoxide hydrolases (EH) have been identified or predicted, and 3 structures are available. Although they catalyze the same chemical reaction, sequence similarity is low. To identify conserved regions, all EHs were aligned. Phylogenetic analysis identified 12 homologous families, which were grouped into 2 major superfamilies: the microsomal EH superfamily, which includes the homologous families of Mammalian, Insect, Fungal, and Bacterial EHs, and the cytosolic EH superfamily, which includes Mammalian, Plant, and Bacterial EHs. Bacterial EHs show a high sequence diversity. Based on structure comparison of three known structures from Agrobacterium radiobacter AD1 (cytosolic EH), Aspergillus niger (microsomal EH), Mus musculus (cytosolic EH), and multisequence alignment and phylogenetic analysis of 95 EHs, the modular architecture of this enzyme family was analyzed. Although core and cap domain are highly conserved, the structural differences between the EHs are restricted to only two loops: the NC-loop connecting the core and the cap and the cap-loop, which is inserted into the cap domain. EHs were assigned to either of three clusters based on loop length. By using this classification, core and cap region of all EHs, NC-loops and cap-loops of 78% and 89% of all EHs, respectively, could be modeled. Representative models are available from the Lipase Engineering Database, http://www.led.uni-stuttgart.de.     

Stereoselectivities of microbial epoxide hydrolases

Epoxide hydrolases from bacterial and fungal sources are highly versatile biocatalysts for the asymmetric hydrolysis of epoxides on a preparative scale. Besides kinetic resolution, which yields the corresponding enantiomerically enriched vicinal diol and the remaining nonconverted epoxide, enantioconvergent processes are also possible, which lead to the formation of a single enantiomeric diol from a racemic oxirane. The data available to date indicate that the enantioselectivities of enzymes from certain microbial sources can be correlated to the substitutional pattern of various types of substrates: red yeasts (e.g. Rhodotorula or Rhodosporidium sp.) give best enantioselectivities with monosubstituted oxiranes; fungal cells (e.g. from Aspergillus and Beauveria sp.) are best suited for styrene oxide-type substrates; bacterial enzymes, on the other hand (in particular from Actinomycetes such as Rhodococcus and Nocardia sp.) are the biocatalysts of choice for more highly substituted 2,2- and 2,3-disubstituted epoxides.     

Synthetic applications of epoxide hydrolases

There have been several recent advances in the area of biocatalysed hydrolytic kinetic resolution of epoxides using 'newly discovered' enzymes (i.e. epoxide hydrolases). These biocatalysts, two of which will become commercially available in the near future, appear to be highly promising tools for fine organic synthesis, as they enable the preparation of various epoxides and/or their corresponding diols in enantiopure form.     

Bacterial epoxide hydrolases of opposite enantiopreference

Epoxide hydrolases of matching opposite enantiopreference were found among various Actinomyces spp. While (S)-2,2-disubstituted oxiranes were hydrolyzed by Rhodococcus and Nocardia spp., several strains of methylotrophic bacteria, such as Mycoplana rubra and Methylobacterium spp., exhibited a preference for the (R)-enantiomers. Thus, the stereochemical course of the reaction can be controlled by a simple choice of the appropriate enzyme source.     

Role of epoxide hydrolases in lipid metabolism

Epoxide hydrolases (EH), enzymes present in all living organisms, transform epoxide-containing lipids to 1,2-diols by the addition of a molecule of water. Many of these oxygenated lipid substrates have potent biological activities: host defense, control of development, regulation of blood pressure, inflammation, and pain. In general, the bioactivity of these natural epoxides is significantly reduced upon metabolism to diols. Thus, through the regulation of the titer of lipid epoxides, EHs have important and diverse biological roles with profound effects on the physiological state of the host organism. This review will discuss the biological activity of key lipid epoxides in mammals. In addition, the use of EH specific inhibitors will be highlighted as possible therapeutic disease interventions.     

Peptidyl-urea based inhibitors of soluble epoxide hydrolases

We prepared a series of amino acid derived cyclohexyl and adamantyl ureas and tested them as inhibitors of the human soluble epoxide hydrolase, and obtained very potent compounds (K(I)=15nM) that are >10-fold more soluble than previously described sEH inhibitors. While our lead compound 2 showed low apparent bioavailability in dogs and rats, this series of compounds revealed that sEH inhibitor structures could accept large groups that could lead to better orally available drugs.     

Immunochemical characterization of human lung epoxide hydrolases

Immunochemical techniques were used to investigate the biochemical properties of human lung epoxide hydrolases. Two epoxide hydrolases with different immunoreactive properties were identified. These two epoxide hydrolases were found in both cytosolic and microsomal cell fractions. Immunotitration of enzyme activity showed that enzymes that catalyze the hydration of benzo(a)pyrene 4,5-oxide react with antiserum to rat microsomal epoxide hydrolase; those that hydrate trans-stilbene oxide do not. Immunotitration and Western blot experiments showed that microsomal and cytosolic benzo(a)pyrene 4,5-oxide hydrolases have significant structural homology. Immunohistochemical staining of human lung benzo(a)pyrene 4,5-oxide hydrolase showed that the enzyme is localized primarily in the bronchial epithelium. No cell type-specific localization was observed. An enzyme-linked immunosorbent assay was developed which allows direct quantitation of benzo(a)pyrene 4,5-oxide hydrolase protein. Levels of enzyme protein detected by this assay correlated well with enzyme levels determined by substrate conversion assays.     

Putative lipoproteins of Streptococcus agalactiae identified by bioinformatic genome analysis

Streptococcus agalactiae is a significant pathogen causing invasive disease in neonates and thus an understanding of the molecular basis of the pathogenicity of this organism is of importance. N-terminal lipidation is a major mechanism by which bacteria can tether proteins to membranes. Lipidation is directed by the presence of a cysteine-containing 'lipobox' within specific signal peptides and this feature has greatly facilitated the bioinformatic identification of putative lipoproteins. We have designed previously a taxon-specific pattern (G+LPP) for the identification of Gram-positive bacterial lipoproteins, based on the signal peptides of experimentally verified lipoproteins (Sutcliffe I.C. and Harrington D.J. Microbiology 148: 2065-2077). Patterns searches with this pattern and other bioinformatic methods have been used to identify putative lipoproteins in the recently published genomes of S. agalactiae strains 2603/V and NEM316. A core of 39 common putative lipoproteins was identified, along with 5 putative lipoproteins unique to strain 2603/V and 2 putative lipoproteins unique to strain NEM316. Thus putative lipoproteins represent ca. 2% of the S. agalactiae proteome. As in other Gram-positive bacteria, the largest functional category of S. agalactiae lipoproteins is that predicted to comprise of substrate binding proteins of ABC transport systems. Other roles include lipoproteins that appear to participate in adhesion (including the previously characterised Lmb protein), protein export and folding, enzymes and several species-specific proteins of unknown function. These data suggest lipoproteins may have significant roles that influence the virulence of this important pathogen.     

Kinetic resolution of racemic leukotriene A4 by mammalian cytosolic epoxide hydrolases

Cytosols of rat and guinea pig liver and of human placenta were screened for their capacity to catalyze the conversion of racemic leukotriene A4 into 5S, 12R-dihydroxy-(Z,E,E,Z)-6,8,10,14-eicosatetraenoic acid (leukotriene B4). The epoxide hydrolase activities showed some specificity for the 5S,6S-oxido-(E,E,Z,Z)-7,9,11,14-eicosatetraenoic acid (LTA4) and produced mixtures of leukotriene B4 and its enantiomer containing up to 78-87% of leukotriene B4.     

Novel microbial epoxide hydrolases for biohydrolysis of glycidyl derivatives

Microbial isolates from biofilters and petroleum-polluted bioremediation sites were screened for the presence of enantioselective epoxide hydrolases active towards tert-butyl glycidyl ether, benzyl glycidyl ether, and allyl glycidyl ether. Out of 270 isolated strains, which comprised bacteria, yeasts, and filamentous fungi, four were selected based on the enantioselectivities of their epoxide hydrolases determined in biotransformation reactions. The enzyme of Aspergillus niger M200 preferentially hydrolyses (S)-tert-butyl glycidyl ether to (S)-3-tert-butoxy-1,2-propanediol with a relatively high enantioselectivity (the enantiomeric ratio E is about 30 at a reaction temperature of 28 °C). Epoxide hydrolases of Rhodotorula mucilaginosa M002 and Rhodococcus fascians M022 hydrolyse benzyl glycidyl ether with relatively low enantioselectivities, the former reacting predominantly with the (S)-enantiomer, the latter preferring the (R)-enantiomer. Enzymatic hydrolysis of allyl glycidyl ether by Cryptococcus laurentii M001 proceeds with low enantioselectivity (E = 3). (R)-tert-Butyl glycidyl ether with an enantiomeric excess (ee) of over 99%, and (S)-3-tert-butoxy-1,2-propanediol with an ee-value of 86% have been prepared on a gram-scale using whole cells of A. niger M200. An enantiomeric ratio of approximately 100 has been determined under optimised biotransformation conditions with the partially purified epoxide hydrolase from A. niger M200. The regioselectivity of this enzyme was determined to be total for both (S)-tert-butyl glycidyl ether and (R)-tert-butyl glycidyl ether.     

用生物信息学方法预测猪链球菌2型05ZYH33株的脂蛋白

[目的]鉴别已公布基因组序列的SS2 05ZYH33株的脂蛋白(Lpp).[方法]首先从Genbank获取由 SS2 05ZYH33基因组推测的蛋白质氨基酸序列,然后利用Lpp预测软件PrositeScan和DOLOP预测05ZYH33株的Lpp,采用SignalP 3.0 HMM、PrediSi、Phobius、LipoP-HMM和TMHMMversion2.0分析预测的Lpp的信号肽,然后经"多数票决法"确定Lpp;最后利用InterProScan和BlastP服务器对鉴定的Lpp进行功能分析.[结果]鉴定出34种Lpp,占SS2致病株05ZYH33蛋白质组的1.555%.结构与功能分析结果表明,最大的一类Lpp是ABC运输蛋白的底物结合蛋白,共有16种,其中YP_001197586、YP_001197918、YP_001198449、YP_001199435、YP_001197482、YP_001199452和YP_001198914等7种基因可与其他成分组成完整的ABC运输蛋白操纵子,这些Lpp在SS2获取糖、氨基酸和金属离子等营养物质方面具有重要作用.YP_001197698与无乳链球菌的黏附素Lmb和化脓链球菌的黏附素Lbp及YP_001198710与肺炎链球菌的SlrA高度同源,推测它们与黏附功能有关,为SS2新的毒力因子;其他Lpp包括酶、参与蛋白质折叠过程的Lpp及功能未知的Lpp.[结论]这些资料提示Lpp在SS2的生理和致病性方面具有重要作用.     

Cyclopropyl oxiranes: reversible inhibitors of cytosolic and microsomal epoxide hydrolases

A series of aryl- and alkyl-substituted cyclopropyl oxiranes were synthesized as potential suicide inhibitors of mouse liver epoxide hydrolase (EH). The inhibitory potency of each compound and its corresponding alkene precursor was determined with mouse liver EHs using [3H]-cis-stilbene oxide as substrate for microsomal EH (mEH) and for glutathione-S-transferase, and using [3H]-trans-stilbene oxide for cytosolic EH (cEH). The cyclopropyl oxiranes all showed low (26-60% at 5 X 10(-4) M) inhibition of glutathione transferase and moderate inhibition (I50 = 5 X 10(-4) to 6 X 10(-6) M) for cEH and mEH. cis-Phenylcyclopropyl oxirane had an I50 for mEH near that for a commonly used inhibitor, 1,1,1-trichloropropene oxide. Inhibition appeared competitive and reversible, and the cyclopropyl oxiranes appeared to function as alternate substrates. Absence of irreversible inhibition is evidence against a strongly electrophilic epoxide-opening mechanism involving a cyclopropyl carbinyl-homoallyl cation rearrangement. Instead, a concerted mechanism is favored, in which electrophilic opening and hydroxide attack occur in a concerted fashion.     

Radiometric assays for mammalian epoxide hydrolases and glutathione S-transferase

A number of epoxides, including cis- and trans-stilbene oxides, were assayed as substrates for epoxide hydrolases (EHs) by gas-liquid chromatography. Radiolabeled stilbene oxides were prepared by sodium borotritide reduction of desyl chloride followed by ring closure with base treatment. Rapid radiometric assays for EHs were performed by differential partitioning of the epoxide into dodecane, while the product diol remained in the aqueous phase. Glutathione (GSH) transferase was similarly assayed by partitioning the epoxide and diol, if formed metabolically, into 1-hexanol, while the GSH conjugate was retained in the aqueous phase. The cytosolic EH rapidly hydrates the trans isomer while the cis is very poorly hydrated. In contrast, the cis is a better substrate for the microsomal EH than the trans. GSH transferase utilized both epoxides as substrates, but conjugation is faster with the cis isomer. Cytosolic EH activity is high in mouse but very low in rat and guinea pig. Microsomal EH activity, in contrast, is highest in guinea pig, intermediate in rat, and the lowest in mouse. GSH transferase activity, which is high in all three species, can be inhibited by chalcone, with an I₅₀ of 3.1 × 10^?5m. These assays facilitate the rapid evaluation and direct comparison of epoxide-metabolizing systems in cell homogenates used in short-term mutagenicity assays, cell or organ culture, and possibly in vivo.     

Human-specific nonsense mutations identified by genome sequence comparisons

The comparative study of the human and chimpanzee genomes may shed light on the genetic ingredients for the evolution of the unique traits of humans. Here, we present a simple procedure to identify human-specific nonsense mutations that might have arisen since the human–chimpanzee divergence. The procedure involves collecting orthologous sequences in which a stop codon of the human sequence is aligned to a non-stop codon in the chimpanzee sequence and verifying that the latter is ancestral by finding homologs in other species without a stop codon. Using this procedure, we identify nine genes (CML2, FLJ14640, MT1L, NPPA, PDE3B, SERPINA13, TAP2, UIP1, and ZNF277) that would produce human-specific truncated proteins resulting in a loss or modification of the function. The premature terminations of CML2, MT1L, and SERPINA13 genes appear to abolish the original function of the encoded protein because the mutation removes a major part of the known active site in each case. The other six mutated genes are either known or presumed to produce functionally modified proteins. The mutations of five genes (CML2, FLJ14640, MT1L, NPPA, TAP2) are known or predicted to be polymorphic in humans. In these cases, the stop codon alleles are more prevalent than the ancestral allele, suggesting that the mutant alleles are approaching fixation since their emergence during the human evolution. The findings support the notion that functional modification or inactivation of genes by nonsense mutation is a part of the process of adaptive evolution and acquisition of species-specific features. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Novel estrogen-related genes and potential biomarkers of ovarian endometriosis identified by differential expression analysis

In the search for novel biomarkers of endometriosis, we selected 152 genes from the GeneLogic database based on results of genome-wide expression analysis of ovarian endometriosis, plus 20 genes related to estrogen metabolism and action. We then performed low-density array analysis of these 172 genes on 11 ovarian endometriosis samples and 9 control endometrium samples. Principal component analysis of the gene expression levels showed clear separation between the endometriosis and control groups. We identified 78 genes as differentially expressed. Based on Ingenuity pathway analysis, these differentially expressed genes were arranged into groups according to biological function. These analyses revealed that 32 differentially expressed genes are estrogen related, 23 of which have not been reported previously in connection with endometriosis. Functional annotation showed that 25 and 22 genes are associated with the biological terms "secreted" and "extracellular region", respectively. Differential expression of 4 out of 5 genes related to estrogen metabolism and action (ESR1, ESR2, PGR and BGN) was also confirmed by immunohistochemistry. Our study thus reveals differential expression of several genes that have not previously been associated with endometriosis and that encode potential novel biomarkers and drug targets.     

Comparative analysis of a cryptic thienamycin-like gene cluster identified in Streptomyces flavogriseus by genome mining

In silico database searches allowed the identification in the S. flavogriseus ATCC 33331 genome of a carbapenem gene cluster highly related to the S. cattleya thienamycin one. This is the second cluster found for a complex highly substituted carbapenem. Comparative analysis revealed that both gene clusters display a high degree of synteny in gene organization and in protein conservation. Although the cluster appears to be silent under our laboratory conditions, the putative metabolic product was predicted from bioinformatics analyses using sequence comparison tools. These data, together with previous reports concerning epithienamycins production by S. flavogriseus strains, suggest that the cluster metabolic product might be a thienamycin-like carbapenem, possibly the epimeric epithienamycin. This finding might help in understanding the biosynthetic pathway to thienamycin and other highly substituted carbapenems. It also provides another example of genome mining in Streptomyces sequenced genomes as a powerful approach for novel antibiotic discovery.     

The database of epoxide hydrolases and haloalkane dehalogenases: one structure, many functions

The epoxide hydrolases and haloalkane dehalogenases database (EH/HD) integrates sequence and structure of a highly diverse protein family, including mainly the Asp-hydrolases of EHs and HDs but also proteins, such as Ser-hydrolases non-heme peroxidases, prolyl iminopetidases and 2-hydroxymuconic semialdehyde hydrolases. These proteins have a highly conserved structure, but display a remarkable diversity in sequence and function. A total of 305 protein entries were assigned to 14 homologous families, forming two superfamilies. Annotated multisequence alignments and phylogenetic trees are provided for each homologous family and superfamily. Experimentally derived structures of 19 proteins are superposed and consistently annotated. Sequence and structure of all 305 proteins were systematically analysed. Thus, deeper insight is gained into the role of a highly conserved sequence motifs and structural elements. AVAILABILITY: The EH/HD database is available at http://www.led.uni-stuttgart.de     

Expression of microsomal and cytosolic epoxide hydrolases in cultured rat hepatocytes and hepatoma cell lines

Microsomal epoxide hydrolase activity, determined using benzpyrene 4,5-oxide and styrene 7,8-oxide, increased in cultured hepatocytes compared to freshly isolated cells. In contrast, cytosolic epoxide hydrolase activity, assayed using trans-stilbene oxide, had decreased 80% by 24 hr and was barely detectable after 96 hr in culture. There was no difference in enzyme activity between freshly isolated hepatocytes and the two rat hepatoma cell lines McA-RH 7777 and H4-II-E, when styrene 7,8-oxide was used as substrate. However, benzpyrene 4,5-oxide hydrolase activity of the McA-RH 7777 and H4-II-E cell lines were 55 and 10%, respectively, of freshly isolated hepatocytes. These results show that hepatoma cell lines provide a suitable system for studying the regulation of both the microsomal and cytosolic epoxide hydrolase enzymes.     

Genetic variants in epoxide hydrolases modify the risk of oligozoospermia and asthenospermia in Han-Chinese population

Objectives

Epoxide hydrolases are involved in detoxifying and excreting the environmental chemicals, which are associated with decreased semen quality and male infertility. We hypothesized that polymorphisms in epoxide hydrolases may be associated with risk of oligozoospermia and asthenospermia.

Design and methods

In this study, 468 fertile controls and 672 idiopathic male infertile patients were recruited. SNPstream and TaqMan assay were used to genotype four single nucleotide polymorphisms in EPHX1 and EPHX2. The semen analysis was performed by computer-assisted semen analysis system.

Results

Our results demonstrated that rs1042064 of EPHX2 was significantly associated with decreased risk of oligozoospermia (OR = 0.65, 95% CI: 0.44–0.98) and asthenospermia (OR = 0.66, 95% CI: 0.46–0.94).

Conclusions

Our results provided evidence that genetic variants in epoxide hydrolases may modify the risk of oligozoospermia and asthenospermia in Han-Chinese population.