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Sample records for adohcy hydrolase inhibitors

  1. A molecular model for the active site of S-adenosyl- l-homocysteine hydrolase

    NASA Astrophysics Data System (ADS)

    Yeh, Jerry C.; Borchardt, Ronald T.; Vedani, Angelo

    1991-06-01

    S-adenosyl- l-homocysteine hydrolase (AdoHcy hydrolase, EC 3.3.1.1.), a specific target for antiviral drug design, catalyzes the hydrolysis of AdoHcy to adenosine (Ado) and homocysteine (Hcy) as well as the synthesis of AdoHcy from Ado and Hcy. The enzyme isolated from different sources has been shown to contain tightly bound NAD+. Based on the 2.0 Å-resolution X-ray crystal structure of dogfish lactate dehydrogenase (LDH), which is functionally homologous to AdoHcy hydrolase, and the primary sequence of rat liver AdoHcy hydrolase, we have derived a molecular model of an extended active site for AdoHcy hydrolase. The computational mutation was performed using the software MUTAR (Yeh et al., University of Kansas, Lawrence), followed by molecular mechanics optimizations using the programs AMBER (Singh et al., University of California, San Francisco) and YETI (Vedani, University of Kansas). Solvation of the model structure was achieved by use of the program SOLVGEN (Jacober, University of Kansas); 56 water molecules were explicitly included in all refinements. Some of these may be involved in the catalytic reaction. We also studied a model of the complex of AdoHcy hydrolase with NAD+, as well as the ternary complexes of the redox reaction catalyzed by AdoHcy hydrolase and has been used to differentiate the relative binding strength of inhibitors.

  2. Potent Urea and Carbamate Inhibitors of Soluble Epoxide Hydrolases

    NASA Astrophysics Data System (ADS)

    Morisseau, Christophe; Goodrow, Marvin H.; Dowdy, Deanna; Zheng, Jiang; Greene, Jessica F.; Sanborn, James R.; Hammock, Bruce D.

    1999-08-01

    The soluble epoxide hydrolase (sEH) plays a significant role in the biosynthesis of inflammation mediators as well as xenobiotic transformations. Herein, we report the discovery of substituted ureas and carbamates as potent inhibitors of sEH. Some of these selective, competitive tightbinding inhibitors with nanomolar Ki values interacted stoichiometrically with the homogenous recombinant murine and human sEHs. These inhibitors enhance cytotoxicity of trans-stilbene oxide, which is active as the epoxide, but reduce cytotoxicity of leukotoxin, which is activated by epoxide hydrolase to its toxic diol. They also reduce toxicity of leukotoxin in vivo in mice and prevent symptoms suggestive of acute respiratory distress syndrome. These potent inhibitors may be valuable tools for testing hypotheses of involvement of diol and epoxide lipids in chemical mediation in vitro or in vivo systems.

  3. Peripheral FAAH and soluble epoxide hydrolase inhibitors are synergistically antinociceptive.

    PubMed

    Sasso, Oscar; Wagner, Karen; Morisseau, Christophe; Inceoglu, Bora; Hammock, Bruce D; Piomelli, Daniele

    2015-07-01

    We need better medicines to control acute and chronic pain. Fatty acid amide hydrolase (FAAH) and soluble epoxide hydrolase (sEH) catalyze the deactivating hydrolysis of two classes of bioactive lipid mediators--fatty acid ethanolamides (FAEs) and epoxidized fatty acids (EpFAs), respectively--which are biogenetically distinct but share the ability to attenuate pain responses and inflammation. In these experiments, we evaluated the antihyperalgesic activity of small-molecule inhibitors of FAAH and sEH, administered alone or in combination, in two pain models: carrageenan-induced hyperalgesia in mice and streptozocin-induced allodynia in rats. When administered separately, the sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl)urea (TPPU) and the peripherally restricted FAAH inhibitor URB937 were highly active in the two models. The combination TPPU plus URB937 was markedly synergistic, as assessed using isobolographic analyses. The results of these experiments reveal the existence of a possible functional crosstalk between FAEs and EpFAs in regulating pain responses. Additionally, the results suggest that combinations of sEH and FAAH inhibitors might be exploited therapeutically to achieve greater analgesic efficacy.

  4. Sulfonyl Fluoride Inhibitors of Fatty Acid Amide Hydrolase

    PubMed Central

    Alapafuja, Shakiru O.; Nikas, Spyros P.; Bharatan, Indu; Shukla, Vidyanand G.; Nasr, Mahmoud L.; Bowman, Anna L.; Zvonok, Nikolai; Li, Jing; Shi, Xiaomeng; Engen, John R.; Makriyannis, Alexandros

    2013-01-01

    Sulfonyl fluorides are known to inhibit esterases. Early work from our laboratory has identified hexadecyl sulfonylfluoride (AM374) as a potent in vitro and in vivo inhibitor of fatty acid amide hydrolase (FAAH). We now report on later generation sulfonyl fluoride analogs that exhibit potent and selective inhibition of FAAH. Using recombinant rat and human FAAH we show that 5-(4-hydroxyphenyl)pentanesulfonyl fluoride (AM3506) has similar inhibitory activity for both the rat and the human enzyme, while rapid dilution assays and mass spectrometry analysis suggest that the compound is a covalent modifier for FAAH and inhibits its action in an irreversible manner. Our SAR results are highlighted by molecular docking of key analogs. PMID:23083016

  5. Discovery of enantioselectivity of urea inhibitors of soluble epoxide hydrolase.

    PubMed

    Manickam, Manoj; Pillaiyar, Thanigaimalai; Boggu, PullaReddy; Venkateswararao, Eeda; Jalani, Hitesh B; Kim, Nam-Doo; Lee, Seul Ki; Jeon, Jang Su; Kim, Sang Kyum; Jung, Sang-Hun

    2016-07-19

    Soluble epoxide hydrolase (sEH) hydrolyzes epoxyeicosatrienoic acids (EETs) in the metabolic pathway of arachidonic acid and has been considered as an important therapeutic target for chronic diseases such as hypertension, diabetes and inflammation. Although many urea derivatives are known as sEH inhibitors, the enantioselectivity of the inhibitors is not highlighted in spite of the stereoselective hydrolysis of EETs by sEH. In an effort to explore the importance of enantioselectivity in the urea scaffold, a series of enantiomers with the stereocenter adjacent to the urea nitrogen atom were prepared. The selectivity of enantiomers of 1-(α-alkyl-α-phenylmethyl)-3-(3-phenylpropyl)ureas showed wide range differences up to 125 fold with the low IC50 value up to 13 nM. The S-configuration with planar phenyl and small alkyl groups at α-position is crucial for the activity and selectivity. However, restriction of the free rotation of two α-groups with indan-1-yl or 1,2,3,4-tetrahydronaphthalen-1-yl moiety abolishes the selectivity between the enantiomers, despite the increase in activity up to 13 nM. The hydrophilic group like sulfonamido group at para position of 3-phenylpropyl motif of 1-(α-alkyl-α-phenylmethyl-3-(3-phenylpropyl)urea improves the activity as well as enantiomeric selectivity. All these ureas are proved to be specific inhibitor of sEH without inhibition against mEH.

  6. Evaluation of NHS Carbamates as a Potent and Selective Class of Endocannabinoid Hydrolase Inhibitors

    PubMed Central

    2013-01-01

    Monoacylglycerol lipase (MAGL) is a principal metabolic enzyme responsible for hydrolyzing the endogenous cannabinoid (endocannabinoid) 2-arachidonoylglycerol (2-AG). Selective inhibitors of MAGL offer valuable probes to further understand the enzyme’s function in biological systems and may lead to drugs for treating a variety of diseases, including psychiatric disorders, neuroinflammation, and pain. N-Hydroxysuccinimidyl (NHS) carbamates have recently been identified as a promising class of serine hydrolase inhibitors that shows minimal cross-reactivity with other proteins in the proteome. Here, we explore NHS carbamates more broadly and demonstrate their potential as inhibitors of endocannabinoid hydrolases and additional enzymes from the serine hydrolase class. We extensively characterize an NHS carbamate 1a (MJN110) as a potent, selective, and in-vivo-active MAGL inhibitor. Finally, we demonstrate that MJN110 alleviates mechanical allodynia in a rat model of diabetic neuropathy, marking NHS carbamates as a promising class of MAGL inhibitors. PMID:23731016

  7. Discovery of Triterpenoids as Reversible Inhibitors of α/β-hydrolase Domain Containing 12 (ABHD12)

    PubMed Central

    Parkkari, Teija; Haavikko, Raisa; Laitinen, Tuomo; Navia-Paldanius, Dina; Rytilahti, Roosa; Vaara, Miia; Lehtonen, Marko; Alakurtti, Sami; Yli-Kauhaluoma, Jari; Nevalainen, Tapio; Savinainen, Juha R.; Laitinen, Jarmo T.

    2014-01-01

    Background α/β-hydrolase domain containing (ABHD)12 is a recently discovered serine hydrolase that acts in vivo as a lysophospholipase for lysophosphatidylserine. Dysfunctional ABHD12 has been linked to the rare neurodegenerative disorder called PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, cataract). In vitro, ABHD12 has been implicated in the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG). Further studies on ABHD12 function are hampered as no selective inhibitor have been identified to date. In contrast to the situation with the other endocannabinoid hydrolases, ABHD12 has remained a challenging target for inhibitor development as no crystal structures are available to facilitate drug design. Methodology/Principal Findings Here we report the unexpected discovery that certain triterpene-based structures inhibit human ABHD12 hydrolase activity in a reversible manner, the best compounds showing submicromolar potency. Based on structure activity relationship (SAR) data collected for 68 natural and synthetic triterpenoid structures, a pharmacophore model has been constructed. A pentacyclic triterpene backbone with carboxyl group at position 17, small hydrophobic substituent at the position 4, hydrogen bond donor or acceptor at position 3 accompanied with four axial methyl substituents was found crucial for ABHD12 inhibitor activity. Although the triterpenoids typically may have multiple protein targets, we witnessed unprecedented selectivity for ABHD12 among the metabolic serine hydrolases, as activity-based protein profiling of mouse brain membrane proteome indicated that the representative ABHD12 inhibitors did not inhibit other serine hydrolases, nor did they target cannabinoid receptors. Conclusions/Significance We have identified reversibly-acting triterpene-based inhibitors that show remarkable selectivity for ABHD12 over other metabolic serine hydrolases. Based on SAR data, we have constructed the first pharmacophore

  8. DEVELOPMENT OF METABOLICALLY STABLE INHIBITORS OF MAMMALIAN MICROSOMAL EPOXIDE HYDROLASE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of xenobiotics such as polyaromatic toxicants. Additionally, polymorphism studies have underlined a potential role of this enzyme in relation to a number of diseases, such as emphysema, spontaneous abortion, eclampsia ...

  9. Discovery of novel leukotriene A4 hydrolase inhibitors based on piperidine and piperazine scaffolds.

    PubMed

    Sandanayaka, Vincent; Mamat, Bjorn; Bhagat, Nikhil; Bedell, Louis; Halldorsdottir, Gudrun; Sigthorsdottir, Heida; Andrésson, Thorkell; Kiselyov, Alex; Gurney, Mark; Singh, Jasbir

    2010-05-01

    Novel piperidine and piperazine derivatives have been designed and tested as inhibitors of LTA(4) hydrolase (LTA(4)H). Most potent compounds showed good potency in both enzymatic and functional human whole blood assay. Crystallography studies further confirmed observed structure-activity relationship and LTA(4)H binding mode for analogs from the piperidine series.

  10. Pyrazole phenylcyclohexylcarbamates as inhibitors of human fatty acid amide hydrolases (FAAH).

    PubMed

    Aghazadeh Tabrizi, Mojgan; Baraldi, Pier Giovanni; Ruggiero, Emanuela; Saponaro, Giulia; Baraldi, Stefania; Romagnoli, Romeo; Martinelli, Adriano; Tuccinardi, Tiziano

    2015-06-05

    Fatty acid amide hydrolase (FAAH) inhibitors have gained attention as potential therapeutic targets in the management of neuropathic pain. Here, we report a series of pyrazole phenylcyclohexylcarbamate derivatives standing on the known carbamoyl FAAH inhibitor URB597. Structural modifications led to the recognition of compound 22 that inhibited human recombinant FAAH (hrFAAH) in the low nanomolar range (IC50 = 11 nM). The most active compounds of this series showed significant selectivity toward monoacylglycerol lipase (MAGL) enzyme. In addition, molecular modeling and reversibility behavior of the new class of FAAH inhibitors are presented in this article.

  11. N-aryl 2-aryloxyacetamides as a new class of fatty acid amide hydrolase (FAAH) inhibitors.

    PubMed

    Sunduru, Naresh; Svensson, Mona; Cipriano, Mariateresa; Marwaha, Sania; Andersson, C David; Svensson, Richard; Fowler, Christopher J; Elofsson, Mikael

    2017-12-01

    Fatty acid amide hydrolase (FAAH) is a promising target for the development of drugs to treat neurological diseases. In search of new FAAH inhibitors, we identified 2-(4-cyclohexylphenoxy)-N-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide, 4g, with an IC50 of 2.6 µM as a chemical starting point for the development of potent FAAH inhibitors. Preliminary hit-to-lead optimisation resulted in 2-(4-phenylphenoxy)-N-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide, 4i, with an IC50 of 0.35 µM.

  12. α-Ketoheterocycle-Based Inhibitors of Fatty Acid Amide Hydrolase (FAAH)

    PubMed Central

    2011-01-01

    A summary of the initial discovery and characterization of the enzyme fatty acid amide hydrolase (FAAH), and the subsequent advancement of an important class of competitive, reversible, potent, and selective inhibitors is presented. Initially explored using substrate-inspired inhibitors bearing electrophilic carbonyls, the examination of α-ketoheterocyle-based inhibitors of FAAH with the benefit of a unique activity-based protein-profiling (ABPP)-based proteome-wide selectivity assay, a powerful in vivo biomarker-based in vivo screen, and subsequent retrospective X-ray cocrystal structures with the enzyme, is summarized. These efforts defined the impact of the central activating heterocycle and its key substituents, provided key simplifications in the C2 acyl side chain and clear interpretations for the unique role and subsequent optimization of the central activating heterocycle, and established the basis for the recent further conformational constraints in the C2 acyl side chain, providing potent, long-acting, orally active FAAH inhibitors. PMID:22639704

  13. Inhibitors of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase: New Targets for Future Antidepressants

    PubMed Central

    Ogawa, Shintaro; Kunugi, Hiroshi

    2015-01-01

    Cannabis and analogs of Δ9-tetrahydrocannabinol have been used for therapeutic purposes, but their therapeutic use remains limited because of various adverse effects. Endogenous cannabinoids have been discovered, and dysregulation of endocannabinoid signaling is implicated in the pathophysiology of major depressive disorder (MDD). Recently, endocannabinoid hydrolytic enzymes such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) have become new therapeutic targets in the treatment of MDD. Several FAAH or MAGL inhibitors are reported to have no cannabimimetic side effects and, therefore, are new potential therapeutic options for patients with MDD who are resistant to first-line antidepressants (selective serotonin and serotonin-norepinephrine reuptake inhibitors). In this review, we focus on the possible relationships between MDD and the endocannabinoid system as well as the inhibitors’ therapeutic potential. MAGL inhibitors may reduce inflammatory responses through activation of cannabinoid receptor type 2. In the hypothalamic–pituitary–adrenal axis, repeated FAAH inhibitor administration may be beneficial for reducing circulating glucocorticoid levels. Both FAAH and MAGL inhibitors may contribute to dopaminergic system regulation. Recently, several new inhibitors have been developed with strong potency and selectivity. FAAH inhibitor, MAGL inhibitor, or dual blocker use would be promising new treatments for MDD. Further pre-clinical studies and clinical trials using these inhibitors are warranted. PMID:26630956

  14. Aryl Piperazinyl Ureas as Inhibitors of Fatty Acid Amide Hydrolase (FAAH) in Rat, Dog, and Primate.

    PubMed

    Keith, John M; Apodaca, Rich; Tichenor, Mark; Xiao, Wei; Jones, William; Pierce, Joan; Seierstad, Mark; Palmer, James; Webb, Michael; Karbarz, Mark; Scott, Brian; Wilson, Sandy; Luo, Lin; Wennerholm, Michelle; Chang, Leon; Brown, Sean; Rizzolio, Michele; Rynberg, Raymond; Chaplan, Sandra; Breitenbucher, J Guy

    2012-10-11

    A series of aryl piperazinyl ureas that act as covalent inhibitors of fatty acid amide hydrolase (FAAH) is described. A potent and selective (does not inhibit FAAH-2) member of this class, JNJ-40355003, was found to elevate the plasma levels of three fatty acid amides: anandamide, oleoyl ethanolamide, and palmitoyl ethanolamide, in the rat, dog, and cynomolgous monkey. The elevation of the levels of these lipids in the plasma of monkeys suggests that FAAH-2 may not play a significant role in regulating plasma levels of fatty acid ethanolamides in primates.

  15. Discovery and molecular basis of potent noncovalent inhibitors of fatty acid amide hydrolase (FAAH).

    PubMed

    Min, Xiaoshan; Thibault, Stephen T; Porter, Amy C; Gustin, Darin J; Carlson, Timothy J; Xu, Haoda; Lindstrom, Michelle; Xu, Guifen; Uyeda, Craig; Ma, Zhihua; Li, Yihong; Kayser, Frank; Walker, Nigel P C; Wang, Zhulun

    2011-05-03

    Fatty acid amide hydrolase (FAAH), an amidase-signature family member, is an integral membrane enzyme that degrades lipid amides including the endogenous cannabinoid anandamide and the sleep-inducing molecule oleamide. Both genetic knock out and pharmacological administration of FAAH inhibitors in rodent models result in analgesic, anxiolytic, and antiinflammatory phenotypes. Targeting FAAH activity, therefore, presents a promising new therapeutic strategy for the treatment of pain and other neurological-related or inflammatory disorders. Nearly all FAAH inhibitors known to date attain their binding potency through a reversible or irreversible covalent modification of the nucleophile Ser241 in the unusual Ser-Ser-Lys catalytic triad. Here, we report the discovery and mechanism of action of a series of ketobenzimidazoles as unique and potent noncovalent FAAH inhibitors. Compound 2, a representative of these ketobenzimidazoles, was designed from a series of ureas that were identified from high-throughput screening. While urea compound 1 is characterized as an irreversible covalent inhibitor, the cocrystal structure of FAAH complexed with compound 2 reveals that these ketobenzimidazoles, though containing a carbonyl moiety, do not covalently modify Ser241. These inhibitors achieve potent inhibition of FAAH activity primarily from shape complementarity to the active site and through numerous hydrophobic interactions. These noncovalent compounds exhibit excellent selectivity and good pharmacokinetic properties. The discovery of this distinctive class of inhibitors opens a new avenue for modulating FAAH activity through nonmechanism-based inhibition.

  16. Discovery of Leukotriene A4 Hydrolase Inhibitors Using Metabolomics Biased Fragment Crystallography

    SciTech Connect

    Davies, D.; Mamat, B; Magnusson, O; Christensen, J; Haraldsson, M; Mishra, R; Pease, B; Hansen, E; Singh, J; et. al.

    2009-01-01

    We describe a novel fragment library termed fragments of life (FOL) for structure-based drug discovery. The FOL library includes natural small molecules of life, derivatives thereof, and biaryl protein architecture mimetics. The choice of fragments facilitates the interrogation of protein active sites, allosteric binding sites, and protein-protein interaction surfaces for fragment binding. We screened the FOL library against leukotriene A4 hydrolase (LTA4H) by X-ray crystallography. A diverse set of fragments including derivatives of resveratrol, nicotinamide, and indole were identified as efficient ligands for LTA4H. These fragments were elaborated in a small number of synthetic cycles into potent inhibitors of LTA4H representing multiple novel chemotypes for modulating leukotriene biosynthesis. Analysis of the fragment-bound structures also showed that the fragments comprehensively recapitulated key chemical features and binding modes of several reported LTA4H inhibitors.

  17. A Systems Pharmacology Perspective on the Clinical Development of Fatty Acid Amide Hydrolase Inhibitors for Pain

    PubMed Central

    Benson, N; Metelkin, E; Demin, O; Li, G L; Nichols, D; van der Graaf, P H

    2014-01-01

    The level of the endocannabinoid anandamide is controlled by fatty acid amide hydrolase (FAAH). In 2011, PF-04457845, an irreversible inhibitor of FAAH, was progressed to phase II clinical trials for osteoarthritic pain. This article discusses a prospective, integrated systems pharmacology model evaluation of FAAH as a target for pain in humans, using physiologically based pharmacokinetic and systems biology approaches. The model integrated physiological compartments; endocannabinoid production, degradation, and disposition data; PF-04457845 pharmacokinetics and pharmacodynamics, and cannabinoid receptor CB1-binding kinetics. The modeling identified clear gaps in our understanding and highlighted key risks going forward, in particular relating to whether methods are in place to demonstrate target engagement and pharmacological effect. The value of this modeling exercise will be discussed in detail and in the context of the clinical phase II data, together with recommendations to enable optimal future evaluation of FAAH inhibitors. PMID:24429592

  18. Fatty acid amide hydrolase inhibitors confer anti-invasive and antimetastatic effects on lung cancer cells

    PubMed Central

    Winkler, Katrin; Ramer, Robert; Dithmer, Sophie; Ivanov, Igor; Merkord, Jutta; Hinz, Burkhard

    2016-01-01

    Inhibition of endocannabinoid degradation has been suggested as tool for activation of endogenous tumor defense. One of these strategies lies in blockade of fatty acid amide hydrolase (FAAH) which catalyzes the degradation of endocannabinoids (anandamide [AEA], 2-arachidonoylglycerol [2-AG]) and endocannabinoid-like substances (N-oleoylethanolamine [OEA], N-palmitoylethanolamine [PEA]). This study addressed the impact of two FAAH inhibitors (arachidonoyl serotonin [AA-5HT], URB597) on A549 lung cancer cell metastasis and invasion. LC-MS analyses revealed increased levels of FAAH substrates (AEA, 2-AG, OEA, PEA) in cells incubated with either FAAH inhibitor. In athymic nude mice FAAH inhibitors were shown to elicit a dose-dependent antimetastatic action yielding a 67% and 62% inhibition of metastatic lung nodules following repeated administration of 15 mg/kg AA-5HT and 5 mg/kg URB597, respectively. In vitro, a concentration-dependent anti-invasive action of either FAAH inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). Using siRNA approaches, a causal link between the TIMP-1-upregulating and anti-invasive action of FAAH inhibitors was confirmed. Moreover, knockdown of FAAH by siRNA was shown to confer decreased cancer cell invasiveness and increased TIMP-1 expression. Inhibitor experiments point toward a role of CB2 and transient receptor potential vanilloid 1 in conferring anti-invasive effects of FAAH inhibitors and FAAH siRNA. Finally, antimetastatic and anti-invasive effects were confirmed for all FAAH substrates with AEA and OEA causing a TIMP-1-dependent anti-invasive action. Collectively, the present study provides first-time proof for an antimetastatic action of FAAH inhibitors. As mechanism of its anti-invasive properties an upregulation of TIMP-1 was identified. PMID:26930716

  19. Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii

    PubMed Central

    Kemp, Louise E.; Rusch, Marion; Adibekian, Alexander; Bullen, Hayley E.; Graindorge, Arnault; Freymond, Céline; Rottmann, Matthias; Braun-Breton, Catherine; Baumeister, Stefan; Porfetye, Arthur T.; Vetter, Ingrid R.; Hedberg, Christian; Soldati-Favre, Dominique

    2013-01-01

    In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein. PMID:23913689

  20. Piperazine and piperidine carboxamides and carbamates as inhibitors of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL).

    PubMed

    Korhonen, Jani; Kuusisto, Anne; van Bruchem, John; Patel, Jayendra Z; Laitinen, Tuomo; Navia-Paldanius, Dina; Laitinen, Jarmo T; Savinainen, Juha R; Parkkari, Teija; Nevalainen, Tapio J

    2014-12-01

    The key hydrolytic enzymes of the endocannabinoid system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), are potential targets for various therapeutic applications. In this paper, we present more extensively the results of our previous work on piperazine and piperidine carboxamides and carbamates as FAAH and MAGL inhibitors. The best compounds of these series function as potent and selective MAGL/FAAH inhibitors or as dual FAAH/MAGL inhibitors at nanomolar concentrations. This study revealed that MAGL inhibitors should comprise leaving-groups with a conjugate acid pKa of 8-10, while diverse leaving groups are tolerated for FAAH inhibitors.

  1. Structure-Based Optimization of Arylamides as Inhibitors of Soluble Epoxide Hydrolase

    SciTech Connect

    Eldrup, Anne B.; Soleymanzadeh, Fariba; Taylor, Steven J.; Muegge, Ingo; Farrow, Neil A.; Joseph, David; McKellop, Keith; Man, Chuk C.; Kukulka, Alison; De Lombaert, Stephane

    2009-11-04

    Inhibition of soluble epoxide hydrolase (sEH) is hypothesized to lead to an increase in circulating levels of epoxyeicosatrienoic acids, resulting in the potentiation of their in vivo pharmacological properties. As part of an effort to identify inhibitors of sEH with high and sustained plasma exposure, we recently performed a high throughput screen of our compound collection. The screen identified N-(3,3-diphenyl-propyl)-nicotinamide as a potent inhibitor of sEH. Further profiling of this lead revealed short metabolic half-lives in microsomes and rapid clearance in the rat. Consistent with these observations, the determination of the in vitro metabolic profile of N-(3,3-diphenyl-propyl)-nicotinamide in rat liver microsomes revealed extensive oxidative metabolism and a propensity for metabolite switching. Lead optimization, guided by the analysis of the solid-state costructure of N-(3,3-diphenyl-propyl)-nicotinamide bound to human sEH, led to the identification of a class of potent and selective inhibitors. An inhibitor from this class displayed an attractive in vitro metabolic profile and high and sustained plasma exposure in the rat after oral administration.

  2. Human Intestinal Raf Kinase Inhibitor Protein (RKIP) Catalyzes Prasugrel as a Bioactivation Hydrolase.

    PubMed

    Kazui, Miho; Ogura, Yuji; Hagihara, Katsunobu; Kubota, Kazuishi; Kurihara, Atsushi

    2016-01-01

    Prasugrel is a thienopyridine antiplatelet prodrug that undergoes rapid hydrolysis in vivo to a thiolactone metabolite by human carboxylesterase-2 (hCE2) during gastrointestinal absorption. The thiolactone metabolite is further converted to a pharmacologically active metabolite by cytochrome P450 isoforms. The aim of the current study was to elucidate hydrolases other than hCE2 involved in the bioactivation step of prasugrel in human intestine. Using size-exclusion column chromatography of a human small intestinal S9 fraction, another peak besides the hCE2 peak was observed to have prasugrel hydrolyzing activity, and this protein was found to have a molecular weight of about 20 kDa. This prasugrel hydrolyzing protein was successfully purified from a monkey small intestinal cytosolic fraction by successive four-step column chromatography and identified as Raf-1 kinase inhibitor protein (RKIP) by liquid chromatography-tandem mass spectrometry. Second, we evaluated the enzymatic kinetic parameters for prasugrel hydrolysis using recombinant human RKIP and hCE2 and estimated the contributions of these two hydrolyzing enzymes to the prasugrel hydrolysis reaction in human intestine, which were approximately 40% for hRKIP and 60% for hCE2. Moreover, prasugrel hydrolysis was inhibited by anti-hRKIP antibody and carboxylesterase-specific chemical inhibitor (bis p-nitrophenyl phosphate) by 30% and 60%, respectively. In conclusion, another protein capable of hydrolyzing prasugrel to its thiolactone metabolite was identified as RKIP, and this protein may play a significant role with hCE2 in prasugrel bioactivation in human intestine. RKIP is known to have diverse functions in many intracellular signaling cascades, but this is the first report describing RKIP as a hydrolase involved in drug metabolism.

  3. In vitro metabolism of the mammalian soluble epoxide hydrolase inhibitor, 1-cyclohexyl-3-dodecyl-urea.

    PubMed

    Watanabe, Takaho; Morisseau, Christophe; Newman, John W; Hammock, Bruce D

    2003-07-01

    The metabolism of the soluble epoxide hydrolase (sEH) inhibitor, 1-cyclohexyl-3-dodecyl-urea (CDU), was studied in rat and human hepatic microsomes. The microsomal metabolism of CDU enhanced sEH inhibition potency of the reaction mixture and resulted in the formation of several metabolites. During the course of this study, a sensitive and specific high-performance liquid chromatography with tandem mass spectrometry analytical method was developed to investigate simultaneously the production of these metabolites. In both rat and human hepatic microsomes, CDU was ultimately transformed into the corresponding omega-carboxylate; however, the rodent tissue appeared to perform this transformation more rapidly. After a 60-min incubation in rat hepatic microsomes, the percentage of residual CDU, the omega-carboxylate, and the intermediary omega-hydroxyl were about 20%, 20%, and 50%, respectively. Carbon monoxide inhibited the metabolism of CDU by rat hepatic microsomes, suggesting that the initial step is catalyzed by cytochrome P450. Further metabolism was enhanced by the addition of NAD, suggesting that dehydrogenases are associated with intermediate metabolic steps. Regardless, the ultimate product of microsomal metabolism, 12-(3-cyclohexyl-ureido)-dodecanoic acid, is also an excellent sEH inhibitor with several hundred-fold higher solubility, supporting the hypothesis that CDU has prodrug characteristics. These findings will facilitate the rational design and optimization of sEH inhibitors with better physical properties and improved metabolic stability.

  4. Optimization of Amide-Based Inhibitors of Soluble Epoxide Hydrolase with Improved Water Solubility

    PubMed Central

    Kim, In-Hae; Heirtzler, Fenton R.; Morisseau, Christophe; Nishi, Kosuke; Tsai, Hsing-Ju; Hammock, Bruce D.

    2006-01-01

    Soluble epoxide hydrolase (sEH) plays an important role in the metabolism of endogenous chemical mediators involved in the regulation of blood pressure and inflammation. 1,3-Disubstituted ureas with a polar group located on the fifth atom from the carbonyl group of urea function are active inhibitors of sEH both in vitro and in vivo. However, their limited solubility in water and relatively high melting point lead to difficulties in formulating the compounds and poor in vivo efficacy. To improve these physical properties, the effect of structural modification of the urea pharmacophore on the inhibition potencies, water solubilities, octanol/water partition coefficients (log P), and melting points of a series of compounds was evaluated. For murine sEH, no loss of inhibition potency was observed when the urea pharmacophore was modified to an amide function, while for human sEH 2.5-fold decreased inhibition was obtained in the amide compounds. In addition, a NH group on the right side of carbonyl group of the amide pharmacophore substituted with an adamantyl group (such as compound 14) and a methylene carbon present between the adamantyl and amide groups were essential to produce potent inhibition of sEH. The resulting amide inhibitors have 10–30-fold better solubility and lower melting point than the corresponding urea compounds. These findings will facilitate synthesis of sEH inhibitors that are easier to formulate and more bioavailable. PMID:15887969

  5. Rational Design of Potent and Selective Inhibitors of an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa.

    PubMed

    Kitamura, Seiya; Hvorecny, Kelli L; Niu, Jun; Hammock, Bruce D; Madden, Dean R; Morisseau, Christophe

    2016-05-26

    The virulence factor cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is secreted by Pseudomonas aeruginosa and is the founding member of a distinct class of epoxide hydrolases (EHs) that triggers the catalysis-dependent degradation of the CFTR. We describe here the development of a series of potent and selective Cif inhibitors by structure-based drug design. Initial screening revealed 1a (KB2115), a thyroid hormone analog, as a lead compound with low micromolar potency. Structural requirements for potency were systematically probed, and interactions between Cif and 1a were characterized by X-ray crystallography. On the basis of these data, new compounds were designed to yield additional hydrogen bonding with residues of the Cif active site. From this effort, three compounds were identified that are 10-fold more potent toward Cif than our first-generation inhibitors and have no detectable thyroid hormone-like activity. These inhibitors will be useful tools to study the pathological role of Cif and have the potential for clinical application.

  6. Discovery of bile salt hydrolase inhibitors using an efficient high-throughput screening system.

    PubMed

    Smith, Katie; Zeng, Ximin; Lin, Jun

    2014-01-01

    The global trend of restricting the use of antibiotic growth promoters (AGP) in animal production necessitates the need to develop valid alternatives to maintain productivity and sustainability of food animals. Previous studies suggest inhibition of bile salt hydrolase (BSH), an intestinal bacteria-produced enzyme that exerts negative impact on host fat digestion and utilization, is a promising approach to promote animal growth performance. To achieve the long term goal of developing novel alternatives to AGPs, in this study, a rapid and convenient high-throughput screening (HTS) system was developed and successfully used for identification of BSH inhibitors. With the aid of a high-purity BSH from a chicken Lactobacillus salivarius strain, we optimized various screening conditions (e.g. BSH concentration, reaction buffer pH, incubation temperature and length, substrate type and concentration) and establish a precipitation-based screening approach to identify BSH inhibitors using 96-well or 384-well microplates. A pilot HTS was performed using a small compound library comprised of 2,240 biologically active and structurally diverse compounds. Among the 107 hits, several promising and potent BSH inhibitors (e.g. riboflavin and phenethyl caffeate) were selected and validated by standard BSH activity assay. Interestingly, the HTS also identified a panel of antibiotics as BSH inhibitor; in particular, various tetracycline antibiotics and roxarsone, the widely used AGP, have been demonstrated to display potent inhibitory effect on BSH. Together, this study developed an efficient HTS system and identified several BSH inhibitors with potential as alternatives to AGP. In addition, the findings from this study also suggest a new mode of action of AGP for promoting animal growth.

  7. Discovery of Bile Salt Hydrolase Inhibitors Using an Efficient High-Throughput Screening System

    PubMed Central

    Smith, Katie; Zeng, Ximin; Lin, Jun

    2014-01-01

    The global trend of restricting the use of antibiotic growth promoters (AGP) in animal production necessitates the need to develop valid alternatives to maintain productivity and sustainability of food animals. Previous studies suggest inhibition of bile salt hydrolase (BSH), an intestinal bacteria-produced enzyme that exerts negative impact on host fat digestion and utilization, is a promising approach to promote animal growth performance. To achieve the long term goal of developing novel alternatives to AGPs, in this study, a rapid and convenient high-throughput screening (HTS) system was developed and successfully used for identification of BSH inhibitors. With the aid of a high-purity BSH from a chicken Lactobacillus salivarius strain, we optimized various screening conditions (e.g. BSH concentration, reaction buffer pH, incubation temperature and length, substrate type and concentration) and establish a precipitation-based screening approach to identify BSH inhibitors using 96-well or 384-well microplates. A pilot HTS was performed using a small compound library comprised of 2,240 biologically active and structurally diverse compounds. Among the 107 hits, several promising and potent BSH inhibitors (e.g. riboflavin and phenethyl caffeate) were selected and validated by standard BSH activity assay. Interestingly, the HTS also identified a panel of antibiotics as BSH inhibitor; in particular, various tetracycline antibiotics and roxarsone, the widely used AGP, have been demonstrated to display potent inhibitory effect on BSH. Together, this study developed an efficient HTS system and identified several BSH inhibitors with potential as alternatives to AGP. In addition, the findings from this study also suggest a new mode of action of AGP for promoting animal growth. PMID:24454844

  8. Ingestion of the epoxide hydrolase inhibitor AUDA modulates immune responses of the mosquito, Culex quinquefasciatus during blood feeding.

    PubMed

    Xu, Jiawen; Morisseau, Christophe; Yang, Jun; Lee, Kin Sing Stephen; Kamita, Shizuo G; Hammock, Bruce D

    2016-09-01

    Epoxide hydrolases (EHs) are enzymes that play roles in metabolizing xenobiotic epoxides from the environment, and in regulating lipid signaling molecules, such as juvenile hormones in insects and epoxy fatty acids in mammals. In this study we fed mosquitoes with an epoxide hydrolase inhibitor AUDA during artificial blood feeding, and we found the inhibitor increased the concentration of epoxy fatty acids in the midgut of female mosquitoes. We also observed ingestion of AUDA triggered early expression of defensin A, cecropin A and cecropin B2 at 6 h after blood feeding. The expression of cecropin B1 and gambicin were not changed more than two fold compared to controls. The changes in gene expression were transient possibly because more than 99% of the inhibitor was metabolized or excreted at 42 h after being ingested. The ingestion of AUDA also affected the growth of bacteria colonizing in the midgut, but did not affect mosquito longevity, fecundity and fertility in our laboratory conditions. When spiked into the blood, EpOMEs and DiHOMEs were as effective as the inhibitor AUDA in reducing the bacterial load in the midgut, while EETs rescued the effects of AUDA. Our data suggest that epoxy fatty acids from host blood are immune response regulators metabolized by epoxide hydrolases in the midgut of female mosquitoes, inhibition of which causes transient changes in immune responses, and affects growth of microbes in the midgut.

  9. Anti-Ulcer Efficacy of Soluble Epoxide Hydrolase Inhibitor TPPU on Diclofenac-Induced Intestinal Ulcers

    PubMed Central

    Goswami, Sumanta Kumar; Wan, Debin; Yang, Jun; Trindade da Silva, Carlos A.; Morisseau, Christophe; Kodani, Sean D.; Yang, Guang-Yu; Inceoglu, Bora

    2016-01-01

    Proton pump inhibitors such as omeprazole (OME) reduce the severity of gastrointestinal (GI) ulcers induced by nonsteroidal anti-inflammatory drugs (NSAIDs) but can also increase the chance of dysbiosis. The aim of this study was to test the hypothesis that preventive use of a soluble epoxide hydrolase inhibitor (sEHI) such as TPPU can decrease NSAID-induced ulcers by increasing anti-inflammatory epoxyeicosatrienoic acids (EETs). Dose- [10, 30, and 100 mg/kg, by mouth (PO)] and time-dependent (6 and 18 hours) ulcerative effects of diclofenac sodium (DCF, an NSAID) were studied in the small intestine of Swiss Webster mice. Dose-dependent effects of TPPU (0.001–0.1 mg/kg per day for 7 days, in drinking water) were evaluated in DCF-induced intestinal toxicity and compared with OME (20 mg/kg, PO). In addition, the effect of treatment was studied on levels of Hb in blood, EETs in plasma, inflammatory markers such as myeloperoxidase (MPO) in intestinal tissue homogenates, and tissue necrosis factor-α (TNF-α) in serum. DCF dose dependently induced ulcers that were associated with both a significant (P < 0.05) loss of Hb and an increase in the level of MPO and TNF-α, with severity of ulceration highest at 18 hours. Pretreatment with TPPU dose dependently prevented ulcer formation by DCF, increased the levels of epoxy fatty acids, including EETs, and TPPU’s efficacy was comparable to OME. TPPU significantly (P < 0.05) reversed the effect of DCF on the level of Hb, MPO, and TNF-α. Thus sEHI might be useful in the management of NSAID-induced ulcers. PMID:26989141

  10. α-Ketoheterocycle Inhibitors of Fatty Acid Amide Hydrolase: Exploration of Conformational Constraints in the Acyl Side Chain

    PubMed Central

    Duncan, Katharine K.; Otrubova, Katerina; Boger, Dale L.

    2014-01-01

    A series of α-ketooxazoles containing heteroatoms embedded within conformational constraints in the C2 acyl side chain of 2 (OL-135) were synthesized and evaluated as inhibitors of fatty acid amide hydrolase (FAAH). The studies reveal that the installation of a heteroatom (O) in the conformational constraint is achievable, although the potency of these novel derivatives is reduced slightly relative to 2 and the analogous 1,2,3,4-tetrahydronaphthalene series. Interestingly, both enantiomers (R and S) of the candidate inhibitors bearing a chiral center adjacent to the electrophilic carbonyl were found to effectively inhibit FAAH. PMID:24690529

  11. Discovery of potent inhibitors of soluble epoxide hydrolase by combinatorial library design and structure-based virtual screening.

    PubMed

    Xing, Li; McDonald, Joseph J; Kolodziej, Steve A; Kurumbail, Ravi G; Williams, Jennifer M; Warren, Chad J; O'Neal, Janet M; Skepner, Jill E; Roberds, Steven L

    2011-03-10

    Structure-based virtual screening was applied to design combinatorial libraries to discover novel and potent soluble epoxide hydrolase (sEH) inhibitors. X-ray crystal structures revealed unique interactions for a benzoxazole template in addition to the conserved hydrogen bonds with the catalytic machinery of sEH. By exploitation of the favorable binding elements, two iterations of library design based on amide coupling were employed, guided principally by the docking results of the enumerated virtual products. Biological screening of the libraries demonstrated as high as 90% hit rate, of which over two dozen compounds were single digit nanomolar sEH inhibitors by IC(50) determination. In total the library design and synthesis produced more than 300 submicromolar sEH inhibitors. In cellular systems consistent activities were demonstrated with biochemical measurements. The SAR understanding of the benzoxazole template provides valuable insights into discovery of novel sEH inhibitors as therapeutic agents.

  12. ADMINISTRATION OF A SUBSTITUTED ADAMANTLY-UREA INHIBITOR OF THE SOLUBLE EPOXIDE HYDROLASE PROTECTS THE KIDNEY FROM DAMAGE IN HYPERTENSIVE GOTO-KAKIZAKI RATS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hypertension and type II diabetes are co-morbid diseases that lead to the development of nephropathy. Soluble epoxide hydrolase (sEH) inhibitors are reported to provide protection from renal injury. We hypothesized that the sEH inhibitor 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) protects ...

  13. The fatty-acid amide hydrolase inhibitor URB597 does not affect triacylglycerol hydrolysis in rat tissues.

    PubMed

    Clapper, Jason R; Duranti, Andrea; Tontini, Andrea; Mor, Marco; Tarzia, Giorgio; Piomelli, Daniele

    2006-11-01

    The O-arylcarbamate URB597 (cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester; also referred to as KDS-4103) is a potent inhibitor of fatty-acid amide hydrolase (FAAH), an intracellular serine hydrolase responsible for the inactivation of the endogenous cannabinoid anandamide. URB597 demonstrates a remarkable degree of selectivity for FAAH over other serine hydrolases (e.g. cholinesterases) or other components of the endocannabinoid system (e.g. cannabinoid receptors). However, in a proteomic-based selectivity screen based on the displacement of fluorophosphonate-rhodamine (FPR) from mouse brain proteins, it was recently shown that URB597 prevents FPR binding to triacylglycerol hydrolase (TGH) with a median inhibitory concentration of 192nM. To determine whether this effect correlates with inhibition of TGH activity, we investigated the ability of URB597 to inhibit triolein hydrolysis in rat liver and heart tissues, which are rich in TGH, as well as white adipose tissue (WAT), which is rich in adipose triacylglycerol lipase (TGL) and hormone-sensitive lipase. The results show that URB597 does not affect triolein hydrolysis in any of these tissues at concentrations as high as 10microM, whereas it inhibits FAAH activity at low nanomolar concentrations. Moreover, intraperitoneal (i.p.) administration of URB597 at doses that maximally inhibit FAAH in vivo (0.3-3mgkg(-1)) exerts no effect on triolein hydrolysis and tissue triacylglycerol (TAG) levels in rat liver, heart or WAT. The results indicate that URB597, while potent at inhibiting FAAH, does not affect TGH and TGL activities in rat tissues.

  14. Rational Design of Fatty Acid Amide Hydrolase Inhibitors that Act by Covalently Bonding to Two Active Site Residues

    PubMed Central

    Otrubova, Katerina; Brown, Monica; McCormick, Michael S.; Han, Gye W.; O’Neal, Scott T.; Cravatt, Benjamin F.; Stevens, Raymond C.; Lichtman, Aron H.; Boger, Dale L.

    2013-01-01

    The design and characterization of α-ketoheterocycle fatty acid amide hydrolase (FAAH) inhibitors are disclosed that additionally and irreversibly target a cysteine (Cys269) found in the enzyme cytosolic port while maintaining the reversible covalent Ser241 attachment responsible for their rapid and initially reversible enzyme inhibition. Two α-ketooxazoles (3 and 4) containing strategically placed electrophiles at the C5 position of the pyridyl substituent of 2 (OL-135) were prepared and examined as inhibitors of FAAH. Consistent with the observed time-dependent non-competitive inhibition, the co-crystal X-ray structure of 3 bound to a humanized variant of rat FAAH revealed that 3 was not only covalently bound to the active site catalytic nucleophile Ser241 as a deprotonated hemiketal, but also to Cys269 through the pyridyl C5-substituent, thus providing an inhibitor with dual covalent attachment in the enzyme active site. In vivo characterization of the prototypical inhibitors in mice demonstrate that they raise endogenous brain levels of FAAH substrates to a greater extent and for a much longer duration (>6 h) than the reversible inhibitor 2, indicating that the inhibitors accumulate and persist in the brain to completely inhibit FAAH for a prolonged period. Consistent with this behavior and the targeted irreversible enzyme inhibition, 3 reversed cold allodynia in the chronic constriction injury model of neuropathic pain in mice for a sustained period (>6 h) beyond that observed with the reversible inhibitor 2, providing effects that were unchanged over the 1–6 h time course monitored. PMID:23581831

  15. Binding and Inactivation Mechanism of a Humanized Fatty Acid Amide Hydrolase by [alpha]-Ketoheterocycle Inhibitors Revealed from Cocrystal Structures

    SciTech Connect

    Mileni, Mauro; Garfunkle, Joie; DeMartino, Jessica K.; Cravatt, Benjamin F.; Boger, Dale L.; Stevens, Raymond C.

    2010-08-17

    The cocrystal X-ray structures of two isomeric {alpha}-ketooxazole inhibitors (1 (OL-135) and 2) bound to fatty acid amide hydrolase (FAAH), a key enzymatic regulator of endocannabinoid signaling, are disclosed. The active site catalytic Ser241 is covalently bound to the inhibitors electrophilic carbonyl groups, providing the first structures of FAAH bound to an inhibitor as a deprotonated hemiketal mimicking the enzymatic tetrahedral intermediate. The work also offers a detailed view of the oxyanion hole and an exceptional 'in-action' depiction of the unusual Ser-Ser-Lys catalytic triad. These structures capture the first picture of inhibitors that span the active site into the cytosolic port providing new insights that help to explain FAAH's interaction with substrate leaving groups and their role in modulating inhibitor potency and selectivity. The role for the activating central heterocycle is clearly defined and distinguished from that observed in prior applications with serine proteases, reconciling the large electronic effect of attached substituents found unique to this class of inhibitors with FAAH. Additional striking active site flexibility is seen upon binding of the inhibitors, providing insights into the existence of a now well-defined membrane access channel with the disappearance of a spatially independent portion of the acyl chain-binding pocket. Finally, comparison of the structures of OL-135 (1) and its isomer 2 indicates that they bind identically to FAAH, albeit with reversed orientations of the central activating heterocycle, revealing that the terminal 2-pyridyl substituent and the acyl chain phenyl group provide key anchoring interactions and confirming the distinguishing role of the activating oxazole.

  16. X-ray Crystallographic Analysis of α-Ketoheterocycle Inhibitors Bound to a Humanized Variant of Fatty Acid Amide Hydrolase

    PubMed Central

    Mileni, Mauro; Garfunkle, Joie; Ezzili, Cyrine; Kimball, F. Scott; Cravatt, Benjamin F.; Stevens, Raymond C.; Boger, Dale L.

    2009-01-01

    Three cocrystal X-ray structures of the α-ketoheterocycle inhibitors 3–5 bound to a humanized variant of fatty acid amide hydrolase (FAAH) are disclosed and comparatively discussed alongside those of 1 (OL-135) and its isomer 2. These five X-ray structures systematically probe each of the three active site regions key to substrate or inhibitor binding: (1) the conformationally mobile acyl chain-binding pocket and membrane access channel responsible for fatty acid amide substrate and inhibitor acyl chain binding, (2) the atypical active site catalytic residues and surrounding oxyanion hole that covalently binds the core of the α-ketoheterocycle inhibitors captured as deprotonated hemiketals mimicking the tetrahedral intermediate of the enzyme catalyzed reaction, and (3) the cytosolic port and its uniquely important imbedded ordered water molecules and a newly identified anion binding site. The detailed analysis of their key active site interactions and their implications on the interpretation of the available structure–activity relationships are discussed providing important insights for future design. PMID:19924997

  17. O-(triazolyl)methyl carbamates as a novel and potent class of fatty acid amide hydrolase (FAAH) inhibitors.

    PubMed

    Colombano, Giampiero; Albani, Clara; Ottonello, Giuliana; Ribeiro, Alison; Scarpelli, Rita; Tarozzo, Glauco; Daglian, Jennifer; Jung, Kwang-Mook; Piomelli, Daniele; Bandiera, Tiziano

    2015-02-01

    Inhibition of fatty acid amide hydrolase (FAAH) activity is under investigation as a valuable strategy for the treatment of several disorders, including pain and drug addiction. A number of potent FAAH inhibitors belonging to different chemical classes have been disclosed to date; O-aryl carbamates are one of the most representative families. In the search for novel FAAH inhibitors, a series of O-(1,2,3-triazol-4-yl)methyl carbamate derivatives were designed and synthesized exploiting a copper- catalyzed [3+2] cycloaddition reaction between azides and alkynes (click chemistry). Exploration of the structure-activity relationships within this new class of compounds identified potent inhibitors of both rat and human FAAH with IC50 values in the single-digit nanomolar range. In addition, these derivatives showed improved stability in rat plasma and kinetic solubility in buffer with respect to the lead compound. Based on the results of the study, the novel analogues identified can be considered to be promising starting point for the development of new FAAH inhibitors with improved drug-like properties.

  18. The macamide N-3-methoxybenzyl-linoleamide is a time-dependent fatty acid amide hydrolase (FAAH) inhibitor.

    PubMed

    Almukadi, Haifa; Wu, Hui; Böhlke, Mark; Kelley, Charles J; Maher, Timothy J; Pino-Figueroa, Alejandro

    2013-10-01

    The Peruvian plant Lepidium meyenii (Maca) has been shown to possess neuroprotective activity both in vitro and in vivo. Previous studies have also demonstrated the activity of the pentane extract and its macamides, the most representative lipophilic constituents of Maca, in the endocannabinoid system as fatty acid amide hydrolase (FAAH) inhibitors. One of the most active macamides, N-3-methoxybenzyl-linoleamide, was studied to determine its mechanism of interaction with FAAH and whether it has inhibitory activity on mono-acyl glycerol lipase (MAGL), the second enzyme responsible for endocannabinoid degradation. Macamide concentrations from 1 to 100 μM were tested using FAAH and MAGL inhibitor assay methods and showed no effect on MAGL. Tests with other conditions were performed in order to characterize the inhibitory mechanism of FAAH inhibition. N-3-methoxybenzyl-linoleamide displayed significant time-dependent and dose-dependent FAAH inhibitory activity. The mechanism of inhibition was most likely irreversible or slowly reversible. These results suggest the potential application of macamides isolated from Maca as FAAH inhibitors, as they might act on the central nervous system to provide analgesic, anti-inflammatory, or neuroprotective effects, by modulating the release of neurotransmitters.

  19. Development and characterization of endocannabinoid hydrolases FAAH and MAGL inhibitors bearing a benzotriazol-1-yl carboxamide scaffold.

    PubMed

    Morera, Ludovica; Labar, Geoffray; Ortar, Giorgio; Lambert, Didier M

    2012-11-01

    A series of (1H-benzo[d][1,2,3]triazol-1-yl)(4-benzylpiperazin-1-yl)methanones and of (1H-benzo[d][1,2,3]triazol-1-yl)(4-phenylpiperazin-1-yl)methanones has been prepared and tested on human fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). In the benzylpiperazinyl series, compound 29 (ML30) exhibited an IC(50) value of 0.54 nM on MAGL, combined with a 1000-fold selectivity versus FAAH, while compounds 11 and 16 acted as potent dual FAAH-MAGL inhibitors (IC(50)<10 nM). In the phenylpiperazinyl series, compounds 37, 38, 42, and 43 displayed IC(50) values against MAGL in the nanomolar range, whilst being between one and two orders of magnitude less potent on the FAAH, while compounds 31 and 32 were potent FAAH inhibitors (IC(50)<20 nM) and over 12-fold selective versus MAGL. The key structural determinants driving the structure-activity relationships were explored by the minimization of the inhibitors inside the active site of both enzymes.

  20. (4-Phenoxyphenyl)tetrazolecarboxamides and related compounds as dual inhibitors of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL).

    PubMed

    Holtfrerich, Angela; Hanekamp, Walburga; Lehr, Matthias

    2013-05-01

    Inhibitors of the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), the principle enzymes involved in the degradation of endogenous cannabinoids like anandamide and 2-arachidonoylglycerol, have potential utility in the treatment of several disorders including pain, inflammation and anxiety. In the present study, the effectivity and selectivity of eight known FAAH and MAGL inhibitors for inhibition of the appropriate enzyme were measured applying in vitro assays, which work under comparable conditions. Because many of the known FAAH and MAGL inhibitors simply consist of a lipophilic scaffold to which a heterocyclic system is bound, furthermore, different heterocyclic structures were evaluated for their contribution to enzyme inhibition by attaching them to the same lipophilic backbone, namely 4-phenoxybenzene. One of the most active compound synthesized during this investigation was N,N-dimethyl-5-(4-phenoxyphenyl)-2H-tetrazole-2-carboxamide (16) (IC50 FAAH: 0.012 μM; IC50 MAGL: 0.028 μM). This inhibitor was systematically modified in the lipophilic 4-phenoxyphenyl region. Structure-activity relationship studies revealed that the inhibitory potency against FAAH and MAGL, respectively, could still be increased by replacement of the phenoxy residue of 16 by 3-chlorophenoxy (45) or pyrrol-1-yl groups (49). Finally, the tetrazolecarboxamide 16 and some related compounds were tested for metabolic stability with rat liver S9 fractions showing that these kind of FAAH/MAGL inhibitors are readily inactivated by cleavage of the bond between the tetrazole ring and its carboxamide substituent.

  1. Crystal structures of Mycobacterium tuberculosis S-adenosyl-L-homocysteine hydrolase in ternary complex with substrate and inhibitors

    SciTech Connect

    Reddy, Manchi C.M.; Kuppan, Gokulan; Shetty, Nishant D.; Owen, Joshua L.; Ioerger, Thomas R.; Sacchettini, James C.

    2009-12-01

    S-adenosylhomocysteine hydrolase (SAHH) is a ubiquitous enzyme that plays a central role in methylation-based processes by maintaining the intracellular balance between S-adenosylhomocysteine (SAH) and S-adenosylmethionine. We report the first prokaryotic crystal structure of SAHH, from Mycobacterium tuberculosis (Mtb), in complex with adenosine (ADO) and nicotinamide adenine dinucleotide. Structures of complexes with three inhibitors are also reported: 3{prime}-keto aristeromycin (ARI), 2-fluoroadenosine, and 3-deazaadenosine. The ARI complex is the first reported structure of SAHH complexed with this inhibitor, and confirms the oxidation of the 3{prime} hydroxyl to a planar keto group, consistent with its prediction as a mechanism-based inhibitor. We demonstrate the in vivo enzyme inhibition activity of the three inhibitors and also show that 2-fluoradenosine has bactericidal activity. While most of the residues lining the ADO-binding pocket are identical between Mtb and human SAHH, less is known about the binding mode of the homocysteine (HCY) appendage of the full substrate. We report the 2.0 {angstrom} resolution structure of the complex of SAHH cocrystallized with SAH. The most striking change in the structure is that binding of HCY forces a rotation of His363 around the backbone to flip out of contact with the 5{prime} hydroxyl of the ADO and opens access to a nearby channel that leads to the surface. This complex suggests that His363 acts as a switch that opens up to permit binding of substrate, then closes down after release of the cleaved HCY. Differences in the entrance to this access channel between human and Mtb SAHH are identified.

  2. In vitro and in vivo metabolism of N-adamantyl substituted urea-based soluble epoxide hydrolase inhibitors.

    PubMed

    Liu, Jun-Yan; Tsai, Hsing-Ju; Morisseau, Christophe; Lango, Jozsef; Hwang, Sung Hee; Watanabe, Takaho; Kim, In-Hae; Hammock, Bruce D

    2015-12-15

    N,N'-disubstituted urea-based soluble epoxide hydrolase (sEH) inhibitors are promising therapeutics for hypertension, inflammation, and pain in multiple animal models. The drug absorption and pharmacological efficacy of these inhibitors have been reported extensively. However, the drug metabolism of these inhibitors is not well described. Here we reported the metabolic profile and associated biochemical studies of an N-adamantyl urea-based sEH inhibitor 1-adamantan-1-yl-3-(5-(2-(2-ethoxyethoxy)ethoxy)pentyl)urea (AEPU) in vitro and in vivo. The metabolites of AEPU were identified by interpretation of liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and/or NMR. In vitro, AEPU had three major positions for phase I metabolism including oxidations on the adamantyl moiety, urea nitrogen atoms, and cleavage of the polyethylene glycol chain. In a rodent model, the metabolites from the hydroxylation on the adamantyl group and nitrogen atom were existed in blood while the metabolites from cleavage of polyethylene glycol chain were not found in urine. The major metabolite found in rodent urine was 3-(3-adamantyl-ureido)-propanoic acid, a presumably from cleavage and oxidation of the polyethylene glycol moiety. All the metabolites found were active but less potent than AEPU at inhibiting human sEH. Furthermore, cytochrome P450 (CYP) 3A4 was found to be a major enzyme mediating AEPU metabolism. In conclusion, the metabolism of AEPU resulted from oxidation by CYP could be shared with other N-adamantyl-urea-based compounds. These findings suggest possible therapeutic roles for AEPU and new strategies for drug design in this series of possible drugs.

  3. Synthesis and QSAR of Fatty Acid Amide Hydrolase Inhibitors: Modulation at the N-Portion of Biphenyl-3-yl Alkylcarbamates

    PubMed Central

    Mor, Marco; Lodola, Alessio; Rivara, Silvia; Vacondio, Federica; Duranti, Andrea; Tontini, Andrea; Sanchini, Silvano; Piersanti, Giovanni; Clapper, Jason R.; King, Alvin R.; Tarzia, Giorgio; Piomelli, Daniele

    2013-01-01

    Alkylcarbamic acid biphenyl-3-yl esters are a class of fatty acid amide hydrolase (FAAH) inhibitors that comprises cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester (URB597), a compound with analgesic, anxiolytic-like and antidepressant-like properties in rat and mouse models. Here, we extended the structure-activity relationships (SARs) for this class of compounds by replacing the cyclohexyl ring of the parent compound cyclohexylcarbamic acid biphenyl-3-yl ester (URB524) (IC50, for FAAH = 63 nM) with a selected set of substituents of different size, shape, flexibility and lipophilicity. Docking experiments and Linear Interaction Energy (LIE) calculations indicated that the N-terminal group of O-arylcarbamates fits within the lipophilic region of the substrate-binding site, mimicking the arachidonoyl chain of anandamide. Significant potency improvements were observed for the β-naphthylmethyl derivative 4q (IC50 = 5.3 nM) and its 3′-carbamoylbiphenyl-3-yl ester 4z (URB880, IC50 = 0.63 nM), indicating that shape complementarity and hydrogen bonds are crucial to obtain highly potent inhibitors. PMID:18507372

  4. Effect of a Soluble Epoxide Hydrolase Inhibitor, UC1728, on LPS-Induced Uveitis in the Rabbit

    PubMed Central

    McLellan, Gillian J.; Aktas, Zeynep; Hennes-Beean, Elizabeth; Kolb, Aaron W.; Larsen, Inna V.; Schmitz, Emily J.; Clausius, Hilary R.; Yang, Jun; Hwang, Sung Hee; Morisseau, Christophe; Inceoglu, Bora; Hammock, Bruce D.; Brandt, Curtis R.

    2016-01-01

    Cytochrome P450 epoxygenase isozymes convert free arachidonic acid into eicosanoids named epoxyeicosatrienoic acids (EETs) that have roles in regulating inflammation. EETs are rapidly converted to dihydroxyeicosatrienoic acids (DiHETs) by soluble epoxide hydrolase (sEH). Little is known about the potential role of these metabolites in uveitis, but conversion of EETs to DiHETs could contribute to the inflammation. We tested a potent and orally available inhibitor of sEH for its ability to reduce ocular inflammation in a rabbit LPS-induced model of uveitis. Rabbits were treated by subcutaneous injection with the sEH inhibitor (UC1728, 3 mg/kg), or the vehicle control (PEG400) and uveitis was assessed at 6, 24 and 48 h post-intracameral LPS injection using a modified Hackett-McDonald scoring system. Eyes treated by intra-cameral injection of PBS, or by aseptic preparation served as further controls. Signs of inflammation in this model were mild and transient. Treatment with UC1728 did not significantly reduce inflammation compared to animals treated with the PEG400 vehicle. Blood levels of UC1728 were a thousand fold higher than the in vitro determined inhibitory potency (IC50) of the compound suggesting a significant degree of inhibition of sEH in the rabbit. The lack of efficacy suggests that sEH or its substrates the EETs may not be involved in mediating inflammation in this model of uveitis. PMID:28066796

  5. Discovering Drugs with DNA-Encoded Library Technology: From Concept to Clinic with an Inhibitor of Soluble Epoxide Hydrolase.

    PubMed

    Belyanskaya, Svetlana L; Ding, Yun; Callahan, James F; Lazaar, Aili L; Israel, David I

    2017-03-09

    DNA-encoded chemical library technology was developed with the vision of its becoming a transformational platform for drug discovery. The hope was that a new paradigm for the discovery of low-molecular-weight drugs would be enabled by combining the vast molecular diversity achievable with combinatorial chemistry, the information-encoding attributes of DNA, the power of molecular biology, and a streamlined selection-based discovery process. Here, we describe the discovery and early clinical development of GSK2256294, an inhibitor of soluble epoxide hydrolase (sEH, EPHX2), by using encoded-library technology (ELT). GSK2256294 is an orally bioavailable, potent and selective inhibitor of sEH that has a long half life and produced no serious adverse events in a first-time-in-human clinical study. To our knowledge, GSK2256294 is the first molecule discovered from this technology to enter human clinical testing and represents a realization of the vision that DNA-encoded chemical library technology can efficiently yield molecules with favorable properties that can be readily progressed into high-quality drugs.

  6. Omeprazole increases the efficacy of a soluble epoxide hydrolase inhibitor in a PGE2 induced pain model

    PubMed Central

    Goswami, Sumanta Kumar; Inceoglu, Bora; Yang, Jun; Wan, Debin; Kodani, Sean D.; da Silva, Carlos Antonio Trindade; Morisseau, Christophe; Hammock, Bruce D.

    2015-01-01

    Epoxyeicosatrienoic acids (EETs) are potent endogenous analgesic metabolites produced from arachidonic acid by cytochrome P450s (P450s). Metabolism of EETs by soluble epoxide hydrolase (sEH) reduces their activity, while their stabilization by sEH inhibition decreases both inflammatory and neuropathic pain. Here, we tested the complementary hypothesis that increasing the level of EETs through induction of P450s by omeprazole (OME), can influence pain related signaling by itself, and potentiate the anti-hyperalgesic effect of sEH inhibitor. Rats were treated with OME (100 mg/kg/day, p.o., 7 days), sEH inhibitor TPPU (3 mg/kg/day, p.o.) and OME (100 mg/kg/day, p.o., 7 days) + TPPU (3 mg/kg/day, p.o., last 3 days of OME dose) dissolved in vehicle PEG400, and their effect on hyperalgesia (increased sensitivity to pain) induced by PGE2 was monitored. While OME treatment by itself exhibited variable effects on PGE2 induced hyperalgesia, it strongly potentiated the effect of TPPU in the same assay. The significant decrease in pain with OME + TPPU treatment correlated with the increased levels of EETs in plasma and increased activities of P450 1A1 and P450 1A2 in liver microsomes. The results show that reducing catabolism of EETs with a sEH inhibitor yielded a stronger analgesic effect than increasing generation of EETs by OME, and combination of both yielded the strongest pain reducing effect under the condition of this study. PMID:26522832

  7. Effects of Fatty Acid Amide Hydrolase (FAAH) Inhibitors in Non-Human Primate Models of Nicotine Reward and Relapse

    PubMed Central

    Justinova, Zuzana; Panlilio, Leigh V; Moreno-Sanz, Guillermo; Redhi, Godfrey H; Auber, Alessia; Secci, Maria E; Mascia, Paola; Bandiera, Tiziano; Armirotti, Andrea; Bertorelli, Rosalia; Chefer, Svetlana I; Barnes, Chanel; Yasar, Sevil; Piomelli, Daniele; Goldberg, Steven R

    2015-01-01

    Inhibition of the enzyme fatty acid amide hydrolase (FAAH) counteracts reward-related effects of nicotine in rats, but it has not been tested for this purpose in non-human primates. Therefore, we studied the effects of the first- and second-generation O-arylcarbamate-based FAAH inhibitors, URB597 (cyclohexyl carbamic acid 3'-carbamoyl-3-yl ester) and URB694 (6-hydroxy-[1,1'-biphenyl]-3-yl-cyclohexylcarbamate), in squirrel monkeys. Both FAAH inhibitors: (1) blocked FAAH activity in brain and liver, increasing levels of endogenous ligands for cannabinoid and α-type peroxisome proliferator-activated (PPAR-α) receptors; (2) shifted nicotine self-administration dose–response functions in a manner consistent with reduced nicotine reward; (3) blocked reinstatement of nicotine seeking induced by reexposure to either nicotine priming or nicotine-associated cues; and (4) had no effect on cocaine or food self-administration. The effects of FAAH inhibition on nicotine self-administration and nicotine priming-induced reinstatement were reversed by the PPAR-α antagonist, MK886. Unlike URB597, which was not self-administered by monkeys in an earlier study, URB694 was self-administered at a moderate rate. URB694 self-administration was blocked by pretreatment with an antagonist for either PPAR-α (MK886) or cannabinoid CB1 receptors (rimonabant). In additional experiments in rats, URB694 was devoid of THC-like or nicotine-like interoceptive effects under drug-discrimination procedures, and neither of the FAAH inhibitors induced dopamine release in the nucleus accumbens shell—consistent with their lack of robust reinforcing effects in monkeys. Overall, both URB597 and URB694 show promise for the initialization and maintenance of smoking cessation because of their ability to block the rewarding effects of nicotine and prevent nicotine priming-induced and cue-induced reinstatement. PMID:25754762

  8. Effects of Fatty Acid Amide Hydrolase (FAAH) Inhibitors in Non-Human Primate Models of Nicotine Reward and Relapse.

    PubMed

    Justinova, Zuzana; Panlilio, Leigh V; Moreno-Sanz, Guillermo; Redhi, Godfrey H; Auber, Alessia; Secci, Maria E; Mascia, Paola; Bandiera, Tiziano; Armirotti, Andrea; Bertorelli, Rosalia; Chefer, Svetlana I; Barnes, Chanel; Yasar, Sevil; Piomelli, Daniele; Goldberg, Steven R

    2015-08-01

    Inhibition of the enzyme fatty acid amide hydrolase (FAAH) counteracts reward-related effects of nicotine in rats, but it has not been tested for this purpose in non-human primates. Therefore, we studied the effects of the first- and second-generation O-arylcarbamate-based FAAH inhibitors, URB597 (cyclohexyl carbamic acid 3'-carbamoyl-3-yl ester) and URB694 (6-hydroxy-[1,1'-biphenyl]-3-yl-cyclohexylcarbamate), in squirrel monkeys. Both FAAH inhibitors: (1) blocked FAAH activity in brain and liver, increasing levels of endogenous ligands for cannabinoid and α-type peroxisome proliferator-activated (PPAR-α) receptors; (2) shifted nicotine self-administration dose-response functions in a manner consistent with reduced nicotine reward; (3) blocked reinstatement of nicotine seeking induced by reexposure to either nicotine priming or nicotine-associated cues; and (4) had no effect on cocaine or food self-administration. The effects of FAAH inhibition on nicotine self-administration and nicotine priming-induced reinstatement were reversed by the PPAR-α antagonist, MK886. Unlike URB597, which was not self-administered by monkeys in an earlier study, URB694 was self-administered at a moderate rate. URB694 self-administration was blocked by pretreatment with an antagonist for either PPAR-α (MK886) or cannabinoid CB1 receptors (rimonabant). In additional experiments in rats, URB694 was devoid of THC-like or nicotine-like interoceptive effects under drug-discrimination procedures, and neither of the FAAH inhibitors induced dopamine release in the nucleus accumbens shell--consistent with their lack of robust reinforcing effects in monkeys. Overall, both URB597 and URB694 show promise for the initialization and maintenance of smoking cessation because of their ability to block the rewarding effects of nicotine and prevent nicotine priming-induced and cue-induced reinstatement.

  9. Piperazinyl carbamate fatty acid amide hydrolase inhibitors and transient receptor potential channel modulators as "dual-target" analgesics.

    PubMed

    Maione, Sabatino; Costa, Barbara; Piscitelli, Fabiana; Morera, Enrico; De Chiaro, Maria; Comelli, Francesca; Boccella, Serena; Guida, Francesca; Verde, Roberta; Ortar, Giorgio; Di Marzo, Vincenzo

    2013-10-01

    We showed previously that inhibiting fatty acid amide hydrolase (FAAH), an endocannabinoid degrading enzyme, and transient receptor potential vanilloid type-1 (TRPV1) channels with the same molecule, the naturally occurring N-arachidonoyl-serotonin (AA-5-HT), produces more efficacious anti-nociceptive and anti-hyperalgesic actions than the targeting of FAAH or TRPV1 alone. We also reported the synthesis of some piperazinyl carbamates as "dual" FAAH inhibitors and either antagonists at TRPV1 or agonists/desensitizers of the transient receptor potential ankyrin type-1 (TRPA1) cannel, another target for analgesic drugs. We investigated here if two such compounds, the FAAH/TRPV1 blocker OMDM198 and the FAAH inhibitor/TRPA1 agonist, OMDM202, exert anti-nociceptive actions in the formalin test of pain in mice, and through what mechanism. Both compounds inhibited the second phase of the response to formalin, the effect being maximal at 3 mg/kg, i.p. Antagonism of CB1 or CB2 receptors with AM251 or AM630 (1 mg/kg, i.p.), respectively, reversed this effect. A TRPV1 agonist, palvanil (0.1 mg/kg, i.p.), also reversed the analgesic effect of OMDM198. OMDM202 action was also antagonized by a per se inactive dose of the selective TRPA1 blocker, AP-18 (0.05 mg/kg, i.p.), but not by a TRPV1 antagonist. AP-18 at higher doses (0.1-0.2 mg/kg) inhibited both the first and second phase of the formalin response. The effects of OMDM198 and OMDM202 were accompanied by elevation of anandamide levels in the spinal cord. OMDM198 (0.1-5.0 mg/kg, i.p.) also reversed carrageenan-induced oedema and thermal hyperalgesia in mice with efficacy similar to that of AA-5-HT. These data suggest that "dual" fatty acid amide hydrolase and transient receptor potential channel modulators should be clinically evaluated as novel analgesics.

  10. Proteomic Analysis of Oesophagostomum dentatum (Nematoda) during Larval Transition, and the Effects of Hydrolase Inhibitors on Development

    PubMed Central

    Ondrovics, Martina; Silbermayr, Katja; Mitreva, Makedonka; Young, Neil D.; Razzazi-Fazeli, Ebrahim; Gasser, Robin B.; Joachim, Anja

    2013-01-01

    In this study, in vitro drug testing was combined with proteomic and bioinformatic analyses to identify and characterize proteins involved in larval development of Oesophagostomum dentatum, an economically important parasitic nematode. Four hydrolase inhibitors ο-phenanthroline, sodium fluoride, iodoacetamide and 1,2-epoxy-3-(pnitrophenoxy)-propane (EPNP) significantly inhibited (≥90%) larval development. Comparison of the proteomic profiles of the development-inhibited larvae with those of uninhibited control larvae using two-dimensional gel electrophoresis, and subsequent MALDI-TOF mass spectrometric analysis identified a down-regulation of 12 proteins inferred to be involved in various larval developmental processes, including post-embryonic development and growth. Furthermore, three proteins (i.e. intermediate filament protein B, tropomyosin and peptidyl-prolyl cis-trans isomerase) inferred to be involved in the moulting process were down-regulated in moulting- and development-inhibited O. dentatum larvae. This first proteomic map of O. dentatum larvae provides insights in the protein profile of larval development in this parasitic nematode, and significantly improves our understanding of the fundamental biology of its development. The results and the approach used might assist in developing new interventions against parasitic nematodes by blocking or disrupting their key biological pathways. PMID:23717515

  11. Single administration of soluble epoxide hydrolase inhibitor suppresses neuroinflammation and improves neuronal damage after cardiac arrest in mice.

    PubMed

    Taguchi, Noriko; Nakayama, Shin; Tanaka, Makoto

    2016-10-01

    Cardiac arrest (CA) causes ischemia-reperfusion injury in the whole body among victims. Especially in the brain, inflammation and neuronal cell death can lead to irreversible dysfunction. Our goal was to determine whether a single administration of soluble epoxide hydrolase inhibitor (AS2586144-CL) has a neuroprotective effect and decreases the inflammatory response after CA and cardiopulmonary resuscitation (CPR). Global cerebral ischemia was induced in male C57BL/6 mice with 8min of CA. Thirty minutes after recovery of spontaneous circulation, the mice were randomly assigned to three groups and administered AS2586144-CL: 1mg/kg (n=25), 10mg/kg (n=25), or 0mg/kg (vehicle, n=25). At 6 and 7 days after CA/CPR, behavioral tests were conducted and brains were removed for histological evaluation. Analysis of histological damage 7 days after CA/CPR revealed that 10mg/kg of AS2586144-CL protected neurons, and suppressed cytokine production and microglial migration into the hippocampus. Two hours after CA/CPR, 10mg/kg of AS2586144-CL suppressed serum tumor necrosis factor-α and hippocampal nuclear factor κB expression. Our data show that 10mg/kg of AS2586144-CL administered following CA/CPR suppresses inflammation and decreases neuronal damage.

  12. Soluble epoxide hydrolase inhibitor 1-trifluoromethoxyphenyl-3- (1-propionylpiperidin-4-yl) urea attenuates bleomycin-induced pulmonary fibrosis in mice.

    PubMed

    Zhou, Yong; Yang, Jun; Sun, Guo-Ying; Liu, Tian; Duan, Jia-Xi; Zhou, Hui-Fang; Lee, Kin Sing; Hammock, Bruce D; Fang, Xiang; Jiang, Jian-Xin; Guan, Cha-Xiang

    2016-02-01

    Epoxyeicosatrienoic acids (EETs), the metabolites of arachidonic acid derived from the cytochrome P450 (CYP450) epoxygenases, are mainly metabolized by soluble epoxide hydrolase (sEH) to their corresponding diols. EETs but not their diols, have anti-inflammatory properties and inhibition of sEH might provide protective effects against inflammatory fibrosis. We test the effects of a selected sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), on bleomycin-induced pulmonary fibrosis (PF) in mice. A mouse model of PF was established by intratracheal injection of bleomycin and TPPU was administered for 21 days after bleomycin injection. We found TPPU treatment improved the body weight loss and survival rate of bleomycin-stimulated mice. Histological examination showed that TPPU treatment alleviated bleomycin-induced inflammation and maintained the alveolar structure of the pulmonary tissues. TPPU also decreased the bleomycin-induced deposition of collagen and the expression of procollagen I mRNA in lung tissues of mice. TPPU decreased the transforming growth factor-β1 (TGF-β1), interleukin-1β (IL-1β) and IL-6 levels in the serum of bleomycin-stimulated mice. Furthermore, TPPU inhibited the proliferation and collagen synthesis of mouse fibroblasts and partially reversed TGF-β1-induced α-smooth muscle actin expression. Our results indicate that the inhibition of sEH attenuates bleomycin-induced inflammation and collagen deposition and therefore prevents bleomycin-induced PF in a mouse model.

  13. Activation of TRPA1 channels by the fatty acid amide hydrolase inhibitor 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597).

    PubMed

    Niforatos, Wende; Zhang, Xu-Feng; Lake, Marc R; Walter, Karl A; Neelands, Torben; Holzman, Thomas F; Scott, Victoria E; Faltynek, Connie R; Moreland, Robert B; Chen, Jun

    2007-05-01

    As a member of the transient receptor potential (TRP) ion channel superfamily, the ligand-gated ion channel TRPA1 has been implicated in nociceptive function and pain states. The endogenous ligands that activate TRPA1 remain unknown. However, various agonists have been identified, including environmental irritants (e.g., acrolein) and ingredients of pungent natural products [e.g., allyl isothiocyanate (ITC), cinnamaldehyde, allicin, and gingerol]. In general, these agents are either highly reactive, nonselective, or not potent or efficacious, significantly limiting their utilities in the study of TRPA1 channel properties and biological functions. In a search for novel TRPA1 agonists, we identified 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597), a potent and systemically active inhibitor of fatty acid amide hydrolase (FAAH). This enzyme is responsible for anandamide degradation and therefore has been pursued as an antinociceptive and antiepileptic drug target. Using Ca(2+) influx assays and patch-clamp techniques, we demonstrated that URB597 could activate heterologously expressed human and rat TRPA1 channels, whereas two other FAAH inhibitors (i.e., URB532 and Compound 7) had no effect. When applied to inside-out membrane patches expressing rat TRPA1, URB597 elicited single-channel activities with a unitary conductance of 40 pS. Furthermore, URB597 activated TRPA1 channels endogenously expressed in a population of rat dorsal root ganglion neurons that also responded to ITC. In contrast to its effect on TRPA1, URB597 inhibited TRPM8 and had no effects on TRPV1 or TRPV4. Thus, we conclude that URB597 is a novel agonist of TRPA1 and probably activates the channel through a direct gating mechanism.

  14. Soluble Epoxide Hydrolase Inhibitors Reduce the Development of Atherosclerosis in Apolipoprotein E-Knockout Mouse Model

    PubMed Central

    Ulu, Arzu; Davis, Benjamin B.; Tsai, Hsing-Ju; Kim, In-Hae; Morisseau, Christophe; Inceoglu, Bora; Fiehn, Oliver; Hammock, Bruce D.; Weiss, Robert H.

    2008-01-01

    In order to determine whether sEH inhibitors influence atherosclerotic lesion formation, we utilized an established murine model of accelerated atherogenesis, ApoE knockout (−/−) mice. The sEH inhibitor, 1-adamantan-3-(5-(2-(2-ethylethoxy)ethoxy)pentyl)urea (AEPU) was delivered in drinking water. All animals were fed an atherogenic diet while simultaneously infused with angiotensin II by osmotic minipump to induce atherosclerosis. In AEPU-treated animals, there was a 53% reduction in atherosclerotic lesions in the descending aortae as compared to control aortae. AEPU and its major metabolites were detected in the plasma of animals which received it. As expected from the inhibition of sEH, a significant increase in linoleic and arachidonic acid epoxides, as well as an increase in individual 11,12-EET/DHET and 14,15-EET/DHET ratios, were observed. The reduction in atherosclerotic lesion area was inversely correlated with 11,12- and 14,15- EET/DHET ratios, suggesting that the reduction corresponds to the inhibition of sEH. Our data suggest that orally-available sEH inhibitors may be useful in the treatment of patients with atherosclerotic cardiovascular disease. PMID:18791465

  15. Optimization of the Central Heterocycle of α-Ketoheterocycle Inhibitors of Fatty Acid Amide Hydrolase

    PubMed Central

    Garfunkle, Joie; Ezzili, Cyrine; Rayl, Thomas J.; Hochstatter, Dustin G.; Hwang, Inkyu; Boger, Dale L.

    2008-01-01

    The synthesis and evaluation of a refined series of α-ketoheterocycles based on the oxazole 2 (OL-135) incorporating systematic changes in the central heterocycle bearing a key set of added substituents are described. The nature of the central heterocycle, even within the systematic and minor perturbations explored herein, significantly influenced the inhibitor activity: 1,3,4-oxadiazoles and 1,2,4-oxadiazoles 9 > tetrazoles, the isomeric 1,2,4-oxadiazoles 10, 1,3,4-thiadiazoles > oxazoles including 2 > 1,2-diazines > thiazoles > 1,3,4-triazoles. Most evident in these trends is the observation that introduction of an additional heteroatom at position 4 (oxazole numbering, N > O > CH) substantially increases activity that may be attributed to a reduced destabilizing steric interaction at the FAAH active site. Added heterocycle substituents displaying well defined trends may be utilized to enhance the inhibitor potency and, more significantly, to enhance the inhibitor selectivity. These trends, exemplified herein, emerge from both enhancements in the FAAH activity and simultaneous disruption of binding affinity for competitive off-target enzymes. PMID:18630870

  16. 1,3-Disubstituted Ureas Functionalized with Ether Groups are Potent Inhibitors of the Soluble Epoxide Hydrolase with Improved Pharmacokinetic Properties

    PubMed Central

    Kim, In-Hae; Tsai, Hsing-Ju; Nishi, Kosuke; Kasagami, Takeo; Morisseau, Christophe; Hammock, Bruce D.

    2008-01-01

    Soluble epoxide hydrolase (sEH) is a therapeutic target for treating hypertension and inflammation. 1,3-Disubstituted ureas functionalized with an ether group are potent sEH inhibitors. However, their relatively low metabolic stability leads to poor pharmacokinetic properties. To improve their bioavailability, we investigated the effect of incorporating various polar groups on the ether function on the inhibition potencies, physical properties, in vitro metabolic stability, and pharmacokinetic properties. The structure-activity relationship (SAR) studies showed that a hydrophobic linker between the urea group and the ether function is necessary to keep their potency. In addition, urea-ether inhibitors having a polar group such as diethylene glycol or morpholine significantly improved their physical properties and metabolic stability without any loss of inhibitory potency. Furthermore, improved pharmacokinetic properties in murine and canine models were obtained with the resulting inhibitors. These findings will facilitate the usage of sEH inhibitors in animal models of hypertension and inflammation. PMID:17894481

  17. Lipid sulfates and sulfonates are allosteric competitive inhibitors of the N-terminal phosphatase activity of the mammalian soluble epoxide hydrolase.

    PubMed

    Tran, Katherine L; Aronov, Pavel A; Tanaka, Hiromasa; Newman, John W; Hammock, Bruce D; Morisseau, Christophe

    2005-09-13

    The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), a homodimeric enzyme with each monomer containing two domains with distinct activities. The C-terminal domain, containing the epoxide hydrolase activity (Cterm-EH), is involved in the metabolism of arachidonic acid epoxides, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, and inflammation. We recently demonstrated that the N-terminal domain contains a Mg2+-dependent lipid phosphate phosphatase activity (Nterm-phos). However, the biological role of this activity is unknown. The inability of known phosphatase inhibitors to inhibit the Nterm-phos constitutes a significant barrier to the elucidation of its function. We describe herein sulfate, sulfonate, and phosphonate lipids as novel potent inhibitors of Nterm-phos. These compounds are allosteric competitive inhibitors with K(I) in the hundred nanomolar range. These inhibitors may provide a valuable tool to investigate the biological role of the Nterm-phos. We found that polyisoprenyl phosphates are substrates of Nterm-phos, suggesting a possible role in sterol synthesis or inflammation. Furthermore, some of these compounds inhibit the C-terminal sEH activity through a noncompetitive inhibition mechanism involving a new binding site on the C-terminal domain. This novel site may play a role in the natural in vivo regulation of epoxide hydrolysis by sEH.

  18. URB597, an inhibitor of fatty acid amide hydrolase, reduces hyperalgesia in diabetic rats.

    PubMed

    Hasanein, Parisa; Parviz, Mohsen; Keshavarz, Mansoor; Roohbakhsh, Ali

    2009-06-01

    Diabetic rats display increased pain responses after injection of formalin into the paw or thermal stimulation of the tail, suggesting the presence of hyperalgesia. In this study, we investigated the efficacy of URB597 (0.1, 0.3, and 0.5 mg/kg, i.p.), an inhibitor of endocannabinoids metabolism, on 2 models of experimental hyperalgesia in streptozotocin (STZ)-induced diabetic rats. Animals were divided into control, URB597-treated control (0.1, 0.3, and 0.5 mg/kg), diabetic, and URB597-treated diabetic (0.1, 0.3, and 0.5 mg/kg) groups. Formalin and tail-flick tests were performed 4 and 8 weeks after the onset of hyperglycemia, respectively. Diabetes caused significant hyperalgesia during these tests. URB597 (0.3 and 0.5 mg/kg) reversed chemical and thermal hyperalgesia in diabetic rats. Administration of URB597 at a dose of 0.1 mg/kg did not alter pain-related behaviors in control and diabetic groups compared with those of the respective control groups. URB597 treatment did not affect body weight or plasma glucose level of treated animals compared with nontreated animals. This study shows that increasing endocannabinoid neurotransmission with URB597 displays efficacy in chemical and thermal models of diabetic hyperalgesia. It also suggests that URB597 is a promising tool for treatment of painful diabetic neuropathy.

  19. Chemistry around imidazopyrazine and ibuprofen: discovery of novel fatty acid amide hydrolase (FAAH) inhibitors.

    PubMed

    De Wael, Frédéric; Muccioli, Giulio G; Lambert, Didier M; Sergent, Thérèse; Schneider, Yves-Jacques; Rees, Jean-François; Marchand-Brynaert, Jacqueline

    2010-09-01

    Based on the imidazo-[1,2-a]-pyrazin-3-(7H)-one scaffold, a dual action prodrug has been designed for combining antioxidant and anti-inflammatory activities, possibly unmasked upon oxidation. The construction of the target-molecule requires two building blocks, namely a 2-amino-1,4-pyrazine and a 2-ketoaldehyde. Attempts to synthesize the 2-ketoaldehyde (5a) derived from ibuprofen failed, but led to the corresponding 2-ketoaldoxime (7a) which could not be condensed with the pyrazine synthons. However, a model compound, i.e. phenylglyoxal aldoxime, reacted well under microwave activation to furnish novel imidazo[1,2-a]-pyrazine-3-(7H)-imine derivatives (18a,b). These heterobicycles behave as antioxidants by inhibiting the lipid peroxidation, and one compound (18b) is endowed with a significant anti-inflammatory effect in a cellular test. Unexpectedly, all the synthetic intermediates derived from ibuprofen are good inhibitors of FAAH, the most active compound (4a) featuring the 1,3-dithian-2-yl motif.

  20. Structure-based optimization of the piperazino-containing 1,3-disubstituted ureas affording sub-nanomolar inhibitors of soluble epoxide hydrolase

    PubMed Central

    Huang, Shao-Xu; Cao, Bin; Morisseau, Christophe; Jin, Yi; Hammock, Bruce D.; Long, Ya-Qiu

    2014-01-01

    The inhibition of the soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of hypertension, inflammation and other cardiovascular disorders. Piperazino functionality as the tertiary pharmacophore remarkably improved the drug-like profile of the 1,3-disubstituted urea sEH inhibitors. However, the potency was more dependent on the overall best balance of the hydrophilicity and lipophilicity. Based on the sEH-inhibitor complex structure, further structural optimization on the piperazino-containing 1,3-disubstituted urea scaffold was conducted for an improved potency. The 1-adamantylacetamide and para-phenylcarbonyl group were identified to be an optimal primary pharmacophore and secondary pharmacophore motif, respectively, generating sub-nanomolar sEH inhibitors with favorable water solubility. PMID:25225606

  1. Structure-based optimization of the piperazino-containing 1,3-disubstituted ureas affording sub-nanomolar inhibitors of soluble epoxide hydrolase.

    PubMed

    Huang, Shao-Xu; Cao, Bin; Morisseau, Christophe; Jin, Yi; Hammock, Bruce D; Long, Ya-Qiu

    2012-03-01

    The inhibition of the soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of hypertension, inflammation and other cardiovascular disorders. Piperazino functionality as the tertiary pharmacophore remarkably improved the drug-like profile of the 1,3-disubstituted urea sEH inhibitors. However, the potency was more dependent on the overall best balance of the hydrophilicity and lipophilicity. Based on the sEH-inhibitor complex structure, further structural optimization on the piperazino-containing 1,3-disubstituted urea scaffold was conducted for an improved potency. The 1-adamantylacetamide and para-phenylcarbonyl group were identified to be an optimal primary pharmacophore and secondary pharmacophore motif, respectively, generating sub-nanomolar sEH inhibitors with favorable water solubility.

  2. Crystal Structure of Fatty Acid Amide Hydrolase Bound to the Carbamate Inhibitor URB597: Discovery of a Deacylating Water Molecule and Insight into Enzyme Inactivation

    SciTech Connect

    Mileni, Mauro; Kamtekar, Satwik; Wood, David C.; Benson, Timothy E.; Cravatt, Benjamin F.; Stevens, Raymond C.

    2010-08-12

    The endocannabinoid system regulates a wide range of physiological processes including pain, inflammation, and cognitive/emotional states. URB597 is one of the best characterized covalent inhibitors of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH). Here, we report the structure of the FAAH-URB597 complex at 2.3 {angstrom} resolution. The structure provides insights into mechanistic details of enzyme inactivation and experimental evidence of a previously uncharacterized active site water molecule that likely is involved in substrate deacylation. This water molecule is part of an extensive hydrogen-bonding network and is coordinated indirectly to residues lining the cytosolic port of the enzyme. In order to corroborate our hypothesis concerning the role of this water molecule in FAAH's catalytic mechanism, we determined the structure of FAAH conjugated to a urea-based inhibitor, PF-3845, to a higher resolution (2.4 {angstrom}) than previously reported. The higher-resolution structure confirms the presence of the water molecule in a virtually identical location in the active site. Examination of the structures of serine hydrolases that are non-homologous to FAAH, such as elastase, trypsin, or chymotrypsin, shows a similarly positioned hydrolytic water molecule and suggests a functional convergence between the amidase signature enzymes and serine proteases.

  3. Discovery of the first dual inhibitor of the 5-lipoxygenase-activating protein and soluble epoxide hydrolase using pharmacophore-based virtual screening

    PubMed Central

    Temml, Veronika; Garscha, Ulrike; Romp, Erik; Schubert, Gregor; Gerstmeier, Jana; Kutil, Zsofia; Matuszczak, Barbara; Waltenberger, Birgit; Stuppner, Hermann; Werz, Oliver; Schuster, Daniela

    2017-01-01

    Leukotrienes (LTs) are pro-inflammatory lipid mediators derived from arachidonic acid (AA) with roles in inflammatory and allergic diseases. The biosynthesis of LTs is initiated by transfer of AA via the 5-lipoxygenase-activating protein (FLAP) to 5-lipoxygenase (5-LO). FLAP inhibition abolishes LT formation exerting anti-inflammatory effects. The soluble epoxide hydrolase (sEH) converts AA-derived anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatetraenoic acids (di-HETEs). Its inhibition consequently also counteracts inflammation. Targeting both LT biosynthesis and the conversion of EETs with a dual inhibitor of FLAP and sEH may represent a novel, powerful anti-inflammatory strategy. We present a pharmacophore-based virtual screening campaign that led to 20 hit compounds of which 4 targeted FLAP and 4 were sEH inhibitors. Among them, the first dual inhibitor for sEH and FLAP was identified, N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N’-(3,4-dichlorophenyl)urea with IC50 values of 200 nM in a cell-based FLAP test system and 20 nM for sEH activity in a cell-free assay. PMID:28218273

  4. Discovery of the first dual inhibitor of the 5-lipoxygenase-activating protein and soluble epoxide hydrolase using pharmacophore-based virtual screening

    NASA Astrophysics Data System (ADS)

    Temml, Veronika; Garscha, Ulrike; Romp, Erik; Schubert, Gregor; Gerstmeier, Jana; Kutil, Zsofia; Matuszczak, Barbara; Waltenberger, Birgit; Stuppner, Hermann; Werz, Oliver; Schuster, Daniela

    2017-02-01

    Leukotrienes (LTs) are pro-inflammatory lipid mediators derived from arachidonic acid (AA) with roles in inflammatory and allergic diseases. The biosynthesis of LTs is initiated by transfer of AA via the 5-lipoxygenase-activating protein (FLAP) to 5-lipoxygenase (5-LO). FLAP inhibition abolishes LT formation exerting anti-inflammatory effects. The soluble epoxide hydrolase (sEH) converts AA-derived anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatetraenoic acids (di-HETEs). Its inhibition consequently also counteracts inflammation. Targeting both LT biosynthesis and the conversion of EETs with a dual inhibitor of FLAP and sEH may represent a novel, powerful anti-inflammatory strategy. We present a pharmacophore-based virtual screening campaign that led to 20 hit compounds of which 4 targeted FLAP and 4 were sEH inhibitors. Among them, the first dual inhibitor for sEH and FLAP was identified, N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N’-(3,4-dichlorophenyl)urea with IC50 values of 200 nM in a cell-based FLAP test system and 20 nM for sEH activity in a cell-free assay.

  5. Structure-activity relationship studies on 1-heteroaryl-3-phenoxypropan-2-ones acting as inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase: replacement of the activated ketone group by other serine traps.

    PubMed

    Sundermann, Tom; Hanekamp, Walburga; Lehr, Matthias

    2016-08-01

    Cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are serine hydrolases. cPLA2α is involved in the generation of pro-inflammatory lipid mediators, FAAH terminates the anti-inflammatory effects of endocannabinoids. Therefore, inhibitors of these enzymes may represent new drug candidates for the treatment of inflammation. We have reported that certain 1-heteroarylpropan-2-ones are potent inhibitors of cPLA2α and FAAH. The serine reactive ketone group of these compounds, which is crucial for enzyme inhibition, is readily metabolized resulting in inactive alcohol derivatives. In order to obtain metabolically more stable inhibitors, we replaced this moiety by α-ketoheterocyle, cyanamide and nitrile serine traps. Investigations on activity and metabolic stability of these substances revealed that in all cases an increased metabolic stability was accompanied by a loss of inhibitory potency against cPLA2α and FAAH, respectively.

  6. Discovery and Optimization of Piperidyl-1,2,3-Triazole Ureas as Potent, Selective, and In Vivo-Active Inhibitors of Alpha/Beta-Hydrolase Domain Containing 6 (ABHD6)

    PubMed Central

    Hsu, Ku-Lung; Tsuboi, Katsunori; Chang, Jae Won; Whitby, Landon R.; Speers, Anna E.; Pugh, Holly; Cravatt, Benjamin F.

    2014-01-01

    Alpha/beta-hydrolase domain containing 6 (ABHD6) is a transmembrane serine hydrolase that hydrolyzes the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) to regulate certain forms of cannabinoid receptor-dependent signaling in the nervous system. The full spectrum of ABHD6 metabolic activities and functions is currently unknown and would benefit from selective, in vivo-active inhibitors. Here, we report the development and characterization of an advanced series of irreversible (2-substituted)-piperidyl-1,2,3-triazole urea inhibitors of ABHD6, including compounds KT182 and KT203, which show exceptional potency and selectivity in cells (< 5 nM) and, at equivalent doses in mice (1 mg kg-1), served as systemic and peripherally-restricted ABHD6 inhibitors, respectively. We also describe an orally-bioavailable ABHD6 inhibitor KT185 that displays excellent selectivity against other brain and liver serine hydrolases in vivo. We thus describe several chemical probes for biological studies of ABHD6, including brain-penetrant and peripherally-restricted inhibitors that should prove of value for interrogating ABHD6 function in animal models. PMID:24152295

  7. Attenuation of persistent pain-related behavior by fatty acid amide hydrolase (FAAH) inhibitors in a rat model of HIV sensory neuropathy

    PubMed Central

    Nasirinezhad, Farinaz; Jergova, Stanislava; Pearson, James P.; Sagen, Jacqueline

    2015-01-01

    Distal sensory neuropathies are a hallmark of HIV infections and can result in persistent and disabling pain despite advances in antiretroviral therapies. HIV-sensory neuropathic (HIV-SN) pain may be amenable to cannabinoid treatment, but currently available agonist treatments are limited by untoward side effects and potential for abuse in this patient population. Fatty acid amide hydrolase (FAAH) inhibitors may offer an alternative approach by inhibiting the degradation of endocannabinoids with purportedly fewer untoward CNS side effects. In order to evaluate this potential approach in the management of HIV-SN pain, the recombinant HIV envelope protein gp120 was applied epineurally to the rat sciatic nerve to induce an HIV-SN-like pain syndrome. Two distinct FAAH inhibitory compounds, URB597 and PF-3845 were tested, and contrasted with standard antinociceptive gabapentin or vehicle treatment, for attenuation of tactile allodynia, cold allodynia, and mechanical hyperalgesia. Both FAAH inhibitors markedly reduced cold and tactile allodynia with limited anti-hyperalgesic effects. Peak antinociceptive effects produced by both agents were more modest than gabapentin in reducing tactile allodynia with similar potency ranges. URB597 produced comparable cold anti-allodynic effects to gabapentin, and the effects of both FAAH inhibitors were longer lasting than gabapentin. To assess the contribution of cannabinoid receptors in these antinociceptive effects, CB1 antagonist AM251 or CB2 antagonist SR144528 were tested in conjunction with FAAH inhibitors. Results suggested a contribution of both CB1- and CB2-mediated effects, particularly in reducing tactile allodynia. In summary, these findings support inhibition of endocannabinoid degradation as a promising target for management of disabling persistent HIV-SN pain syndromes. PMID:25486617

  8. Attenuation of persistent pain-related behavior by fatty acid amide hydrolase (FAAH) inhibitors in a rat model of HIV sensory neuropathy.

    PubMed

    Nasirinezhad, Farinaz; Jergova, Stanislava; Pearson, James P; Sagen, Jacqueline

    2015-08-01

    Distal sensory neuropathies are a hallmark of HIV infections and can result in persistent and disabling pain despite advances in antiretroviral therapies. HIV-sensory neuropathic (HIV-SN) pain may be amenable to cannabinoid treatment, but currently available agonist treatments are limited by untoward side effects and potential for abuse in this patient population. Fatty acid amide hydrolase (FAAH) inhibitors may offer an alternative approach by inhibiting the degradation of endocannabinoids with purportedly fewer untoward CNS side effects. In order to evaluate this potential approach in the management of HIV-SN pain, the recombinant HIV envelope protein gp120 was applied epineurally to the rat sciatic nerve to induce an HIV-SN-like pain syndrome. Two distinct FAAH inhibitory compounds, URB597 and PF-3845 were tested, and contrasted with standard antinociceptive gabapentin or vehicle treatment, for attenuation of tactile allodynia, cold allodynia, and mechanical hyperalgesia. Both FAAH inhibitors markedly reduced cold and tactile allodynia with limited anti-hyperalgesic effects. Peak antinociceptive effects produced by both agents were more modest than gabapentin in reducing tactile allodynia with similar potency ranges. URB597 produced comparable cold anti-allodynic effects to gabapentin, and the effects of both FAAH inhibitors were longer lasting than gabapentin. To assess the contribution of cannabinoid receptors in these antinociceptive effects, CB1 antagonist AM251 or CB2 antagonist SR144528 were tested in conjunction with FAAH inhibitors. Results suggested a contribution of both CB1- and CB2-mediated effects, particularly in reducing tactile allodynia. In summary, these findings support inhibition of endocannabinoid degradation as a promising target for management of disabling persistent HIV-SN pain syndromes.

  9. Fluoride-mediated capture of a noncovalent bound state of a reversible covalent enzyme inhibitor: X-ray crystallographic analysis of an exceptionally potent α-ketoheterocycle inhibitor of fatty acid amide hydrolase.

    PubMed

    Mileni, Mauro; Garfunkle, Joie; Ezzili, Cyrine; Cravatt, Benjamin F; Stevens, Raymond C; Boger, Dale L

    2011-03-23

    Two cocrystal X-ray structures of the exceptionally potent α-ketoheterocycle inhibitor 1 (K(i) = 290 pM) bound to a humanized variant of rat fatty acid amide hydrolase (FAAH) are disclosed, representing noncovalently and covalently bound states of the same inhibitor with the enzyme. Key to securing the structure of the noncovalently bound state of the inhibitor was the inclusion of fluoride ion in the crystallization conditions that is proposed to bind the oxyanion hole precluding inhibitor covalent adduct formation with stabilization of the tetrahedral hemiketal. This permitted the opportunity to detect important noncovalent interactions stabilizing the binding of the inhibitor within the FAAH active site independent of the covalent reaction. Remarkably, noncovalently bound 1 in the presence of fluoride appears to capture the active site in the same "in action" state with the three catalytic residues Ser241-Ser217-Lys142 occupying essentially identical positions observed in the covalently bound structure of 1, suggesting that this technique of introducing fluoride may have important applications in structural studies beyond inhibiting substrate or inhibitor oxyanion hole binding. Key insights to emerge from the studies include the observations that noncovalently bound 1 binds in its ketone (not gem diol) form, that the terminal phenyl group in the acyl side chain of the inhibitor serves as the key anchoring interaction overriding the intricate polar interactions in the cytosolic port, and that the role of the central activating heterocycle is dominated by its intrinsic electron-withdrawing properties. These two structures are also briefly compared with five X-ray structures of α-ketoheterocycle-based inhibitors bound to FAAH recently disclosed.

  10. Fluoride-Mediated Capture of a Noncovalent Bound State of a Reversible Covalent Enzyme Inhibitor: X-ray Crystallographic Analysis of an Exceptionally Potent α-Ketoheterocycle Inhibitor of Fatty Acid Amide Hydrolase

    PubMed Central

    Mileni, Mauro; Garfunkle, Joie; Ezzili, Cyrine; Cravatt, Benjamin F.; Stevens, Raymond C.; Boger, Dale L.

    2011-01-01

    Two cocrystal X-ray structures of the exceptionally potent α-ketoheterocycle inhibitor 1 (Ki = 290 pM) bound to a humanized variant of rat fatty acid amide hydrolase (FAAH) are disclosed, representing noncovalently and covalently bound states of the same inhibitor with the enzyme. Key to securing the structure of the noncovalently bound state of the inhibitor was the inclusion of fluoride ion in the crystallization conditions that is proposed to bind the oxyanion hole precluding inhibitor covalent adduct formation with stabilization of the tetrahedral hemiketal. This permitted the opportunity to detect important noncovalent interactions stabilizing the binding of the inhibitor within the FAAH active site independent of the covalent reaction. Remarkably, noncovalently bound 1 in the presence of fluoride appears to capture the active site in the same “in action” state with the three catalytic residues Ser241–Ser217–Lys142 occupying essentially identical positions observed in the covalently bound structure of 1, suggesting that this technique of introducing fluoride may have important applications in structural studies beyond inhibiting substrate or inhibitor oxyanion hole binding. Key insights to emerge from the studies include the observations that noncovalently bound 1 binds in its ketone (not gem diol) form, that the terminal phenyl group in the acyl side chain of the inhibitor serves as the key anchoring interaction overriding the intricate polar interactions in the cytosolic port, and that the role of the central activating heterocycle is dominated by its intrinsic electron-withdrawing properties. These two structures are also briefly compared with five X-ray structures of α-ketoheterocycle-based inhibitors bound to FAAH recently disclosed. PMID:21355555

  11. Selective inhibition and selective induction of multiple microsomal epoxide hydrolases.

    PubMed

    Guenthner, T M

    1986-03-01

    The inhibition in vitro and induction in vivo of microsomal trans-stilbene oxide hydrolase have been studied. This microsomal epoxide hydrolase activity is distinguishable from the previously well-defined microsomal arene oxide hydrolase by a number of catalytic criteria. Two substituted chalcone oxides, 4-phenylchalcone oxide and 4'-phenylchalcone oxide, are potent inhibitors of microsomal trans-stilbene oxide hydrolase, but have no apparent activity against benzo[a]pyrene 4,5-oxide hydrolase. Conversely, compounds that are potent inhibitors of benzo[a]pyrene 4,5-oxide hydrolase, including styrene oxide, cyclohexene oxide, and trichloropropene oxide, inhibit microsomal trans-stilbene oxide hydrolase only at very high (millimolar) concentrations. The chalcone oxides inhibit microsomal trans-stilbene oxide hydrolase noncompetitively, and have micromolar or nanomolar affinity constants for the enzyme. Attempts were made to induce microsomal trans-stilbene oxide hydrolase in vivo. Compounds that induced microsomal benzo[a]pyrene 4,5-oxide hydrolase levels in mice did not simultaneously induce trans-stilbene oxide hydrolase levels. Clofibrate was an exception; it induced levels of both enzymes to a small but statistically significant degree. The two microsomal hydrolase activities have, therefore, very different catalytic sites and appear to be under separate genetic control. 4-Phenylchalcone oxide and 4'-phenylchalcone oxide are selective inhibitors of microsomal trans-stilbene oxide hydrolase and may prove to be very useful in assessing the involvement of this enzyme in the metabolism of endogenous or xenobiotic epoxides.

  12. Potent Natural Soluble Epoxide Hydrolase Inhibitors from Pentadiplandra brazzeana Baillon: Synthesis, Quantification, and Measurement of Biological Activities In Vitro and In Vivo

    PubMed Central

    Kitamura, Seiya; Morisseau, Christophe; Inceoglu, Bora; Kamita, Shizuo G.; De Nicola, Gina R.; Nyegue, Maximilienne; Hammock, Bruce D.

    2015-01-01

    We describe here three urea-based soluble epoxide hydrolase (sEH) inhibitors from the root of the plant Pentadiplandra brazzeana. The concentration of these ureas in the root was quantified by LC-MS/MS, showing that 1, 3-bis (4-methoxybenzyl) urea (MMU) is the most abundant (42.3 μg/g dry root weight). All of the ureas were chemically synthesized, and their inhibitory activity toward recombinant human and recombinant rat sEH was measured. The most potent compound, MMU, showed an IC50 of 92 nM via fluorescent assay and a Ki of 54 nM via radioactivity-based assay on human sEH. MMU effectively reduced inflammatory pain in a rat nociceptive pain assay. These compounds are among the most potent sEH inhibitors derived from natural sources. Moreover, inhibition of sEH by these compounds may mechanistically explain some of the therapeutic effects of P. brazzeana. PMID:25659109

  13. Bioactive lipid profiling reveals drug target engagement of a soluble epoxide hydrolase inhibitor in a murine model of tobacco smoke exposure.

    PubMed

    Nording, Malin L; Yang, Jun; Hoang, Laura; Zamora, Vanessa; Uyeminami, Dale; Espiritu, Imelda; Pinkerton, Kent E; Hammock, Bruce D; Luria, Ayala

    2015-04-01

    The inflammatory process underlying chronic obstructive pulmonary disease (COPD) may be caused by tobacco smoke (TS) exposure. Previous studies show that epoxyeicosatrienoic acids (EETs) possess promising anti-inflammatory properties, therefore stabilization of EETs and other fatty acid epoxides through inhibition of soluble epoxide hydrolase (sEH) was investigated in mouse models of acute and sub-chronic inflammation caused by TS exposure. During the entire TS exposure, the potent sEH inhibitor 1-(1-methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS) was given via drinking water. To assess drug target engagement of TUPS, a tandem mass spectrometry method was used for bioactive lipid profiling of a broad range of fatty acid metabolites, including EETs, and their corresponding diols (DHETs) derived from arachidonic acid, as well as epoxides and diols derived from other fatty acids. Several, but not all, plasma epoxide/diol ratios increased in mice treated with sEH inhibitor, compared to non-treated mice suggesting a wider role for sEH involving more fatty acid precursors besides arachidonic acid. This study supports qualitative use of epoxide/diol ratios explored by bioactive lipid profiling to indicate drug target engagement in mouse models of TS exposure relevant to COPD, which may have ramifications for future therapeutic interventions of sEH.

  14. Fatty acid amide hydrolase (FAAH) inhibitors exert pharmacological effects, but lack antinociceptive efficacy in rats with neuropathic spinal cord injury pain.

    PubMed

    Hama, Aldric T; Germano, Peter; Varghese, Matthew S; Cravatt, Benjamin F; Milne, G Todd; Pearson, James P; Sagen, Jacqueline

    2014-01-01

    Amelioration of neuropathic spinal cord injury (SCI) pain is a clinical challenge. Increasing the endocannabinoid anandamide and other fatty acid amides (FAA) by blocking fatty acid amide hydrolase (FAAH) has been shown to be antinociceptive in a number of animal models of chronic pain. However, an antinociceptive effect of blocking FAAH has yet to be demonstrated in a rat model of neuropathic SCI pain. Four weeks following a SCI, rats developed significantly decreased hind paw withdrawal thresholds, indicative of below-level cutaneous hypersensitivity. A group of SCI rats were systemically treated (i.p.) with either the selective FAAH inhibitor URB597 or vehicle twice daily for seven days. A separate group of SCI rats received a single dose (p.o.) of either the selective FAAH inhibitor PF-3845 or vehicle. Following behavioral testing, levels of the FAA N-arachidonoylethanolamide, N-oleoyl ethanolamide and N-palmitoyl ethanolamide were quantified in brain and spinal cord from SCI rats. Four weeks following SCI, FAA levels were markedly reduced in spinal cord tissue. Although systemic treatment with URB597 significantly increased CNS FAA levels, no antinociceptive effect was observed. A significant elevation of CNS FAA levels was also observed following oral PF-3845 treatment, but only a modest antinociceptive effect was observed. Increasing CNS FAA levels alone does not lead to robust amelioration of below-level neuropathic SCI pain. Perhaps utilizing FAAH inhibition in conjunction with other analgesic mechanisms could be an effective analgesic therapy.

  15. Cardioprotective effects of fatty acid amide hydrolase inhibitor URB694, in a rodent model of trait anxiety

    PubMed Central

    Carnevali, Luca; Vacondio, Federica; Rossi, Stefano; Macchi, Emilio; Spadoni, Gilberto; Bedini, Annalida; Neumann, Inga D.; Rivara, Silvia; Mor, Marco; Sgoifo, Andrea

    2015-01-01

    In humans, chronic anxiety represents an independent risk factor for cardiac arrhythmias and sudden death. Here we evaluate in male Wistar rats bred for high (HAB) and low (LAB) anxiety-related behavior, as well as non-selected (NAB) animals, the relationship between trait anxiety and cardiac electrical instability and investigate whether pharmacological augmentation of endocannabinoid anandamide-mediated signaling exerts anxiolytic-like and cardioprotective effects. HAB rats displayed (i) a higher incidence of ventricular tachyarrhythmias induced by isoproterenol, and (ii) a larger spatial dispersion of ventricular refractoriness assessed by means of an epicardial mapping protocol. In HAB rats, acute pharmacological inhibition of the anandamide-degrading enzyme, fatty acid amide hydrolase (FAAH), with URB694 (0.3 mg/kg), (i) decreased anxiety-like behavior in the elevated plus maze, (ii) increased anandamide levels in the heart, (iii) reduced isoproterenol-induced occurrence of ventricular tachyarrhythmias, and (iv) corrected alterations of ventricular refractoriness. The anti-arrhythmic effect of URB694 was prevented by pharmacological blockade of the cannabinoid type 1 (CB1), but not of the CB2, receptor. These findings suggest that URB694 exerts anxiolytic-like and cardioprotective effects in HAB rats, the latter via anandamide-mediated activation of CB1 receptors. Thus, pharmacological inhibition of FAAH might be a viable pharmacological strategy for the treatment of anxiety-related cardiac dysfunction. PMID:26656183

  16. Inhibition of fatty acid amide hydrolase and monoacylglycerol lipase by the anandamide uptake inhibitor VDM11: evidence that VDM11 acts as an FAAH substrate.

    PubMed

    Vandevoorde, Séverine; Fowler, Christopher J

    2005-08-01

    There is some dispute concerning the extent to which the uptake inhibitor VDM11 (N-(4-hydroxy-2-methylphenyl) arachidonoyl amide) is capable of inhibiting the metabolism of the endocannabinoid anandamide (AEA) by fatty acid amide hydrolase (FAAH). In view of a recent study demonstrating that the closely related compound AM404 (N-(4-hydroxyphenyl)arachidonylamide) is a substrate for FAAH, we re-examined the interaction of VDM11 with FAAH. In the presence of fatty acid-free bovine serum albumin (BSA, 0.125% w v(-1)), both AM404 and VDM11 inhibited the metabolism of AEA by rat brain FAAH with similar potencies (IC(50) values of 2.1 and 2.6 microM, respectively). The compounds were about 10-fold less potent as inhibitors of the metabolism of 2-oleoylglycerol (2-OG) by cytosolic monoacylglycerol lipase (MAGL). The potency of VDM11 towards FAAH was dependent upon the assay concentration of fatty acid-free bovine serum albumin (BSA). Thus, in the absence of fatty acid-free BSA, the IC(50) value for inhibition of FAAH was reduced by a factor of about two (from 2.9 to 1.6 microM). A similar reduction in the IC(50) value for the inhibition of membrane bound MAGL by both this compound (from 14 to 6 microM) and by arachidonoyl serinol (from 24 to 13 microM) was seen. An HPLC assay was set up to measure 4-amino-m-cresol, the hypothesised product of FAAH-catalysed VDM11 hydrolysis. 4-Amino-m-cresol was eluted with a retention time of approximately 2.4 min, but showed a time-dependent degradation to compounds eluting at peaks of approximately 5.6 and approximately 8 min. Peaks with the same retention times were also found following incubation of the membranes with VDM11, but were not seen when the membranes were preincubated with the FAAH inhibitors URB597 (3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) and CAY10401 (1-oxazolo[4,5-b]pyridin-2-yl-9-octadecyn-1-one) prior to addition of VDM11. The rate of metabolism of VDM11 was estimated to be roughly 15-20% of that for

  17. Characterisation of (R)-2-(2-Fluorobiphenyl-4-yl)-N-(3-Methylpyridin-2-yl)Propanamide as a Dual Fatty Acid Amide Hydrolase: Cyclooxygenase Inhibitor

    PubMed Central

    Gouveia-Figueira, Sandra; Karlsson, Jessica; Deplano, Alessandro; Hashemian, Sanaz; Svensson, Mona; Fredriksson Sundbom, Marcus; Congiu, Cenzo; Onnis, Valentina; Fowler, Christopher J.

    2015-01-01

    Background Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-2) inhibitors is a promising approach for pain-relief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl)-N-(3-methylpyridin-2-yl)propanamide (Flu-AM1). These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-2 for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-2-derived lipids in intact cells are not known. Methodology/Principal Findings COX inhibition was determined using an oxygraphic method with arachidonic acid and 2-arachidonoylglycerol (2-AG) as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA) as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Both enantiomers inhibited COX-2 in a substrate-selective and time-dependent manner, with IC50 values in the absence of a preincubation phase of: (R)-Flu-AM1, COX-1 (arachidonic acid) 6 μM; COX-2 (arachidonic acid) 20 μM; COX-2 (2-AG) 1 μM; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 μM; COX-2 (arachidonic acid) 10 μM; COX-2 (2-AG) 0.7 μM. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 μM) greatly inhibited the production of prostaglandin D2 and E2 in both unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R)-Flu-AM1 or by 10 μM flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 μM). Conclusions/Significance Both enantiomers of Flu-AM1 are more potent inhibitors of 2-AG compared to arachidonic acid oxygenation by COX-2. Inhibition of COX in lipopolysaccharide + interferon γ- stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the

  18. The fatty acid amide hydrolase inhibitor PF-3845 promotes neuronal survival, attenuates inflammation and improves functional recovery in mice with traumatic brain injury.

    PubMed

    Tchantchou, Flaubert; Tucker, Laura B; Fu, Amanda H; Bluett, Rebecca J; McCabe, Joseph T; Patel, Sachin; Zhang, Yumin

    2014-10-01

    Traumatic brain injury (TBI) is the leading cause of death in young adults in the United States, but there is still no effective agent for treatment. N-arachidonoylethanolamine (anandamide, AEA) is a major endocannabinoid in the brain. Its increase after brain injury is believed to be protective. However, the compensatory role of AEA is transient due to its rapid hydrolysis by the fatty acid amide hydrolase (FAAH). Thus, inhibition of FAAH can boost the endogenous levels of AEA and prolong its protective effect. Using a TBI mouse model, we found that post-injury chronic treatment with PF3845, a selective and potent FAAH inhibitor, reversed TBI-induced impairments in fine motor movement, hippocampus dependent working memory and anxiety-like behavior. Treatment with PF3845 inactivated FAAH activity and enhanced the AEA levels in the brain. It reduced neurodegeneration in the dentate gyrus, and up-regulated the expression of Bcl-2 and Hsp70/72 in both cortex and hippocampus. PF3845 also suppressed the increased production of amyloid precursor protein, prevented dendritic loss and restored the levels of synaptophysin in the ipsilateral dentate gyrus. Furthermore, PF3845 suppressed the expression of inducible nitric oxide synthase and cyclooxygenase-2 and enhanced the expression of arginase-1 post-TBI, suggesting a shift of microglia/macrophages from M1 to M2 phenotype. The effects of PF3845 on TBI-induced behavioral deficits and neurodegeneration were mediated by activation of cannabinoid type 1 and 2 receptors and might be attributable to the phosphorylation of ERK1/2 and AKT. These results suggest that selective inhibition of FAAH is likely to be beneficial for TBI treatment.

  19. Inhibitors of fatty acid amide hydrolase reduce carrageenan-induced hind paw inflammation in pentobarbital-treated mice: comparison with indomethacin and possible involvement of cannabinoid receptors.

    PubMed

    Holt, Sandra; Comelli, Francesca; Costa, Barbara; Fowler, Christopher J

    2005-10-01

    The in vivo effect of inhibitors of fatty acid amide hydrolase (FAAH) upon oedema volume and FAAH activity was evaluated in the carrageenan induced hind paw inflammation model in the mouse. Oedema was measured at two time points, 2 and 4 h, after intraplantar injection of carrageenan to anaesthetised mice. Intraperitoneal (i.p.) injections of the FAAH inhibitor URB597 (0.1, 0.3, 1 and 3 mg kg(-1)) 30 min prior to carrageenan administration, dose-dependently reduced oedema formation. At the 4 h time point, the ED(50) for URB597 was approximately 0.3 mg kg(-1). Indomethacin (5 mg kg(-1) i.p.) completely prevented the oedema response to carrageenan. The antioedema effects of indomethacin and URB597 were blocked by 3 mg kg(-1) i.p. of the CB(2) receptor antagonist SR144528. The effect of URB597 was not affected by pretreatment with the peroxisome proliferator-activated receptor gamma antagonist bisphenol A diglycidyl ether (30 mg kg(-1) i.p.) or the TRPV1 antagonist capsazepine (10 mg kg(-1) i.p.), when oedema was assessed 4 h after carrageenan administration. The CB(1) receptor antagonists AM251 (3 mg kg(-1) i.p.) and rimonabant (0.5 mg kg(-1) i.p.) gave inconsistent effects upon the antioedema effect of URB597. FAAH measurements were conducted ex vivo in the paws, spinal cords and brains of the mice. The activities of FAAH in the paws and spinal cords of the inflamed vehicle-treated mice were significantly lower than the corresponding activities in the noninflamed mice. PMSF treatment almost completely inhibited the FAAH activity in all three tissues, as did the highest dose of URB597 (3 mg kg(-1)) in spinal cord samples, whereas no obvious changes were seen ex vivo for the other treatments. In conclusion, the results show that in mice, treatment with indomethacin and URB597 produce SR144528-sensitive anti-inflammatory effects in the carrageenan model of acute inflammation.

  20. Crystal structure and activity of protein L-isoaspartyl-O-methyltransferase from Vibrio cholerae, and the effect of AdoHcy binding.

    PubMed

    Chatterjee, Tanaya; Mukherjee, Debadrita; Banerjee, Mousumi; Chatterjee, Barun K; Chakrabarti, Pinak

    2015-10-01

    The repair enzyme Protein L-isoaspartyl-O-methyltransferase (PIMT) is widely distributed in various organisms. PIMT catalyzes S-adenosylmethionine (AdoMet) dependent methylation of abnormal L-isoaspartyl residues, formed by the deamidation of asparagines and isomerization of aspartates. We report the crystal structure of PIMT of Vibrio cholerae (VcPIMT), the aetiological agent for cholera, complexed with the demethylated cofactor S-adenosyl-L-homocysteine (AdoHcy) to 2.05 Å resolution. A stretch of residues (39-58), lining the substrate-binding site, is disordered. Urea-induced unfolding free energy for apo and VcPIMT-AdoHcy complex reveals greater stability for the cofactor-bound protein. The kinetic parameters for the methyltransferase activity of the recombinant VcPIMT was determined using a continuous spectrophotometric color-based assay using the peptide substrate [VYP(L-isoD)HA]. The enzyme exhibited activity higher than the Escherichia coli enzyme and closer to those from thermophilic bacteria and the mammalian source. The association constant for substrate binding is 2.29 × 10(6) M(-1), quite similar to that for AdoHcy. The crystal structure and the model of the peptide-bound structure indicate that the majority of the interactions used for cofactor/substrate binding are provided by the main-chain atoms. Evolutionary relationships derived based on a phylogenetic tree constructed using the PIMT sequences are in conformity with the crystal structures of nine AdoHcy-bound PIMTs.

  1. The fatty acid amide hydrolase (FAAH) inhibitor PF-3845 acts in the nervous system to reverse LPS-induced tactile allodynia in mice

    PubMed Central

    Booker, Lamont; Kinsey, Steven G; Abdullah, Rehab A; Blankman, Jacqueline L; Long, Jonathan Z; Ezzili, Cyrine; Boger, Dale L; Cravatt, Benjamin F; Lichtman, Aron H

    2012-01-01

    BACKGROUND AND PURPOSE Inflammatory pain presents a problem of clinical relevance and often elicits allodynia, a condition in which non-noxious stimuli are perceived as painful. One potential target to treat inflammatory pain is the endogenous cannabinoid (endocannabinoid) system, which is comprised of CB1 and CB2 cannabinoid receptors and several endogenous ligands, including anandamide (AEA). Blockade of the catabolic enzyme fatty acid amide hydrolase (FAAH) elevates AEA levels and elicits antinociceptive effects, without the psychomimetic side effects associated with Δ9-tetrahydrocannabinol (THC). EXPERIMENTAL APPROACH Allodynia was induced by intraplantar injection of LPS. Complementary genetic and pharmacological approaches were used to determine the strategy of blocking FAAH to reverse LPS-induced allodynia. Endocannabinoid levels were quantified using mass spectroscopy analyses. KEY RESULTS FAAH (−/−) mice or wild-type mice treated with FAAH inhibitors (URB597, OL-135 and PF-3845) displayed an anti-allodynic phenotype. Furthermore, i.p. PF-3845 increased AEA levels in the brain and spinal cord. Additionally, intraplantar PF-3845 produced a partial reduction in allodynia. However, the anti-allodynic phenotype was absent in mice expressing FAAH exclusively in the nervous system under a neural specific enolase promoter, implicating the involvement of neuronal fatty acid amides (FAAs). The anti-allodynic effects of FAAH-compromised mice required activation of both CB1 and CB2 receptors, but other potential targets of FAA substrates (i.e. µ-opioid, TRPV1 and PPARα receptors) had no apparent role. CONCLUSIONS AND IMPLICATIONS AEA is the primary FAAH substrate reducing LPS-induced tactile allodynia. Blockade of neuronal FAAH reverses allodynia through the activation of both cannabinoid receptors and represents a promising target to treat inflammatory pain. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To

  2. Discovery of 4-[(2S)-2-{[4-(4-chlorophenoxy)phenoxy]methyl}-1-pyrrolidinyl]butanoic acid (DG-051) as a novel leukotriene A4 hydrolase inhibitor of leukotriene B4 biosynthesis.

    PubMed

    Sandanayaka, Vincent; Mamat, Bjorn; Mishra, Rama K; Winger, Jennifer; Krohn, Michael; Zhou, Li-Ming; Keyvan, Monica; Enache, Livia; Sullins, David; Onua, Emmanuel; Zhang, Jun; Halldorsdottir, Gudrun; Sigthorsdottir, Heida; Thorlaksdottir, Audur; Sigthorsson, Gudmundur; Thorsteinnsdottir, Margret; Davies, Douglas R; Stewart, Lance J; Zembower, David E; Andresson, Thorkell; Kiselyov, Alex S; Singh, Jasbir; Gurney, Mark E

    2010-01-28

    Both in-house human genetic and literature data have converged on the identification of leukotriene 4 hydrolase (LTA(4)H) as a key target for the treatment of cardiovascular disease. We combined fragment-based crystallography screening with an iterative medicinal chemistry effort to optimize inhibitors of LTA(4)H. Ligand efficiency was followed throughout our structure-activity studies. As applied within the context of LTA(4)H inhibitor design, the chemistry team was able to design a potent compound 20 (DG-051) (K(d) = 26 nM) with high aqueous solubility (>30 mg/mL) and high oral bioavailability (>80% across species) that is currently undergoing clinical evaluation for the treatment of myocardial infarction and stroke. The structural biology-chemistry interaction described in this paper provides a sound alternative to conventional screening techniques. This is the first example of a gene-to-clinic paradigm enabled by a fragment-based drug discovery effort.

  3. Twisting of glycosidic bonds by hydrolases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Patterns of scissile bond twisting have been found in crystal structures of glycoside hydrolases (GHs) that are complexed with substrates and inhibitors. To estimate the increased potential energy in the substrates that results from this twisting, we have plotted torsion angles for the scissile bond...

  4. 1-(3-biaryloxy-2-oxopropyl)indole-5-carboxylic acids and related compounds as dual inhibitors of human cytosolic phospholipase A2α and fatty acid amide hydrolase.

    PubMed

    Zahov, Stefan; Drews, Andreas; Hess, Mark; Schulze Elfringhoff, Alwine; Lehr, Matthias

    2011-03-07

    Cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are enzymes that have emerged as attractive targets for the development of analgesic and anti-inflammatory drugs. We recently reported that 1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylic acid (5) is a dual inhibitor of cPLA2α and FAAH. Structure-activity relationship studies revealed that substituents at the indole 3- and 5-positions and replacement of the indole scaffold of this compound by other heterocycles strongly influences the inhibitory potency against cPLA2α and FAAH, respectively. Herein we report the effect of variation of the 4-octyl residue of 5 and an exchange of its carboxylic acid moiety by some bioisosteric functional groups. Several of the compounds assayed were favorably active against both enzymes, and could therefore represent agents with improved analgesic and anti-inflammatory qualities in comparison with selective cPLA2 α and FAAH inhibitors.

  5. Biochemical and biological properties of 4-(3-phenyl-[1,2,4] thiadiazol-5-yl)-piperazine-1-carboxylic acid phenylamide, a mechanism-based inhibitor of fatty acid amide hydrolase.

    PubMed

    Karbarz, Mark J; Luo, Lin; Chang, Leon; Tham, Chui-Se; Palmer, James A; Wilson, Sandy J; Wennerholm, Michelle L; Brown, Sean M; Scott, Brian P; Apodaca, Richard L; Keith, John M; Wu, Jiejun; Breitenbucher, James Guy; Chaplan, Sandra R; Webb, Michael

    2009-01-01

    Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme within the amidase-signature family. It catalyzes the hydrolysis of several endogenous biologically active lipids, including anandamide (arachidonoyl ethanolamide), oleoyl ethanolamide, and palmitoyl ethanolamide. These endogenous FAAH substrates have been shown to be involved in a variety of physiological and pathological processes, including synaptic regulation, regulation of sleep and feeding, locomotor activity, pain and inflammation. Here we describe the biochemical and biological properties of a potent and selective FAAH inhibitor, 4-(3-phenyl-[1,2,4]thiadiazol-5-yl)-piperazine-1-carboxylic acid phenylamide (JNJ-1661010). The time-dependence of apparent IC(50) values at rat and human recombinant FAAH, dialysis and mass spectrometry data indicate that the acyl piperazinyl fragment of JNJ-1661010 forms a covalent bond with the enzyme. This bond is slowly hydrolyzed, with release of the piperazinyl fragment and recovery of enzyme activity. The lack of inhibition observed in a rat liver esterase assay suggests that JNJ-1661010 is not a general esterase inhibitor. JNJ-1661010 is >100-fold preferentially selective for FAAH-1 when compared to FAAH-2. JNJ-1661010 dose-dependently increases arachidonoyl ethanolamide, oleoyl ethanolamide, and palmitoyl ethanolamide in the rat brain. The compound attenuates tactile allodynia in the rat mild thermal injury model of acute tissue damage and in the rat spinal nerve ligation (Chung) model of neuropathic pain. JNJ-1661010 also diminishes thermal hyperalgesia in the inflammatory rat carrageenan paw model. These data suggest that FAAH inhibitors with modes of action similar to JNJ-1661010 may be useful clinically as broad-spectrum analgesics.

  6. 1-Heteroaryl-3-phenoxypropan-2-ones as inhibitors of cytosolic phospholipase A₂α and fatty acid amide hydrolase: Effect of the replacement of the ether oxygen with sulfur and nitrogen moieties on enzyme inhibition and metabolic stability.

    PubMed

    Sundermann, Tom; Fabian, Jörg; Hanekamp, Walburga; Lehr, Matthias

    2015-05-15

    Cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are enzymes, which have emerged as attractive targets for the development of analgesic and anti-inflammatory drugs. We recently reported that certain 3-phenoxy-substituted 1-heteroarylpropan-2-ones are inhibitors of cPLA2α and/or FAAH. Starting from 1-[2-oxo-3-(4-phenoxyphenoxy)propyl]indole-5-carboxylic acid (3) and 1-(1H-benzotriazol-1-yl)-3-(4-phenoxyphenoxy)propan-2-one (4), the effect of the replacement of the oxygen in position 3 of the propan-2-one scaffold by sulfur and nitrogen containing moieties on inhibition of cPLA2α and fatty acid amide hydrolase as well as on metabolic stability in rat liver S9 fractions was investigated. As a result of these structure-activity relationship studies it was found that the ether oxygen is of great importance for enzyme inhibitory potency. Replacement by sulfur led to an about 100-fold decrease of enzyme inhibition, nitrogen and substituted nitrogen atoms at this position even resulted in inactivity of the compounds. The effect of the structural variations performed on metabolic stability of the important ketone pharmacophore was partly different in the two series of compounds. While introduction of SO and SO2 significantly increased stability of the ketone against reduction in case of the indole-5-carboxylic acid 3, it had no effect in case of the benzotriazole 4. Further analysis of the metabolism of 3 and 4 in rat liver S9 fractions revealed that the major metabolite of 3 was the alcohol 53 formed by reduction of the keto group. In contrast, in case of 4 beside keto reduction an excessive hydroxylation of the terminal phenoxy group occurred leading to the dihydroxy compound 50. Experiments with enzyme inhibitors showed that the phenylhydroxylation of 4 was catalyzed by tranylcypromine sensitive cytochrome P450 isoforms, while the reduction of the ketone function of 3 and 4 was mainly caused by cytosolic short chain dehydrogenases

  7. Inhibition of mutant KrasG12D-initiated murine pancreatic carcinoma growth by a dual c-Raf and soluble epoxide hydrolase inhibitor t-CUPM.

    PubMed

    Liao, Jie; Hwang, Sung Hee; Li, Haonan; Yang, Yihe; Yang, Jun; Wecksler, Aaron T; Liu, Jun-Yan; Hammock, Bruce D; Yang, Guang-Yu

    2016-02-28

    Mutant Kras and chronic pancreatitis are the most common pathological events involved in human pancreatic cancer. It has been demonstrated that c-Raf is responsible for transmitting signals from mutant Ras to its downstream signals including MEK-ERK and for initiating carcinogenesis. The soluble epoxide hydrolase (sEH), a pro-inflammatory enzyme, generally inactivates anti-inflammatory and anti-pain epoxyeicosatrienoic acids (EETs). Herein, we have synthesized a novel compound of trans-4-{4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-cyclohexyloxy}-pyridine-2-carboxylic acid methylamide (t-CUPM) via modifying the central phenyl ring of sorafenib and confirmed its dual inhibition of sEH and c-Raf by recombinant kinase activity assay. Pharmacokinetic analysis revealed that oral dosing of t-CUPM resulted in higher blood levels than that of sorafenib throughout the complete time course (48 h). The effect of t-CUPM on the inhibition of mutant Kras(G12D)-initiated murine pancreatic cancer cell growth was determined using the mouse pancreatic carcinoma cell model obtained from LSL-Kras(G12D)/Pdx1-Cre mice and showed that t-CUPM significantly inhibited this murine pancreatic carcinoma cell growth both in vitro and in mice in vivo. Inhibition of mutant Kras-transmitted phosphorylations of cRAF/MEK/ERK was demonstrated in these pancreatic cancer cells using Western blot assay and immunohistochemical approach. Modulation of oxylipin profile, particularly increased EETs/DHET ratio by sEH inhibition, was observed in mice treated with t-CUPM. These results indicate that t-CUPM is a highly potential agent to treat pancreatic cancer via simultaneously targeting c-Raf and sEH.

  8. Reducing endocannabinoid metabolism with the fatty acid amide hydrolase inhibitor, URB597, fails to modify reinstatement of morphine-induced conditioned floor preference and naloxone-precipitated morphine withdrawal-induced conditioned floor avoidance.

    PubMed

    McCallum, Amanda L; Limebeer, Cheryl L; Parker, Linda A

    2010-10-01

    The potential of the fatty acid amide hydrolase (FAAH) inhibitor, URB597, to modify drug prime-induced reinstatement of morphine-induced conditioned floor preference or naloxone-precipitated morphine withdrawal-induced conditioned floor avoidance was evaluated. In Experiment 1, morphine-induced conditioned floor preference was established across 4 conditioning trials. Following extinction training (4 trials), rats were pretreated with URB597 or vehicle prior to a morphine prime or a saline prime. Morphine reinstated the previously extinguished floor preference, but URB597 did not modify the strength of the reinstated preference. In Experiment 2, naloxone-precipitated morphine withdrawal-induced conditioned floor avoidance was established across 2 conditioning trials. Following extinction training (14 trials), rats were pretreated with URB597 or vehicle prior to a saline prime or a morphine withdrawal prime. The morphine withdrawal prime reinstated the previously extinguished floor avoidance, but URB597 did not modify the strength of reinstated avoidance. These results suggest that under the conditions in which URB597 promotes extinction (e.g., Manwell et al. (2009)) it does not interfere with drug-induced reinstatement of either conditioned floor preference or avoidance. That is, although activation of the endocannabinoid (eCB) system promotes extinction of aversive learning, it may not prevent reinstatement of that aversion by re-exposure to the aversive treatment.

  9. Alterations in endocannabinoid tone following chemotherapy-induced peripheral neuropathy: Effects of endocannabinoid deactivation inhibitors targeting fatty-acid amide hydrolase and monoacylglycerol lipase in comparison to reference analgesics following cisplatin treatment

    PubMed Central

    Guindon, Josée; Lai, Yvonne; Takacs, Sara M.; Bradshaw, Heather B.; Hohmann, Andrea G.

    2012-01-01

    SUMMARY Cisplatin, a platinum-derived chemotherapeutic agent, produces mechanical and cold allodynia reminiscent of chemotherapy-induced neuropathy in humans. The endocannabinoid system represents a novel target for analgesic drug development. The endocannabinoid consists of endocannabinoids (e.g. anandamide (AEA) and 2-arachidonoylglycerol (2-AG)), cannabinoid receptors (e.g. CB1 and CB2) and the enzymes controlling endocannabinoid synthesis and degradation. AEA is hydrolyzed by fatty-acid amide hydrolase (FAAH) whereas 2-AG is hydrolyzed primarily by monoacylglycerol lipase (MGL). We compared effects of brain permeant (URB597) and impermeant (URB937) inhibitors of FAAH with an irreversible inhibitor of MGL (JZL184) on cisplatin-evoked behavioral hypersensitivities. Endocannabinoid modulators were compared with agents used clinically to treat neuropathy (i.e. the opioid analgesic morphine, the anticonvulsant gabapentin and the tricyclic antidepressant amitriptyline). Cisplatin produced robust mechanical and cold allodynia but did not alter responsiveness to heat. After neuropathy was fully established, groups received acute intraperitoneal (i.p.) injections of vehicle, amitriptyline (30 mg/kg), gabapentin (100 mg/kg), morphine (6 mg/kg), URB597 (0.1 or 1 mg/kg), URB937 (0.1 or 1 mg/kg) or JZL184 (1, 3 or 8 mg/kg). Pharmacological specificity was assessed by coadministering each endocannabinoid modulator with either a CB1 (AM251 3 mg/kg), CB2 (AM630 3 mg/kg), TRPV1 (AMG9810 3 mg/kg) or TRPA1 (HC030031 8 mg/kg) antagonist. Effects of cisplatin on endocannabinoid levels and transcription of receptors (CB1, CB2, TRPV1, TRPA1) and enzymes (FAAH, MGL) linked to the endocannabinoid system were also assessed. URB597, URB937, JZL184 and morphine reversed cisplatin-evoked mechanical and cold allodynia to pre-cisplatin levels. By contrast, gabapentin only partially reversed the neuropathy while amitriptyline, administered acutely, was ineffective. CB1 or CB2 antagonist

  10. Discovery libraries targeting the major enzyme classes: the serine hydrolases.

    PubMed

    Otrubova, Katerina; Srinivasan, Venkat; Boger, Dale L

    2014-08-15

    Two libraries of modestly reactive ureas containing either electron-deficient acyl anilines or acyl pyrazoles were prepared and are reported as screening libraries for candidate serine hydrolase inhibitors. Within each library is a small but powerful subset of compounds that serve as a chemotype fragment screening library capable of subsequent structural diversification. Elaboration of the pyrazole-based ureas provided remarkably potent irreversible inhibitors of fatty acid amide hydrolase (FAAH, apparent Ki=100-200 pM) complementary to those previously disclosed enlisting electron-deficient aniline-based ureas.

  11. Discovery libraries targeting the major enzyme classes: the serine hydrolases

    PubMed Central

    Otrubova, Katerina; Srinivasan, Venkat; Boger, Dale L.

    2014-01-01

    Two libraries of modestly reactive ureas containing either electron-deficient acyl anilines or acyl pyrazoles were prepared and are reported as screening libraries for candidate serine hydrolase inhibitors. Within each library is a small but powerful subset of compounds that serve as a chemotype fragment screening library capable of subsequent diversification. Elaboration of the pyrazole-based ureas provided remarkably potent irreversible structural inhibitors of fatty acid amide hydrolase (FAAH, apparent Ki = 100-200 pM) complementary to those previously disclosed enlisting electron-deficient aniline-based ureas. PMID:25037918

  12. Early postnatal treatment with soluble epoxide hydrolase inhibitor or 15-deoxy-Δ(12,14)-prostagandin J2 prevents prenatal dexamethasone and postnatal high saturated fat diet induced programmed hypertension in adult rat offspring.

    PubMed

    Lu, Pei-Chen; Sheen, Jiunn-Ming; Yu, Hong-Ren; Lin, Yu-Ju; Chen, Chih-Cheng; Tiao, Mao-Meng; Tsai, Ching-Chou; Huang, Li-Tung; Tain, You-Lin

    2016-07-01

    Prenatal dexamethasone (DEX) exposure, postnatal high-fat (HF) intake, and arachidonic acid pathway are closely related to hypertension. We tested whether a soluble epoxide hydrolase (SEH) inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) or 15-deoxy-Δ(12,14)-prostagandin J2 (15dPGJ2) therapy can rescue programmed hypertension in the DEX+HF two-hit model. Four groups of Sprague Dawley rats were studied: control, DEX+HF, AUDA, and 15dPGJ2. Dexamethasone (0.1mg/kg body weight) was intraperitoneally administered to pregnant rats from gestational day 16-22. Male offspring received high-fat diet (D12331, Research Diets) from weaning to 4 months of age. In AUDA group, mother rats received 25mg/L in drinking water during lactation. In the 15dPGJ2 group, male offspring received 15dPGJ2 1.5mg/kg BW by subcutaneous injection once daily for 1 week after birth. We found postnatal HF diet aggravated prenatal DEX-induced programmed hypertension, which was similarly prevented by early treatment with AUDA or 15dPGJ2. The beneficial effects of AUDA and 15d-PGJ2 therapy include inhibition of SEH, increases of renal angiotensin converting enzyme-2 (ACE2) and angiotensin II type 2 receptor (AT2R) protein levels, and restoration of nitric oxide bioavailability. Better understanding of the impact of arachidonic acid pathway in the two-hit model will help prevent programmed hypertension in children exposed to corticosteroids and postnatal HF intake.

  13. Inhibition of Chronic Pancreatitis and Murine Pancreatic Intraepithelial Neoplasia by a Dual Inhibitor of c-RAF and Soluble Epoxide Hydrolase in LSL-KrasG12D/Pdx-1-Cre Mice

    PubMed Central

    LIAO, JIE; HWANG, SUNG HEE; LI, HAONAN; LIU, JUN-YAN; HAMMOCK, BRUCE D.; YANG, GUANG-YU

    2016-01-01

    Mutation of Kirsten rat sarcoma viral oncogene homolog (KRAS) and chronic pancreatitis are the most common pathogenic events involved in human pancreatic carcinogenesis. In the process of long-standing chronic inflammation, aberrant metabolites of arachidonic acid play a crucial role in promoting carcinogenesis, in which the soluble epoxide hydrolase (sEH), as a pro-inflammatory enzyme, generally inactivates anti-inflammatory epoxyeicosatrienoic acids (EETs). Herein, we determined the effect of our newly-synthesized novel compound trans-4-{4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-cyclohexyloxy}-pyridine-2-carboxylic acid methylamide (t-CUPM), a dual inhibitor of sEH and RAF1 proto-oncogene serine/threonine kinase (c-RAF), on inhibiting the development of pancreatitis and pancreatic intraepithelial neoplasia (mPanIN) in LSL-KrasG12D/Pdx1-Cre mice. The results showed that t-CUPM significantly reduced the severity of chronic pancreatitis, as measured by the extent of acini loss, inflammatory cell infiltration and stromal fibrosis. The progression of low-grade mPanIN I to high-grade mPanIN II/III was significantly suppressed. Inhibition of mutant Kras-transmitted phosphorylation of mitogen-activated protein kinase’s kinase/extracellular signal-regulated kinases was demonstrated in pancreatic tissues by western blots. Quantitative real-time polymerase chain reaction analysis revealed that t-CUPM treatment significantly reduced the levels of inflammatory cytokines including tumor necrosis facor-α, monocyte chemoattractant protein-1, as well as vascular adhesion molecule-1, and the levels of Sonic hedgehog and Gli transcription factor (Hedgehog pathway). Analysis of the eicosanoid profile revealed a significant increase of the EETs/dihydroxyeicosatrienoic acids ratio, which further confirmed sEH inhibition by t-CUPM. These results indicate that simultaneous inhibition of sEH and c-RAF by t-CUPM is important in preventing chronic pancreatitis and carcinogenesis

  14. Variants of glycoside hydrolases

    DOEpatents

    Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

    2013-02-26

    The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  15. Variants of glycoside hydrolases

    DOEpatents

    Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

    2011-04-26

    The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  16. Structural and functional attributes of malaria parasite diadenosine tetraphosphate hydrolase

    PubMed Central

    Sharma, Arvind; Yogavel, Manickam; Sharma, Amit

    2016-01-01

    Malaria symptoms are driven by periodic multiplication cycles of Plasmodium parasites in human red blood corpuscles (RBCs). Malaria infection still accounts for ~600,000 annual deaths, and hence discovery of both new drug targets and drugs remains vital. In the present study, we have investigated the malaria parasite enzyme diadenosine tetraphosphate (Ap4A) hydrolase that regulates levels of signalling molecules like Ap4A by hydrolyzing them to ATP and AMP. We have tracked the spatial distribution of parasitic Ap4A hydrolase in infected RBCs, and reveal its unusual localization on the infected RBC membrane in subpopulation of infected cells. Interestingly, enzyme activity assays reveal an interaction between Ap4A hydrolase and the parasite growth inhibitor suramin. We also present a high resolution crystal structure of Ap4A hydrolase in apo- and sulphate- bound state, where the sulphate resides in the enzyme active site by mimicking the phosphate of substrates like Ap4A. The unexpected infected erythrocyte localization of the parasitic Ap4A hydrolase hints at a possible role of this enzyme in purinerigic signaling. In addition, atomic structure of Ap4A hydrolase provides insights for selective drug targeting. PMID:26829485

  17. Effect of alpha lipoic acid on leukotriene A4 hydrolase.

    PubMed

    Torres, María José; Fierro, Angélica; Pessoa-Mahana, C David; Romero-Parra, Javier; Cabrera, Gonzalo; Faúndez, Mario

    2017-03-15

    Leukotriene A4 hydrolase is a soluble enzyme with epoxide hydrolase and aminopeptidase activities catalysing the conversion of leukotriene A4 to leukotriene B4 and the hydrolysis of the peptide proline-glycine-proline. Imbalances in leukotriene B4 synthesis are related to several pathologic conditions. Currently there are no available drugs capable to modulate the synthesis of leukotriene B4 or to block its receptors. Here we show the inhibitory profile of alpha lipoic acid on the activity of leukotriene A4 Hydrolase. Alpha lipoic acid inhibited both activities of the enzyme at concentrations lower than 10μM. The 5-lipoxygenase inhibitor zileuton, or the 5-lipoxygenase activating protein inhibitor MK-886, were unable to inhibit the activity of the enzyme. Acute promyelocytic leukaemia HL-60 cells were differentiated to leukotriene A4 hydrolase expressing neutrophil-like cells. Alpha lipoic acid inhibited the aminopeptidase activity of the cytosolic fraction from neutrophil-like cells but had no effect on the cytosolic fraction from undifferentiated cells. Docking and molecular dynamic approximations revealed that alpha lipoic acid participates in electrostatic interactions with K-565 and R-563, which are key residues for the carboxylate group recognition of endogenous substrates by the enzyme. Alpha lipoic acid is a compound widely used in clinical practice, most of its therapeutic effects are associated with its antioxidants properties, however, antioxidant effect alone is unable to explain all clinical effects observed with alpha lipoic acid. Our results invite to evaluate the significance of the inhibitory effect of alpha lipoic acid on the catalytic activity of leukotriene A4 hydrolase using in vivo models.

  18. Polyglycine hydrolases secreted by pathogenic fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pathogens are known to produce proteases that target host defense proteins. Here we describe polyglycine hydrolases, fungal proteases that selectively cleave glycine-glycine peptide bonds within the polyglycine interdomain linker of targeted plant defense chitinases. Polyglycine hydrolases were puri...

  19. Expanding the Cyanuric Acid Hydrolase Protein Family to the Fungal Kingdom

    PubMed Central

    Dodge, Anthony G.; Preiner, Chelsea S.

    2013-01-01

    The known enzymes that open the s-triazine ring, the cyanuric acid hydrolases, have been confined almost exclusively to the kingdom Bacteria and are all homologous members of the rare cyanuric acid hydrolase/barbiturase protein family. In the present study, a filamentous fungus, Sarocladium sp. strain CA, was isolated from soil by enrichment culturing using cyanuric acid as the sole source of nitrogen. A reverse-genetic approach identified a fungal cyanuric acid hydrolase gene composed of two exons and one intron. The translated spliced sequence was 39 to 53% identical to previously characterized bacterial cyanuric acid hydrolases. The sequence was used to generate a gene optimized for expression in Escherichia coli and encoding an N-terminally histidine-tagged protein. The protein was purified by nickel affinity and anion-exchange chromatography. The purified protein was shown by 13C nuclear magnetic resonance (13C-NMR) to produce carboxybiuret as the product, which spontaneously decarboxylated to yield biuret and carbon dioxide. The protein was very narrow in substrate specificity, showing activity only with cyanuric acid and N-methyl cyanuric acid. Barbituric acid was an inhibitor of enzyme activity. Sequence analysis identified genes with introns in other fungi from the Ascomycota that, if spliced, are predicted to encode proteins with cyanuric acid hydrolase activity. The Ascomycota cyanuric acid hydrolase homologs are most closely related to cyanuric acid hydrolases from Actinobacteria. PMID:24039269

  20. Characterization of multiple epoxide hydrolase activities in mouse liver nuclear envelope.

    PubMed

    Guenthner, T M

    1986-10-01

    A nuclear envelope-associated epoxide hydrolase in mouse liver that hydrates trans-stilbene oxide has been identified and characterized. This epoxide hydrolase is distinct from the enzyme in nuclear envelopes that hydrates benzo[a]pyrene 4,5-oxide and other arene oxides. This distinction was demonstrated by the criteria of pH optima, response to specific inhibitors in vitro, and precipitation by specific antibodies. The new epoxide hydrolase had a pH optimum of 6.8, was poorly inhibited by trichloropropene oxide, was potently inhibited by 4-phenylchalcone oxide, and did not bind to antiserum against benzo[a]pyrene 4,5-oxide hydrolase. This nuclear enzyme is similar in many of its properties to cytosolic and microsomal trans-stilbene oxide hydrolases and may be nuclear envelope-bound form of these other epoxide hydrolases. It differed from these other trans-stilbene oxide hydrolases in that its affinities for both trans-stilbene oxide (measured as apparent Km) and 4-phenylchalcone oxide (measured as I50) were 4- to 20-fold lower than those of either the cytosolic or microsomal forms.

  1. Hydrolysis of the 2',3'-allylic epoxides of allylbenzene, estragole, eugenol, and safrole by both microsomal and cytosolic epoxide hydrolases.

    PubMed

    Luo, G; Qato, M K; Guenthner, T M

    1992-01-01

    2',3'-Allylic epoxide derivatives of allylbenzene and its analogs estragole, eugenol, and safrole were synthesized, and their enzymatic conversion to dihydrodiols by cytosolic and microsomal epoxide hydrolases was examined. All four epoxides were good substrates for both epoxide hydrolases, with Michaelis constants in the low micromolar range. Two putatively selective inhibitors of cytosolic and microsomal epoxide hydrolases, trichloropropylene oxide and nordihydroguaiaretic acid, were used to inhibit the hydrolysis of these allylic epoxides. Minimal selectivity toward either hydrolase was seen with either inhibitor, suggesting that the "selectivity" of these inhibitors is highly substrate-dependent. The susceptibilities of these epoxides to rapid hydrolysis by both epoxide hydrolases may explain their low genotoxic potencies in vivo.

  2. Similarities between catalase and cytosolic epoxide hydrolase.

    PubMed

    Guenthner, T M; Qato, M; Whalen, R; Glomb, S

    1989-01-01

    Cytosolic epoxide hydrolase, measured as trans-stilbene oxide hydrolase activity, was isolated and purified from human and guinea pig liver cytosol. Antiserum to the guinea pig liver preparation reacted strongly with bovine liver catalase. We determined that this lack of selectivity of the antiserum was due to catalase contamination of the epoxide hydrolase preparation. We also determined that several commercial catalase preparations are contaminated with cytosolic epoxide hydrolase. Our human epoxide hydrolase preparation contained no detectable catalase contamination, yet antiserum to this protein also cross-reacted slightly with catalase, indicating some intrinsic similarity between the two enzymes. We conclude that catalase and cytosolic epoxide hydrolase contain some similar immunogenic epitopes, and we surmise that similarities between the subunits of these two enzymes may lead to their partial copurification. Functional similarities between the two enzymes are also demonstrated, as several compounds that inhibit catalase are also shown to inhibit cytosolic epoxide hydrolase activity in the same concentration range and rank order.

  3. Prediction of drug-drug interactions with carbamazepine-10,11-epoxide using a new in vitro assay for epoxide hydrolase inhibition.

    PubMed

    Rosa, Maria; Bonnaillie, Pierre; Chanteux, Hugues

    2016-12-01

    1. Carbamazepine is an antiepileptic drug which is metabolized by CYP3A4 into carbamazepine-10,11-epoxide. This metabolite is then detoxified by epoxide hydrolase. As carbamazepine-10,11-epoxide has been associated with neurotoxicity, it is critical to identify whether a new antiepileptic drug has the potential to inhibit epoxide hydrolase and therefore increase carbamazepine-10,11-epoxide plasma levels. 2. In this study, an in vitro assay was developed to evaluate epoxide hydrolase activity by using carbamazepine-10,11-epoxide as probe substrate. The ability of this assay to predict drug-drug interactions (DDI) at the epoxide hydrolase level was also investigated. 3. To this aim, known inhibitors of epoxide hydrolase for which in vivo data are available were used. Firstly, carbamazepine-10,11-epoxide hydrolase activity was determined in liver microsomes, cytosol and hepatocytes. Thereafter, the IC50 of epoxide hydrolase inhibitors (progabide, valproic acid, valpromide and valnoctamide) was determined in liver microsomes and hepatocytes. Finally, prediction of AUC increase was performed using the in vitro data generated. 4. Interestingly, epoxide hydrolase activity was found to be much higher in human hepatocytes compared to liver microsomes/cytosol. Even though assessed on a limited number of compounds, this study demonstrated that the use of hepatocytes seems to be a more relevant model to assess and predict DDI at the epoxide hydrolase level.

  4. Synthesis, evaluation, and mechanism of N,N,N-trimethyl-D-glucosamine-(1→4)-chitooligosaccharides as selective inhibitors of glycosyl hydrolase family 20 β-N-acetyl-D-hexosaminidases.

    PubMed

    Yang, You; Liu, Tian; Yang, Yongliang; Wu, Qingyue; Yang, Qing; Yu, Biao

    2011-02-11

    GH20 β-N-acetyl-D-hexosaminidases are enzymes involved in many vital processes. Inhibitors that specifically target GH20 enzymes in pests are of agricultural and economic importance. Structural comparison has revealed that the bacterial chitindegrading β-N-acetyl-D-hexosaminidases each have an extra +1 subsite in the active site; this structural difference could be exploited for the development of selective inhibitors. N,N,Ntrimethyl-D-glucosamine (TMG)-chitotriomycin, which contains three GlcNAc residues, is a natural selective inhibitor against bacterial and insect β-N-acetyl-D-hexosaminidases. However, our structural alignment analysis indicated that the two GlcNAc residues at the reducing end might be unnecessary. To prove this hypothesis, we designed and synthesized a series of TMG-chitotriomycin analogues containing one to four GlcNAc units. Inhibitory kinetics and molecular docking showed that TMG-(GlcNAc)(2), is as active as TMG-chitotriomycin [TMG-(GlcNAc)(3)]. The selective inhibition mechanism of TMG-chitotriomycin was also explained.

  5. Radioisotope assay for 1-aminocyclopropane-1-carboxylic acid synthase: s-adenosylhomocysteine analogs as inhibitors of the enzyme involved in plant senescence

    SciTech Connect

    Miura, G.A.; Chiang, P.K.

    1985-01-01

    A simple and rapid radioisotopic assay for 1-aminocyclopropane-1-carboxylic acid (ACC) synthase was developed, an enzyme involved in the biosynthesis of the plant hormone ethylene. The assay utilizes an AG50-X4(NH4 (+)) column which separates S-adenosyl-L-(carboxyl-/sup 14/C)methionine (AdoMet) from the product (/sup 14/C)acc, since the latter is not bound to the resin while (/sup 14/C)adoMet is. As opposed to other assays, this procedure measures ACC directly and does not require further conversion to ethylene. When an enzyme preparation from ripe-tomato fruits (Lycopersicon esculentum Mill) was assayed, an I/sub 50/ of 2.5 + or - 0.8 micrometers for sinefungin and a K/sub m/ of 27 + or - 2 micrometers for AdoMet were obtained; these values were in good agreement with previous previous determinations made with a gas-chromatographic assay. When other nucleosides were tested as inhibitors the following order of decreasing activity was found: sinefungin, S-adenosylhomocysteine (AdoHcy), AdoHcy sulfoxide, S-n-butyladenosine, 3-deaza-adenosylhomocysteine, S-isobutyladenosine, S-isobutyladenosine, S-isobutyl-l-deazaadenosine. In contrast, S-isobutyl-3-deazaadenosine, S-isobutyl-7-deazaadenosine, 3-deazaadenosine, and adenodine were not inhibitory.

  6. Epoxide hydrolase activities and epoxy fatty acids in the mosquito Culex quinquefasciatus

    PubMed Central

    Xu, Jiawen; Morisseau, Christophe; Yang, Jun; Mamatha, Dadala M.

    2015-01-01

    Culex mosquitoes have emerged as important model organisms for mosquito biology, and are disease vectors for multiple mosquito-borne pathogens, including West Nile virus. We characterized epoxide hydrolase activities in the mosquito Culex quinquefasciatus, which suggested multiple forms of epoxide hydrolases were present. We found EH activities on epoxy eicosatrienoic acids (EETs). EETs and other eicosanoids are well-established lipid signaling molecules in vertebrates. We showed EETs can be synthesized in vitro from arachidonic acids by mosquito lysate, and EETs were also detected in vivo both in larvae and adult mosquitoes by LC-MS/MS. The EH activities on EETs can be induced by blood feeding, and the highest activity was observed in the midgut of female mosquitoes. The enzyme activities on EETs can be inhibited by urea-based inhibitors designed for mammalian soluble epoxide hydrolases (sEH). The sEH inhibitors have been shown to play diverse biological roles in mammalian systems, and they can be useful tools to study the function of EETs in mosquitoes. Besides juvenile hormone metabolism and detoxification, insect epoxide hydrolases may also play a role in regulating lipid signaling molecules, such as EETs and other epoxy fatty acids, synthesized in vivo or obtained from blood feeding by female mosquitoes. PMID:25686802

  7. Inhibition of murine erythroleukemia cell differentiation by 3-deazaadenosine.

    PubMed

    Sherman, M L; Shafman, T D; Spriggs, D R; Kufe, D W

    1985-11-01

    Recent studies have demonstrated that 5'-methylthioadenosine, an inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase, blocks induction of murine erythroleukemia cell (MEL) differentiation. The nucleoside analogue 3-deazaadenosine (c3Ado) is both an efficient substrate and a potent inhibitor of AdoHcy hydrolase. The present study was undertaken to determine whether c3Ado would similarly inhibit MEL differentiation. The results demonstrate that c3Ado inhibits induction of MEL differentiation by dimethyl sulfoxide, hexamethylene bisacetamide, butyric acid, and diazapam. c3Ado blocks the appearance of the differentiated MEL phenotype by inhibiting both MEL heme synthesis and transcription of alpha- and beta-globin RNA. The inhibitory effect of c3Ado on MEL differentiation is concentration dependent, reversible, and potentiated by L-homocysteine thiolactone. Furthermore the AdoHcy/AdoMet ratio increases nearly 3.5-fold after 24 h of treatment with 50 microM c3Ado. In contrast, this c3Ado effect is not associated with polyamine depletion or cytostasis. These findings indicate that c3Ado blocks the induction of MEL differentiation at a transcriptional level and that this effect may be related to inhibition of AdoHcy hydrolase.

  8. Distinct rat hepatic microsomal epoxide hydrolases catalyze the hydration of cholesterol 5,6 alpha-oxide and certain xenobiotic alkene and arene oxides.

    PubMed

    Levin, W; Michaud, D P; Thomas, P E; Jerina, D M

    1983-02-01

    Metabolism of cholesterol 5,6 alpha-oxide to the 5,6-glycol is catalyzed by a rat liver microsomal epoxide hydrolase that is distinct from the microsomal epoxide hydrolase that metabolizes a wide range of xenobiotic alkene and arene oxides. The two enzymes are antigenically distinct, and the purified microsomal epoxide hydrolase that metabolizes xenobiotic oxides does not catalyze the hydration of cholesterol 5,6 alpha-oxide. In vivo treatment of rats with inducers of microsomal epoxide hydrolase does not enhance the activity of cholesterol 5,6 alpha-oxide hydrolase and, in some cases, actually depresses enzyme activity in the resultant microsomal preparations. Octene 1,2-oxide and benz[a]anthracene 5,6-oxide, both good substrates for xenobiotic epoxide hydrolase, are not competitive inhibitors of cholesterol oxide hydration by rat liver microsomes. The above results establish the existence of a liver microsomal epoxide hydrolase that is under different regulatory control and that appears to have a different substrate specificity than the well-characterized microsomal epoxide hydrolase involved in the metabolism of a widely diverse group of alkene and arene oxides.

  9. Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition through Activation of PPAR-alpha Nuclear Receptors

    ERIC Educational Resources Information Center

    Mazzola, Carmen; Medalie, Julie; Scherma, Maria; Panlilio, Leigh V.; Solinas, Marcello; Tanda, Gianluigi; Drago, Filippo; Cadet, Jean Lud; Goldberg, Steven R.; Yasar, Sevil

    2009-01-01

    Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB[subscript 1]-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain.…

  10. Hydrolase-like properties of a cofactor-independent dioxygenase.

    PubMed

    Thierbach, Sven; Büldt-Karentzopoulos, Klaudia; Dreiling, Alena; Hennecke, Ulrich; König, Simone; Fetzner, Susanne

    2012-05-29

    Mechanistic promiscuity: The (2-alkyl)-3-hydroxy-4(1H)-quinolone-cleaving dioxygenase Hod has an α/β-hydrolase fold and a Ser/His/Asp triad in its active site. Isatoic anhydride, a suicide substrate of serine hydrolases, inactivates Hod by covalent modification of the active-site serine, thus indicating that the α/β-hydrolase fold can accommodate dioxygenase chemistry without completely abandoning hydrolase-like properties.

  11. Structure and function of polyglycine hydrolases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave polyglycine linkers of targeted plant defense chitinases. Unlike typical endoproteases that cleave a specific peptide bond, these 640 amino acid glycoproteins selectively cleave one of multiple peptide bonds within polyglyci...

  12. Recognition of Corn Defense Chitinases by Fungal Polyglycine Hydrolases.

    PubMed

    Naumann, Todd A; Bakota, Erica L; Price, Neil P J

    2017-04-06

    Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn (Zea mays ssp. mays). These target-specific endoproteases are unusual because they do not cut a specific peptide bond but select one of many Gly-Gly bonds within the polyglycine region. Some Gly-Gly bonds are cleaved frequently while others are never cleaved. Moreover, we have previously shown that PGHs from different fungal pathogens prefer to cleave different Gly-Gly peptide bonds. It is not understood how PGHs selectively cleave the ChitA linker, especially because its polyglycine structure lacks peptide sidechains. To gain insights into this process we synthesized several peptide analogs of ChitA to evaluate them as potential substrates and inhibitors of Es-cmp, a PGH from the plant pathogenic fungus Epicoccum sorghi. Our results showed that part of the PGH recognition site for substrate chitinases is adjacent to the polyglycine linker on the carboxy side. More specifically, four amino acid residues were implicated, each spaced four residues apart on an alpha helix. Moreover, analogous peptides with selective Gly->sarcosine (N-methylglycine) mutations or a specific Ser->Thr mutation retained inhibitor activity but were no longer cleaved by PGH. Additonally, our findings suggest that peptide analogs of ChitA that inhibit PGH activity could be used to strengthen plant defenses. This article is protected by copyright. All rights reserved.

  13. Dienelactone hydrolase from Pseudomonas sp. strain B13.

    PubMed Central

    Ngai, K L; Schlömann, M; Knackmuss, H J; Ornston, L N

    1987-01-01

    Dienelactone hydrolase (EC 3.1.1.45) catalyzes the conversion of cis- or trans-4-carboxymethylenebut-2-en-4-olide (dienelactone) to maleylacetate. An approximately 24-fold purification from extracts of 3-chlorobenzoate-grown Pseudomonas sp. strain B13 yielded a homogeneous preparation of the enzyme. The purified enzyme crystallized readily and proved to be a monomer with a molecular weight of about 30,000. Each dienelactone hydrolase molecule contains two cysteinyl side chains. One of these was readily titrated by stoichiometric amounts of p-chloromercuribenzoate, resulting in inactivation of the enzyme; the inactivation could be reversed by the addition of dithiothreitol. The other cysteinyl side chain appeared to be protected in the native protein against chemical reaction with p-chloromercuribenzoate. The properties of sulfhydryl side chains in dienelactone hydrolase resembled those that have been characterized for bacterial 4-carboxymethylbut-3-en-4-olide (enol-lactone) hydrolases (EC 3.1.1.24), which also are monomers with molecular weights of about 30,000. The amino acid composition of the dienelactone hydrolase resembled the amino acid composition of enol-lactone hydrolase from Pseudomonas putida, and alignment of the NH2-terminal amino acid sequence of the dienelactone hydrolase with the corresponding sequence of an Acinetobacter calcoaceticus enol-lactone hydrolase revealed sequence identity at 8 of the 28 positions. These observations foster the hypothesis that the lactone hydrolases share a common ancestor. The lactone hydrolases differed in one significant property: the kcat of dienelactone hydrolase was 1,800 min-1, an order of magnitude below the kcat observed with enol-lactone hydrolases. The relatively low catalytic activity of dienelactone hydrolase may demand its production at the high levels observed for induced cultures of Pseudomonas sp. strain B13. PMID:3804973

  14. Bacterial Cyanuric Acid Hydrolase for Water Treatment

    PubMed Central

    Yeom, Sujin; Mutlu, Baris R.; Aksan, Alptekin

    2015-01-01

    Di- and trichloroisocyanuric acids are widely used as water disinfection agents, but cyanuric acid accumulates with repeated additions and must be removed to maintain free hypochlorite for disinfection. This study describes the development of methods for using a cyanuric acid-degrading enzyme contained within nonliving cells that were encapsulated within a porous silica matrix. Initially, three different bacterial cyanuric acid hydrolases were compared: TrzD from Acidovorax citrulli strain 12227, AtzD from Pseudomonas sp. strain ADP, and CAH from Moorella thermoacetica ATCC 39073. Each enzyme was expressed recombinantly in Escherichia coli and tested for cyanuric acid hydrolase activity using freely suspended or encapsulated cell formats. Cyanuric acid hydrolase activities differed by only a 2-fold range when comparing across the different enzymes with a given format. A practical water filtration system is most likely to be used with nonviable cells, and all cells were rendered nonviable by heat treatment at 70°C for 1 h. Only the CAH enzyme from the thermophile M. thermoacetica retained significant activity under those conditions, and so it was tested in a flowthrough system simulating a bioreactive pool filter. Starting with a cyanuric acid concentration of 10,000 μM, more than 70% of the cyanuric acid was degraded in 24 h, it was completely removed in 72 h, and a respike of 10,000 μM cyanuric acid a week later showed identical biodegradation kinetics. An experiment conducted with water obtained from municipal swimming pools showed the efficacy of the process, although cyanuric acid degradation rates decreased by 50% in the presence of 4.5 ppm hypochlorite. In total, these experiments demonstrated significant robustness of cyanuric acid hydrolase and the silica bead materials in remediation. PMID:26187963

  15. Expression and characterization of an epoxide hydrolase from Anopheles gambiae with high activity on epoxy fatty acids

    PubMed Central

    Xu, Jiawen; Morisseau, Christophe; Hammock, Bruce D.

    2014-01-01

    In insects, epoxide hydrolases (EHs) play critical roles in the metabolism of xenobiotic epoxides from the food resources and in the regulation of endogenous chemical mediators, such as juvenile hormones. Using the baculovirus expression system, we expressed and characterized an epoxide hydrolase from Anopheles gambiae (AgEH) that is distinct in evolutionary history from insect juvenile hormone epoxide hydrolases (JHEHs). We partially purified the enzyme by ion exchange chromatography and isoelectric focusing. The experimentally determined molecular weight and pI were estimated to be 35kD and 6.3 respectively, different than the theoretical ones. The AgEH had the greatest activity on long chain epoxy fatty acids such as 14,15-epoxyeicosatrienoic acids (14,15-EET) and 9,10-epoxy-12Z-octadecenoic acids (9,10-EpOME or leukotoxin) among the substrates evaluated. Juvenile hormone III, a terpenoid insect growth regulator, was the next best substrate tested. The AgEH showed kinetics comparable to the mammalian soluble epoxide hydrolases, and the activity could be inhibited by AUDA [12-(3-adamantan-1-yl-ureido) dodecanoic acid], a urea-based inhibitor designed to inhibit the mammalian soluble epoxide hydrolases. The rabbit serum generated against the soluble epoxide hydrolase of Mus musculus can both cross-react with natural and denatured forms of the AgEH, suggesting immunologically they are similar. The study suggests there are mammalian sEH homologs in insects, and epoxy fatty acids may be important chemical mediators in insects. PMID:25173592

  16. Diadenosine tetraphosphate hydrolase is part of the transcriptional regulation network in immunologically activated mast cells.

    PubMed

    Carmi-Levy, Irit; Yannay-Cohen, Nurit; Kay, Gillian; Razin, Ehud; Nechushtan, Hovav

    2008-09-01

    We previously discovered that microphthalmia transcription factor (MITF) and upstream stimulatory factor 2 (USF2) each forms a complex with its inhibitor histidine triad nucleotide-binding 1 (Hint-1) and with lysyl-tRNA synthetase (LysRS). Moreover, we showed that the dinucleotide diadenosine tetraphosphate (Ap(4)A), previously shown to be synthesized by LysRS, binds to Hint-1, and as a result the transcription factors are released from their suppression. Thus, transcriptional activity is regulated by Ap(4)A, suggesting that Ap(4)A is a second messenger in this context. For Ap(4)A to be unambiguously established as a second messenger, several criteria have to be fulfilled, including the presence of a metabolizing enzyme. Since several enzymes are able to hydrolyze Ap(4)A, we provided here evidence that the "Nudix" type 2 gene product, Ap(4)A hydrolase, is responsible for Ap(4)A degradation following the immunological activation of mast cells. The knockdown of Ap(4)A hydrolase modulated Ap(4)A accumulation, resulting in changes in the expression of MITF and USF2 target genes. Moreover, our observations demonstrated that the involvement of Ap(4)A hydrolase in gene regulation is not a phenomenon exclusive to mast cells but can also be found in cardiac cells activated with the beta-agonist isoproterenol. Thus, we have provided concrete evidence establishing Ap(4)A as a second messenger in the regulation of gene expression.

  17. Hydrolase activity in the venom of the pupal endoparasitic wasp, Pimpla hypochondriaca.

    PubMed

    Dani, M P; Edwards, J P; Richards, E H

    2005-07-01

    Venom from the pupal endoparasitoid, Pimpla hypochondriaca has previously been shown to contain a mixture of biologically active molecules. Currently, P. hypochondriaca venom was examined for the presence of hydrolase activity. Six hydrolases were consistently detected using the API ZYM semiquantitative colourimetric kit. The main hydrolases detected were; acid phosphatase, beta-glucosidase, esterase, beta-galactosidase, esterase lipase, and lipase. The most rapid and intense colour reaction was detected for acid phosphatase. The pH optimum and the specific activity of venom acid phosphatase was determined using p-nitrophenol phosphate as a substrate and were 4.8 and 0.47 nmol p-nitrophenol/min/microg of venom protein, respectively. The acid phosphatase activity was inhibited in a dose dependent manner by sodium fluoride (IC(50) 4.2 x 10(-4) M), and by cocktail inhibitor 2 (CI 2). P. hypochondriaca venom has previously been shown to display potent cytotoxic activity towards Lacanobia oleracea haemocytes maintained in vitro. The contribution of acid phosphatase in venom to this cytotoxic activity was investigated by titrating venom against CI 2 prior to the addition of L. oleracea haemocytes. The results suggest that, despite the relatively high levels of acid phosphatase activity in venom, venom acid phosphatase plays no role in the antihaemocytic activity of P. hypochondriaca venom in vitro.

  18. Design, Synthesis, and Characterization of α-Ketoheterocycles That Additionally Target the Cytosolic Port Cys269 of Fatty Acid Amide Hydrolase

    PubMed Central

    2015-01-01

    A series of α-ketooxazoles incorporating electrophiles at the C5 position of the pyridyl ring of 2 (OL-135) and related compounds were prepared and examined as inhibitors of fatty acid amide hydrolase (FAAH) that additionally target the cytosolic port Cys269. From this series, a subset of the candidate inhibitors exhibited time-dependent FAAH inhibition and noncompetitive irreversible inactivation of the enzyme, consistent with the targeted Cys269 covalent alkylation or addition, and maintained or enhanced the intrinsic selectivity for FAAH versus other serine hydrolases. A preliminary in vivo assessment demonstrates that these inhibitors raise endogenous brain levels of anandamide and other FAAH substrates upon intraperitoneal (i.p.) administration to mice, with peak levels achieved within 1.5–3 h, and that the elevations of the signaling lipids were maintained >6 h, indicating that the inhibitors effectively reach and remain active in the brain, inhibiting FAAH for a sustained period. PMID:24456116

  19. Cyanuric acid hydrolase: evolutionary innovation by structural concatenation

    PubMed Central

    Peat, Thomas S; Balotra, Sahil; Wilding, Matthew; French, Nigel G; Briggs, Lyndall J; Panjikar, Santosh; Cowieson, Nathan; Newman, Janet; Scott, Colin

    2013-01-01

    The cyanuric acid hydrolase, AtzD, is the founding member of a newly identified family of ring-opening amidases. We report the first X-ray structure for this family, which is a novel fold (termed the ‘Toblerone’ fold) that likely evolved via the concatenation of monomers of the trimeric YjgF superfamily and the acquisition of a metal binding site. Structures of AtzD with bound substrate (cyanuric acid) and inhibitors (phosphate, barbituric acid and melamine), along with mutagenesis studies, allowed the identification of the active site. The AtzD monomer, active site and substrate all possess threefold rotational symmetry, to the extent that the active site possesses three potential Ser–Lys catalytic dyads. A single catalytic dyad (Ser85–Lys42) is hypothesized, based on biochemical evidence and crystallographic data. A plausible catalytic mechanism based on these observations is also presented. A comparison with a homology model of the related barbiturase, Bar, was used to infer the active-site residues responsible for substrate specificity, and the phylogeny of the 68 AtzD-like enzymes in the database were analysed in light of this structure–function relationship. PMID:23651355

  20. Soluble epoxide hydrolase inhibitory activity of anthraquinone components from Aloe.

    PubMed

    Sun, Ya Nan; Kim, Jang Hoon; Li, Wei; Jo, A Reum; Yan, Xi Tao; Yang, Seo Young; Kim, Young Ho

    2015-10-15

    Aloe is a short-stemmed succulent herb widely used in traditional medicine to treat various diseases and as raw material in cosmetics and heath foods. In this study, we isolated and identified two new anthraquinone derivatives, aloinoside C (6) and aloinoside D (7), together with six known compounds from an aqueous dissolved Aloe exudate. Their structures were identified by spectroscopic analysis. The inhibitory effects of the isolated compounds on soluble epoxide hydrolase (sEH) were evaluated. Compounds 1-8 inhibited sEH activity potently, with IC50 values ranging from 4.1±0.6 to 41.1±4.2 μM. A kinetic analysis of compounds 1-8 revealed that the inhibitory actions of compounds 1, 6 and 8 were non-competitive, whereas those of compounds 2-5 and 7 were the mixed-type. Molecular docking increases our understanding of receptor-ligand binding of all compounds. These results demonstrate that compounds 1-8 from Aloe are potential sEH inhibitors.

  1. Soluble epoxide hydrolase: a novel therapeutic target in stroke

    PubMed Central

    Zhang, Wenri; Koerner, Ines P; Noppens, Ruediger; Grafe, Marjorie; Tsai, Hsing-Ju; Morisseau, Christophe; Luria, Ayala; Hammock, Bruce D; Falck, John R; Alkayed, Nabil J

    2009-01-01

    The P450 eicosanoids epoxyeicosatrienoic acids (EETs) are produced in brain and perform important biological functions, including protection from ischemic injury. The beneficial effect of EETs, however, is limited by their metabolism via soluble epoxide hydrolase (sEH). We tested the hypothesis that sEH inhibition is protective against ischemic brain damage in vivo by a mechanism linked to enhanced cerebral blood flow (CBF). We determined expression and distribution of sEH immunoreactivity (IR) in brain, and examined the effect of sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE) on CBF and infarct size after experimental stroke in mice. Mice were administered a single intraperitoneal injection of AUDA-BE (10 mg/kg) or vehicle at 30 mins before 2-h middle cerebral artery occlusion (MCAO) or at reperfusion, in the presence and absence of P450 epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH). Immunoreactivity for sEH was detected in vascular and non-vascular brain compartments, with predominant expression in neuronal cell bodies and processes. 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid butyl ester was detected in plasma and brain for up to 24 h after intraperitoneal injection, which was associated with inhibition of sEH activity in brain tissue. Finally, AUDA-BE significantly reduced infarct size at 24 h after MCAO, which was prevented by MS-PPOH. However, regional CBF rates measured by iodoantipyrine (IAP) autoradiography at end ischemia revealed no differences between AUDA-BE- and vehicle-treated mice. The findings suggest that sEH inhibition is protective against ischemic injury by non-vascular mechanisms, and that sEH may serve as a therapeutic target in stroke. PMID:17440491

  2. Loratadine analogues as MAGL inhibitors.

    PubMed

    Patel, Jayendra Z; Ahenkorah, Stephen; Vaara, Miia; Staszewski, Marek; Adams, Yahaya; Laitinen, Tuomo; Navia-Paldanius, Dina; Parkkari, Teija; Savinainen, Juha R; Walczyński, Krzysztof; Laitinen, Jarmo T; Nevalainen, Tapio J

    2015-04-01

    Compound 12a (JZP-361) acted as a potent and reversible inhibitor of human recombinant MAGL (hMAGL, IC50=46 nM), and was found to have almost 150-fold higher selectivity over human recombinant fatty acid amide hydrolase (hFAAH, IC50=7.24 μM) and 35-fold higher selectivity over human α/β-hydrolase-6 (hABHD6, IC50=1.79 μM). Additionally, compound 12a retained H1 antagonistic affinity (pA2=6.81) but did not show cannabinoid receptor activity, when tested at concentrations ⩽ 10 μM. Hence, compound 12a represents a novel dual-acting pharmacological tool possessing both MAGL-inhibitory and antihistaminergic activities.

  3. Identification of benzofuran central cores for the inhibition of leukotriene A(4) hydrolase.

    PubMed

    Eccles, Wendy; Blevitt, Jonathan M; Booker, Jamila N; Chrovian, Christa C; Crawford, Shelby; de Leon, Aimee Rose; Deng, Xiaohu; Fourie, Anne M; Grice, Cheryl A; Herman, Krystal; Karlsson, Lars; Kearney, Aaron M; Lee-Dutra, Alice; Liang, Jimmy; Luna, Rosa; Pippel, Dan; Rao, Navin; Riley, Jason P; Santillán, Alejandro; Savall, Brad; Tanis, Virginia M; Xue, Xiaohua; Young, Arlene L

    2013-02-01

    Leukotrienes (LT's) are known to play a physiological role in inflammatory immune response. Leukotriene A(4) hydrolase (LTA(4)H) is a cystolic enzyme that stereospecifically catalyzes the transformation of LTA(4) to LTB(4). LTB(4) is a known pro-inflammatory mediator. This paper describes the identification and synthesis of substituted benzofurans as LTH(4)H inhibitors. The benzofuran series demonstrated reduced mouse and human whole blood LTB(4) levels in vitro and led to the identification one analog for advanced profiling. Benzofuran 28 showed dose responsive target engagement and provides a useful tool to explore a LTA(4)H inhibitor for the treatment of inflammatory diseases, such as asthma and inflammatory bowel disease (IBD).

  4. Functional analysis of a group A streptococcal glycoside hydrolase Spy1600 from family 84 reveals it is a β-N-acetylglucosaminidase and not a hyaluronidase

    PubMed Central

    Sheldon, William L.; Macauley, Matthew S.; Taylor, Edward J.; Robinson, Charlotte E.; Charnock, Simon J.; Davies, Gideon J.; Vocadlo, David J.; Black, Gary W.

    2006-01-01

    Group A streptococcus (Streptococcus pyogenes) is the causative agent of severe invasive infections such as necrotizing fasciitis (the so-called ‘flesh eating disease’) and toxic-shock syndrome. Spy1600, a glycoside hydrolase from family 84 of the large superfamily of glycoside hydrolases, has been proposed to be a virulence factor. In the present study we show that Spy1600 has no activity toward galactosaminides or hyaluronan, but does remove β-O-linked N-acetylglucosamine from mammalian glycoproteins – an observation consistent with the inclusion of eukaryotic O-glycoprotein 2-acetamido-2-deoxy-β-D-glucopyranosidases within glycoside hydrolase family 84. Proton NMR studies, structure–reactivity studies for a series of fluorinated analogues and analysis of 1,2-dideoxy-2′-methyl-α-D-glucopyranoso-[2,1-d]-Δ2′-thiazoline as a competitive inhibitor reveals that Spy1600 uses a double-displacement mechanism involving substrate-assisted catalysis. Family 84 glycoside hydrolases are therefore comprised of both prokaryotic and eukaryotic β-N-acetylglucosaminidases using a conserved catalytic mechanism involving substrate-assisted catalysis. Since these enzymes do not have detectable hyaluronidase activity, many family 84 glycoside hydrolases are most likely incorrectly annotated as hyaluronidases. PMID:16822234

  5. Glycoside Hydrolases across Environmental Microbial Communities

    PubMed Central

    Berlemont, Renaud

    2016-01-01

    Across many environments microbial glycoside hydrolases support the enzymatic processing of carbohydrates, a critical function in many ecosystems. Little is known about how the microbial composition of a community and the potential for carbohydrate processing relate to each other. Here, using 1,934 metagenomic datasets, we linked changes in community composition to variation of potential for carbohydrate processing across environments. We were able to show that each ecosystem-type displays a specific potential for carbohydrate utilization. Most of this potential was associated with just 77 bacterial genera. The GH content in bacterial genera is best described by their taxonomic affiliation. Across metagenomes, fluctuations of the microbial community structure and GH potential for carbohydrate utilization were correlated. Our analysis reveals that both deterministic and stochastic processes contribute to the assembly of complex microbial communities. PMID:27992426

  6. A simplified electrostatic model for hydrolase catalysis.

    PubMed

    Pessoa Filho, Pedro de Alcantara; Prausnitz, John M

    2015-07-01

    Toward the development of an electrostatic model for enzyme catalysis, the active site of the enzyme is represented by a cavity whose surface (and beyond) is populated by electric charges as determined by pH and the enzyme's structure. The electric field in the cavity is obtained from electrostatics and a suitable computer program. The key chemical bond in the substrate, at its ends, has partial charges with opposite signs determined from published force-field parameters. The electric field attracts one end of the bond and repels the other, causing bond tension. If that tension exceeds the attractive force between the atoms, the bond breaks; the enzyme is then a successful catalyst. To illustrate this very simple model, based on numerous assumptions, some results are presented for three hydrolases: hen-egg white lysozyme, bovine trypsin and bovine ribonuclease. Attention is given to the effect of pH.

  7. A high throughput fluorescent assay for measuring the activity of fatty acid amide hydrolase.

    PubMed

    Kage, Karen L; Richardson, Paul L; Traphagen, Linda; Severin, Jean; Pereda-Lopez, Ana; Lubben, Thomas; Davis-Taber, Rachel; Vos, Melissa H; Bartley, Diane; Walter, Karl; Harlan, John; Solomon, Larry; Warrior, Usha; Holzman, Thomas F; Faltynek, Connie; Surowy, Carol S; Scott, Victoria E

    2007-03-30

    Fatty acid amide hydrolase (FAAH) is the enzyme responsible for the rapid degradation of fatty acid amides such as the endocannabinoid anandamide. Inhibition of FAAH activity has been suggested as a therapeutic approach for the treatment of chronic pain, depression and anxiety, through local activation of the cannabinoid receptor CB1. We have developed a high throughput screening assay for identification of FAAH inhibitors using a novel substrate, decanoyl 7-amino-4-methyl coumarin (D-AMC) that is cleaved by FAAH to release decanoic acid and the highly fluorescent molecule 7-amino-4-methyl coumarin (AMC). This assay gives an excellent signal window for measuring FAAH activity and, as a continuous assay, inherently offers improved sensitivity and accuracy over previously reported endpoint assays. The assay was validated using a panel of known FAAH inhibitors and purified recombinant human FAAH, then converted to a 384 well format and used to screen a large library of compounds (>600,000 compounds) to identify FAAH inhibitors. This screen identified numerous novel FAAH inhibitors of diverse chemotypes. These hits confirmed using a native FAAH substrate, anandamide, and had very similar rank order potency to that obtained using the D-AMC substrate. Collectively these data demonstrate that D-AMC can be successfully used to rapidly and effectively identify novel FAAH inhibitors for potential therapeutic use.

  8. Superfamily-wide portrait of serine hydrolase inhibition achieved by library-versus-library screening.

    PubMed

    Bachovchin, Daniel A; Ji, Tianyang; Li, Weiwei; Simon, Gabriel M; Blankman, Jacqueline L; Adibekian, Alexander; Hoover, Heather; Niessen, Sherry; Cravatt, Benjamin F

    2010-12-07

    Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (> 80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activity-based assay in a library-versus-library format to screen 70+ SHs against 140+ structurally diverse carbamates. Lead inhibitors were discovered for ∼40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo.

  9. Lysophosphatidylcholine hydrolases of human erythrocytes, lymphocytes, and brain: Sensitive targets of conserved specificity for organophosphorus delayed neurotoxicants

    SciTech Connect

    Vose, Sarah C.; Holland, Nina T.; Eskenazi, Brenda; Casida, John E.

    2007-10-01

    Brain neuropathy target esterase (NTE), associated with organophosphorus (OP)-induced delayed neuropathy, has the same OP inhibitor sensitivity and specificity profiles assayed in the classical way (paraoxon-resistant, mipafox-sensitive hydrolysis of phenyl valerate) or with lysophosphatidylcholine (LysoPC) as the substrate. Extending our earlier observation with mice, we now examine human erythrocyte, lymphocyte, and brain LysoPC hydrolases as possible sensitive targets for OP delayed neurotoxicants and insecticides. Inhibitor profiling of human erythrocytes and lymphocytes gave the surprising result of essentially the same pattern as with brain. Human erythrocyte LysoPC hydrolases are highly sensitive to OP delayed neurotoxicants, with in vitro IC{sub 50} values of 0.13-85 nM for longer alkyl analogs, and poorly sensitive to the current OP insecticides. In agricultural workers, erythrocyte LysoPC hydrolyzing activities are similar for newborn children and their mothers and do not vary with paraoxonase status but have high intersample variation that limits their use as a biomarker. Mouse erythrocyte LysoPC hydrolase activity is also of low sensitivity in vitro and in vivo to the OP insecticides whereas the delayed neurotoxicant ethyl n-octylphosphonyl fluoride inhibits activity in vivo at 1-3 mg/kg. Overall, inhibition of blood LysoPC hydrolases is as good as inhibition of brain NTE as a predictor of OP inducers of delayed neuropathy. NTE and lysophospholipases (LysoPLAs) both hydrolyze LysoPC, yet they are in distinct enzyme families with no sequence homology and very different catalytic sites. The relative contributions of NTE and LysoPLAs to LysoPC hydrolysis and clearance from erythrocytes, lymphocytes, and brain remain to be defined.

  10. Activity of murein hydrolases in synchronized cultures of Escherichia coli.

    PubMed Central

    Hakenbeck, R; Messer, W

    1977-01-01

    Murein hydrolase activities were analyzed in synchronized cultures of Escherichia coli B/r. Cell wall-bound murein hydrolase activities, including the penicillin-sensitive endopeptidase, increased discontinuously during the cell cycle and showed maximum activity at a cell age of 30 to 35 min (generation time, 43 min). Maximum activity was observed at the same time that the rate of cell wall synthesis reached its maximum. These oscillations depended on the termination of replication: no increase in hydrolase activity was found if deoxyribonucleic acid synthesis was inhibited at an early time in the life cycle. In contrast, the activity of another murein hydrolase that was not tightly bound to the membrane (transglycosylase) increased exponentially with time, even when deoxyribonucleic acid synthesis was inhibited. PMID:321419

  11. Hybrid Receptor-Bound/MM-GBSA-Per-residue Energy-Based Pharmacophore Modelling: Enhanced Approach for Identification of Selective LTA4H Inhibitors as Potential Anti-inflammatory Drugs.

    PubMed

    Appiah-Kubi, Patrick; Soliman, Mahmoud

    2017-03-01

    Leukotriene A4 hydrolase has been identified as an enzyme with dual anti- and pro-inflammatory role, thus, the conversion of leukotriene to leukotriene B4 in the initiation stage of inflammation and the removal of the chemotactic Pro-Gly-Pro tripeptide. These findings make leukotriene A4 hydrolase an attractive drug target: suggesting an innovative approach towards the identification and design of novel class of compounds that can selectively inhibit leukotriene B4 synthesis while sparing the aminopeptidase activity. Previous inhibitors block the dual activity of the enzyme. Recently, a small lead molecule inhibitor denoted as ARM1 has been identified to block the hydrolase activity of leukotriene A4 hydrolase whilst sparing the aminopeptidase activity. In this study, a hybrid receptor-bound/MM-GBSA-per-residue energy based pharmacophore modeling approach was implemented to identify potential selective hydrolase inhibitors of leukotriene A4 hydrolase. In this approach, active site residues that favorably contributed to the binding of the bound conformation of ARM1 were derived from MD ensembles and MM/GBSA thermodynamic calculations. These residues were then mapped to key pharmacophore features of ARM1. The generated pharmacophore model was used to search the ZINC database for 3D structures that match the pharmacophore. Five new compounds have been identified and proposed as potential epoxide hydrolase selective inhibitors of leukotriene A4 hydrolase. Molecular docking and MM/GBSA analyses revealed that, these top five lead-like compounds ZINC00142747, ZINC94260794, ZINC01382396, ZINC02508448, and ZINC53994447 showed better binding affinities to the hydrolase active site pocket compared to ARM1. Per-residue energy decomposition analysis revealed that amino acid residues Phe314, Tyr378, Pro382, Trp311, Val367, and Ala377 are key residues critical in the selective inhibition of these hits. Information highlighted in this study may guide the the design the next

  12. Investigation of the mechanism of phosphonoacetaldehyde hydrolase

    SciTech Connect

    Hepburn, T.W.; Olsen, D.B.; Dunaway-Mariano, D.; Mariano, P.S.

    1986-05-01

    The authors are presently studying enzymes which catalyze the formation and cleavage of carbon phosphorous bonds. In 1970 LaNauze et. al. reported the isolation of one enzyme of interest - phosphonoacetaldehyde hydrolase from a mutant of Bacillus cereus. This enzyme catalyzes the hydrolysis of phosphonoaldehyde to acetaldehyde and inorganic phosphate. They have isolated phosphonatase from wild type B. cereus (grown on 2-aminoethylphosphonate as the P/sub i/ source) and have used /sup 1/H-NMR and /sup 31/P-NMR techniques to determine the products of the enzyme reaction as phosphate and acetaldehyde. The mechanism of the enzyme could involve the formation of a Schiff base between phosphonoacetaldehyde and lysine or it might only require Mg/sup + +/, an essential cofactor for activity. To distinguish between these possibilities they have begun to look at the Schiff base formation in more detail. NaBH/sub 4/ was found to inactivate the enzyme in the presence of substrate but not in its absence. This is consistent with results obtained for the enzyme isolated from the mutant bacteria. In addition treatment of the wild type enzyme with tritiated NaBH/sub 4/ resulted in significant incorporation of radiolabel into the protein as compared to the control. These results tentatively suggest that hydrolysis proceeds via a covalent imine intermediate.

  13. Structure of HsaD, a steroid-degrading hydrolase, from Mycobacterium tuberculosis.

    PubMed

    Lack, Nathan; Lowe, Edward D; Liu, Jie; Eltis, Lindsay D; Noble, Martin E M; Sim, Edith; Westwood, Isaac M

    2008-01-01

    Tuberculosis is a major cause of death worldwide. Understanding of the pathogenicity of Mycobacterium tuberculosis has been advanced by gene analysis and has led to the identification of genes that are important for intracellular survival in macrophages. One of these genes encodes HsaD, a meta-cleavage product (MCP) hydrolase that catalyzes the hydrolytic cleavage of a carbon-carbon bond in cholesterol metabolism. This paper describes the production of HsaD as a recombinant protein and, following crystallization, the determination of its three-dimensional structure to 2.35 A resolution by X-ray crystallography at the Diamond Light Source in Oxfordshire, England. To the authors' knowledge, this study constitutes the first report of a structure determined at the new synchrotron facility. The volume of the active-site cleft of the HsaD enzyme is more than double the corresponding active-site volumes of related MCP hydrolases involved in the catabolism of aromatic compounds, consistent with the specificity of HsaD for steroids such as cholesterol. Knowledge of the structure of the enzyme facilitates the design of inhibitors.

  14. Partial purification and substrate specificity of a ubiquitin hydrolase from Saccharomyces cerevisiae.

    PubMed Central

    Agell, N; Ryan, C; Schlesinger, M J

    1991-01-01

    A ubiquitin hydrolase that removes ubiquitin from a multi-ubiquitinated protein has been purified 600-fold from Saccharomyces cerevisiae. Four different ubiquitin-protein conjugates were assayed as substrates during the purification procedure. Enzymic activities that removed ubiquitin from ubiquitinated histone H2A, a ubiquitin-ubiquitin dimer and a ubiquitin-ribosomal fusion protein were separated during the purification from an activity that removed a single ubiquitin molecule linked by an isopeptide bond to a ubiquitinated protein. The size of the native enzyme was 160 kDa, based on its sedimentation in a sucrose gradient, and the subunit molecular mass was estimated to be 160 kDa, based on a profile of proteins eluted in different fractions by thiol-affinity chromatography. The partially purified hydrolase was not inhibited by a variety of protease inhibitors, except for thiol-blocking reagents. The natural substrate for this enzyme may be the polyubiquitin chain containing ubiquitin molecules bound to each other in isopeptide bonds, with one of them linked to a lysine residue of a protein targeted for intracellular proteolysis. Images Fig. 1. Fig. 3. PMID:1847617

  15. Lethal Effect of a Heterologous Murein Hydrolase on Penicillin-Treated Streptococcus sanguis

    PubMed Central

    Horne, Diane; Tomasz, Alexander

    1980-01-01

    Nine strains of Streptococcus sanguis exhibited tolerance to benzylpenicillin: the growth of each strain was susceptible to penicillin with minimal inhibitory concentrations of 0.1 μg/ml or lower, but the bacteriolytic and bactericidal effects were limited in each case. The tolerance of these bacteria was also reflected in the large discrepancies between the minimal inhibitory and minimal bactericidal concentrations for benzylpenicillin. The hypothesis that a natural deficiency of endogenous murein hydrolase (autolysin) in this species accounts for the penicillin tolerance was tested by using a heterologous murein hydrolase, the C-phage-associated lysin. In seven of the strains, addition of the lysin to the culture together with penicillin or other cell wall inhibitors resulted in lysis and rapid loss of viability. The enzyme alone did not appreciably affect normally growing cultures. The irreversible effects of penicillin plus lysin were drastically reduced in the presence of the bacteriostatic agents chloramphenicol and cerulenin. Speculations based on experiments are presented for the mechanisms by which penicillin treatment sensitizes these bacteria to an exogenous lytic enzyme. Similar phenomena requiring cooperation of host factors and penicillin may occur during infection, since somewhat similar although less pronounced results were obtained by addition of human lysozyme to penicillin-treated S. sanguis. PMID:6104471

  16. Crystallization and preliminary X-ray diffraction studies of cyanuric acid hydrolase from Azorhizobium caulinodans.

    PubMed

    Cho, Seunghee; Shi, Ke; Wackett, Lawrence P; Aihara, Hideki

    2013-08-01

    Cyanuric acid is synthesized industrially and forms during the microbial metabolism of s-triazine herbicides. Cyanuric acid is metabolized by some microorganisms via cyanuric acid hydrolase (CAH), which opens the s-triazine ring as a prelude to further metabolism. CAH is a member of the rare cyanuric acid hydrolase/barbiturase family. Here, the crystallization and preliminary X-ray diffraction analysis of CAH from Azorhizobium caulinodans are reported. CAH was cocrystallized with barbituric acid, a close analog of cyanuric acid that is a tight-binding competitive inhibitor. Crystals suitable for X-ray diffraction experiments were grown in conditions containing PEG 8K or magnesium sulfate as precipitants. An X-ray diffraction data set was collected from CAH-barbituric acid crystals to 2.7 Å resolution. The crystals were found to belong to space group I4₁22, with unit-cell parameters a = b = 237.9, c = 105.3 Å, α = β = γ = 90°.

  17. Alteration of the mutagenicity 3,3'-dichlorobenzidine by modifiers of rat hepatic epoxide hydrolase activity

    SciTech Connect

    Iba, M.M.

    1986-03-05

    The involvement of arene oxides in the activation of benzidines was assessed by examining the effect of (I) the epoxide hydrolase inhibitor trichloropropylene oxide (TCPO), (II) purified rat liver microsomal (P) epoxide hydrolase (EH), and (III) pretreatment of rats with phenobarbital (PB) on hepatic Sg- or P-catalyzed mutagenicity of benzidine (BZ) and 3,3'-dichlorobenzidine (DCB) to Salmonella TA 98. When catalyzed by Sg from untreated rats, the mutagenicity of DCB and BZ was 601 +/- 101 and 79 +/- 25 (His/sup +/ revertants/plate) respectively, but was 345 +/- 55 and 226 +/- 30 respectively, when catalyzed by microsomes (P) from untreated rats. PB-pretreatment enhanced the Sg-catalyzed mutagenicity of DCB and BZ (2.3-fold and 1.7-fold, respectively) and the P-catalyzed mutagenicity of DCB (1.7-fold), but totally inhibited the P-catalyzed mutagenicity of BZ. In TCPO-supplemented activating systems from PB-pretreated rats, the mutagenicity of DCB was enhanced in both Sg and P (1.9-fold and 1.6-fold, respectively), whereas that of BZ was unchanged. Added EH enhanced the P-catalyzed mutagenicity of DCB (1.4-fold) but had no effect on that of BZ, suggesting that the activity of the enzyme on DCB metabolites may not be entirely detoxifying. The data suggest that epoxidation may contribute to the activation of DCB but not BZ.

  18. The serine hydrolase ABHD6 Is a critical regulator of the metabolic syndrome.

    PubMed

    Thomas, Gwynneth; Betters, Jenna L; Lord, Caleb C; Brown, Amanda L; Marshall, Stephanie; Ferguson, Daniel; Sawyer, Janet; Davis, Matthew A; Melchior, John T; Blume, Lawrence C; Howlett, Allyn C; Ivanova, Pavlina T; Milne, Stephen B; Myers, David S; Mrak, Irina; Leber, Vera; Heier, Christoph; Taschler, Ulrike; Blankman, Jacqueline L; Cravatt, Benjamin F; Lee, Richard G; Crooke, Rosanne M; Graham, Mark J; Zimmermann, Robert; Brown, H Alex; Brown, J Mark

    2013-10-31

    The serine hydrolase α/β hydrolase domain 6 (ABHD6) has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG) in the brain. However, the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. To address this, we utilized targeted antisense oligonucleotides (ASOs) to selectively knock down ABHD6 in peripheral tissues in order to identify in vivo substrates and understand ABHD6's role in energy metabolism. Here, we show that selective knockdown of ABHD6 in metabolic tissues protects mice from high-fat-diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches, we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes.

  19. Human Valacyclovir Hydrolase/Biphenyl Hydrolase-Like Protein Is a Highly Efficient Homocysteine Thiolactonase

    PubMed Central

    McDonald, Matthew G.; Rademacher, Peter M.; MacCoss, Michael J.; Hsieh, Edward J.; Rettie, Allan E.; Furlong, Clement E.

    2014-01-01

    Homocysteinylation of lysine residues by homocysteine thiolactone (HCTL), a reactive homocysteine metabolite, results in protein aggregation and malfunction, and is a well-known risk factor for cardiovascular, autoimmune and neurological diseases. Human plasma paraoxonase-1 (PON1) and bleomycin hydrolase (Blmh) have been reported as the physiological HCTL detoxifying enzymes. However, the catalytic efficiency of HCTL hydrolysis by Blmh is low and not saturated at 20 mM HCTL. The catalytic efficiency of PON1 for HCTL hydrolysis is 100-fold lower than that of Blmh. A homocysteine thiolactonase (HCTLase) was purified from human liver and identified by mass spectrometry (MS) as the previously described human biphenyl hydrolase-like protein (BPHL). To further characterize this newly described HCTLase activity, BPHL was expressed in Escherichia coli and purified. The sequence of the recombinant BPHL (rBPHL) and hydrolytic products of the substrates HCTL and valacyclovir were verified by MS. We found that the catalytic efficiency (kcat/Km) of rBPHL for HCTL hydrolysis was 7.7 × 104 M−1s−1, orders of magnitude higher than that of PON1 or Blmh, indicating a more significant physiological role for BPHL in detoxifying HCTL. PMID:25333274

  20. First Glycoside Hydrolase Family 2 Enzymes from Thermus antranikianii and Thermus brockianus with β-Glucosidase Activity

    PubMed Central

    Schröder, Carola; Blank, Saskia; Antranikian, Garabed

    2015-01-01

    Two glycoside hydrolase encoding genes (tagh2 and tbgh2) were identified from different Thermus species using functional screening. Based on amino acid similarities, the enzymes were predicted to belong to glycoside hydrolase (GH) family 2. Surprisingly, both enzymes (TaGH2 and TbGH2) showed twofold higher activities for the hydrolysis of nitrophenol-linked β-D-glucopyranoside than of -galactopyranoside. Specific activities of 3,966 U/mg for TaGH2 and 660 U/mg for TbGH2 were observed. In accordance, Km values for both enzymes were significantly lower when β-D-glucopyranoside was used as substrate. Furthermore, TaGH2 was able to hydrolyze cellobiose. TaGH2 and TbGH2 exhibited highest activity at 95 and 90°C at pH 6.5. Both enzymes were extremely thermostable and showed thermal activation up to 250% relative activity at temperatures of 50 and 60°C. Especially, TaGH2 displayed high tolerance toward numerous metal ions (Cu2+, Co2+, Zn2+), which are known as glycoside hydrolase inhibitors. In this study, the first thermoactive GH family 2 enzymes with β-glucosidase activity have been identified and characterized. The hydrolysis of cellobiose is a unique property of TaGH2 when compared to other enzymes of GH family 2. Our work contributes to a broader knowledge of substrate specificities in GH family 2. PMID:26090361

  1. N-terminal domain of soluble epoxide hydrolase negatively regulates the VEGF-mediated activation of endothelial nitric oxide synthase

    PubMed Central

    Hou, Hsin-Han; Hammock, Bruce D.; Su, Kou-Hui; Morisseau, Christophe; Kou, Yu Ru; Imaoka, Susumu; Oguro, Ami; Shyue, Song-Kun; Zhao, Jin-Feng; Lee, Tzong-Shyuan

    2012-01-01

    Aims The mammalian soluble epoxide hydrolase (sEH) has both an epoxide hydrolase and a phosphatase domain. The role of sEH hydrolase activity in the metabolism of epoxyeicosatrienoic acids (EETs) and the activation of endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs) has been well defined. However, far less is known about the role of sEH phosphatase activity in eNOS activation. In the present study, we investigated whether the phosphatase domain of sEH was involved in the eNOS activation in ECs. Methods and results The level of eNOS phosphorylation in aortas is higher in the sEH knockout (sEH−/−) mice than in wild-type mice. In ECs, pharmacological inhibition of sEH phosphatase or overexpressing sEH with an inactive phosphatase domain enhanced vascular endothelial growth factor (VEGF)-induced NO production and eNOS phosphorylation. In contrast, overexpressing the phosphatase domain of sEH prevented the VEGF-mediated NO production and eNOS phosphorylation at Ser617, Ser635, and Ser1179. Additionally, treatment with VEGF induced a c-Src kinase-dependent increase in transient tyrosine phosphorylation of sEH and the formation of a sEH–eNOS complex, which was abolished by treatment with a c-Src kinase inhibitor, PP1, or the c-Src dominant-negative mutant K298M. We also demonstrated that the phosphatase domain of sEH played a key role in VEGF-induced angiogenesis by detecting the tube formation in ECs and neovascularization in Matrigel plugs in mice. Conclusion In addition to epoxide hydrolase activity, phosphatase activity of sEH plays a pivotal role in the regulation of eNOS activity and NO-mediated EC functions. PMID:22072631

  2. Structure of XC6422 from Xanthomonas campestris at 1.6 Å resolution: a small serine α/β-hydrolase

    SciTech Connect

    Yang, Chao-Yu; Chin, Ko-Hsin; Chou, Chia-Cheng; Wang, Andrew H.-J.; Chou, Shan-Ho

    2006-06-01

    The crystal structure of a conserved hypothetical protein from X. campestris has been determined to a resolution of 1.6 Å. The determined X. campestris structure shows that it belongs to the superfamily of serine α/β hydrolase, with an extra strand preceding the first β-strand to lead to extensive subunit interactions in the crystal. XC6422 is a conserved hypothetical protein from Xanthomonas campestris pathovar campestris (Xcc), a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. The protein consists of 220 amino acids and its structure has been determined to 1.6 Å resolution using the multi-wavelength anomalous dispersion (MAD) method. Although it has very low sequence identity to protein sequences in the PDB (less than 20%), the determined structure nevertheless shows that it belongs to the superfamily of serine α/β-hydrolases, with an active site that is fully accessible to solvent owing to the absence of a lid domain. Modelling studies with the serine esterase inhibitor E600 indicate that XC6422 adopts a conserved Ser-His-Asp catalytic triad common to this superfamily and has a preformed oxyanion hole for catalytic activation. These structural features suggest that XC6422 is most likely to be a hydrolase active on a soluble ester or a small lipid. An extra strand preceding the first β-strand in the canonical α/β-hydrolase fold leads to extensive subunit interactions between XC6422 monomers, which may explain why XC6422 crystals of good diffraction quality can grow to dimensions of up to 1.5 mm in a few days.

  3. Soluble Epoxide Hydrolase Inhibitory Activity of Selaginellin Derivatives from Selaginella tamariscina.

    PubMed

    Kim, Jang Hoon; Cho, Chong Woon; Tai, Bui Huu; Yang, Seo Young; Choi, Gug-Seoun; Kang, Jong Seong; Kim, Young Ho

    2015-12-02

    Selaginellin derivatives 1-3 isolated from Selaginella tamariscina were evaluated for their inhibition of soluble epoxide hydrolase (sEH) to demonstrate their potential for the treatment of cardiovascular disease. All selaginellin derivatives (1-3) inhibited sEH enzymatic activity and PHOME hydrolysis, in a dose-dependent manner, with IC50 values of 3.1 ± 0.1, 8.2 ± 2.2, and 4.2 ± 0.2 μM, respectively. We further determined that the derivatives function as non-competitive inhibitors. Moreover, the predicted that binding sites and interaction between 1-3 and sEH were solved by docking simulations. According to quantitative analysis, 1-3 were confirmed to have high content in the roots of S. tamariscina; among them, selaginellin 3 exhibited the highest content of 189.3 ± 0.0 μg/g.

  4. Structural Snapshots for Mechanism‐Based Inactivation of a Glycoside Hydrolase by Cyclopropyl Carbasugars

    PubMed Central

    Adamson, Christopher; Pengelly, Robert J.; Shamsi Kazem Abadi, Saeideh; Chakladar, Saswati; Draper, Jason

    2016-01-01

    Abstract Glycoside hydrolases (GHs) have attracted considerable attention as targets for therapeutic agents, and thus mechanism‐based inhibitors are of great interest. We report the first structural analysis of a carbocyclic mechanism‐based GH inactivator, the results of which show that the two Michaelis complexes are in 2H3 conformations. We also report the synthesis and reactivity of a fluorinated analogue and the structure of its covalently linked intermediate (flattened 2H3 half‐chair). We conclude that these inactivator reactions mainly involve motion of the pseudo‐anomeric carbon atom, knowledge that should stimulate the design of new transition‐state analogues for use as chemical biology tools. PMID:27783466

  5. Structural and Mechanistic Insights into C-P Bond Hydrolysis by Phosphonoacetate Hydrolase

    SciTech Connect

    Agarwal, Vinayak; Borisova, Svetlana A.; Metcalf, William W.; van der Donk, Wilfred A.; Nair, Satish K.

    2011-12-22

    Bacteria have evolved pathways to metabolize phosphonates as a nutrient source for phosphorus. In Sinorhizobium meliloti 1021, 2-aminoethylphosphonate is catabolized to phosphonoacetate, which is converted to acetate and inorganic phosphate by phosphonoacetate hydrolase (PhnA). Here we present detailed biochemical and structural characterization of PhnA that provides insights into the mechanism of C-P bond cleavage. The 1.35 {angstrom} resolution crystal structure reveals a catalytic core similar to those of alkaline phosphatases and nucleotide pyrophosphatases but with notable differences, such as a longer metal-metal distance. Detailed structure-guided analysis of active site residues and four additional cocrystal structures with phosphonoacetate substrate, acetate, phosphonoformate inhibitor, and a covalently bound transition state mimic provide insight into active site features that may facilitate cleavage of the C-P bond. These studies expand upon the array of reactions that can be catalyzed by enzymes of the alkaline phosphatase superfamily.

  6. Catalytic scope of the thiamine-dependent multifunctional enzyme cyclohexane-1,2-dione hydrolase.

    PubMed

    Loschonsky, Sabrina; Waltzer, Simon; Fraas, Sonja; Wacker, Tobias; Andrade, Susana L A; Kroneck, Peter M H; Müller, Michael

    2014-02-10

    The thiamine diphosphate (ThDP)-dependent enzyme cyclohexane-1,2-dione hydrolase (CDH) was expressed in Escherichia coli and purified by affinity chromatography (Ni-NTA). Recombinant CDH showed the same C-C bond-cleavage and C-C bond-formation activities as the native enzyme. Furthermore, we have shown that CDH catalyzes the asymmetric cross-benzoin reaction of aromatic aldehydes and (decarboxylated) pyruvate (up to quantitative conversion, 92-99 % ee). CDH accepts also hydroxybenzaldehydes and nitrobenzaldehydes; these previously have not (or only in rare cases) been known as substrates of other ThDP-dependent enzymes. On a semipreparative scale, sterically demanding 4-(tert-butyl)benzaldehyde and 2-naphthaldehyde were transformed into the corresponding 2-hydroxy ketone products in high yields. Additionally, certain benzaldehydes with electron withdrawing substituents were identified as potential inhibitors of the ligase activity of CDH.

  7. Structural and Mechanistic Insights into C-P Bond Hydrolysis by Phosphonoacetate Hydrolase

    PubMed Central

    Agarwal, Vinayak; Borisova, Svetlana A.; Metcalf, William W.; van der Donk, Wilfred A.; Nair, Satish K.

    2015-01-01

    SUMMARY Bacteria have evolved pathways to metabolize phosphonates as a nutrient source for phosphorus. In Sinorhizobium meliloti 1021, 2-aminoethylphosphonate is catabolized to phosphonoacetate, which is converted to acetate and inorganic phosphate by phosphonoacetate hydrolase (PhnA). Here we present detailed biochemical and structural characterization of PhnA that provides insights into the mechanism of C-P bond cleavage. The 1.35 Å resolution crystal structure reveals a catalytic core similar to those of alkaline phosphatases and nucleotide pyrophosphatases, but with notable differences such as a longer metal-metal distance. Detailed structure-guided analysis of active site residues and four additional co-crystal structures with phosphonoacetate substrate, acetate, phosphonoformate inhibitor, and a covalently-bound transition state mimic, provide insight into active site features that may facilitate cleavage of the C-P bond. These studies expand upon the array of reactions that can be catalyzed by enzymes of the alkaline phosphatase superfamily. PMID:22035792

  8. Inhibiting an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa Protects CFTR.

    PubMed

    Bahl, Christopher D; Hvorecny, Kelli L; Bomberger, Jennifer M; Stanton, Bruce A; Hammock, Bruce D; Morisseau, Christophe; Madden, Dean R

    2015-08-17

    Opportunistic pathogens exploit diverse strategies to sabotage host defenses. Pseudomonas aeruginosa secretes the CFTR inhibitory factor Cif and thus triggers loss of CFTR, an ion channel required for airway mucociliary defense. However, the mechanism of action of Cif has remained unclear. It catalyzes epoxide hydrolysis, but there is no known role for natural epoxides in CFTR regulation. It was demonstrated that the hydrolase activity of Cif is strictly required for its effects on CFTR. A small-molecule inhibitor that protects this key component of the mucociliary defense system was also uncovered. These results provide a basis for targeting the distinctive virulence chemistry of Cif and suggest an unanticipated role of physiological epoxides in intracellular protein trafficking.

  9. Long-term consequences of perinatal fatty acid amino hydrolase inhibition

    PubMed Central

    Wu, Chia-Shan; Morgan, Daniel; Jew, Chris P; Haskins, Chris; Andrews, Mary-Jeanette; Leishman, Emma; Spencer, Corinne M; Czyzyk, Traci; Bradshaw, Heather; Mackie, Ken; Lu, Hui-Chen

    2014-01-01

    Background and PurposeFatty acid amide hydrolase inhibitors show promise as a treatment for anxiety, depression and pain. Here we investigated whether perinatal exposure to URB597, a fatty acid amide hydrolase inhibitor, alters brain development and affects behaviour in adult mice. Experimental ApproachMouse dams were treated daily from gestational day 10.5 to 16.5 with 1, 3 or 10 mg kg−1 URB597. MS was used to measure a panel of endocannabinoids and related lipid compounds and brain development was assessed at embryonic day 16.5. Separate cohorts of mouse dams were treated with 10 mg kg−1 URB597, from gestational day 10.5 to postnatal day 7, and the adult offspring were assessed with a battery of behavioural tests. Key ResultsPerinatal URB597 exposure elevated anandamide and related N-acyl amides. URB597 did not induce signs of toxicity or affect dam weight gain, neurogenesis or axonal development at embryonic day 16.5. It did lead to subtle behavioural deficits in adult offspring, manifested by reduced cocaine-conditioned preference, increased depressive behaviours and impaired working memory. Anxiety levels, motor function and sensory-motor gating were not significantly altered. Conclusions and ImplicationsTaken together, the present results highlight how exposure to elevated levels of anandamide and related N-acyl amides during brain development can lead to subtle alterations in behaviour in adulthood. Linked ArticlesThis article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6 PMID:24730060

  10. Expression of key hydrolases for soy sauce fermentation in Zygosaccharomyces rouxii.

    PubMed

    Yuzuki, Masanobu; Matsushima, Kenichiro; Koyama, Yasuji

    2015-01-01

    Several key hydrolases in soy sauce fermentation such as proteases, peptidases, and glutaminases are supplied by Aspergillus sojae or Aspergillus oryzae. The genes encoding these hydrolases were successfully expressed in salt-tolerant yeast Zygosaccharomyces rouxii. These transformants are expected to supply extra hydrolases during soy sauce fermentation process.

  11. Development of fluorescent substrates for microsomal epoxide hydrolase and application to inhibition studies

    PubMed Central

    Morisseau, Christophe; Bernay, Maud; Escaich, Aurélie; Sanborn, James R.; Lango, Jozsef; Hammock, Bruce D.

    2011-01-01

    The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of numerous xenobiotics. Additionally, it has a potential role in sexual development and bile acid transport, and it is associated with a number of diseases, such as emphysema, spontaneous abortion, eclampsia and several forms of cancer. Toward developing chemical tools to study mEH biological role, we designed and synthesized a series of absorbent and fluorescent substrates. The highest activity for both rat and human mEH was obtained with the fluorescent substrate cyano(6-methoxy-naphthalen-2-yl)methyl glycidyl carbonate (11). An in vitro inhibition assay using this substrate ranked a series of known inhibitors similarly to the assay that used radioactive cis-stilbene oxide, but with a greater discrimination between inhibitors. These results demonstrate that the new fluorescence-based assay is a useful tool for the discovery of structure-activity relationships among mEH inhibitors. Further, this substrate could also be used for the screening chemical library with high accuracy and with a Z' value about 0.7. This new assay permits a significant decrease in labor and cost as well as offering the advantage of a continuous readout. However, it should not be used with crude enzyme preparations due to interfering reactions. PMID:21371418

  12. Structure of the Ubiquitin Hydrolase UCH-L3 Complexed with a Suicide Substrate

    SciTech Connect

    Misaghi, S.; Galardy, P.J.; Meester, W.J.; Ovaa, H.; Ploegh, H.L.; Gaudet, R.

    2009-03-24

    Ubiquitin C-terminal hydrolases (UCHs) comprise a family of small ubiquitin-specific proteases of uncertain function. Although no cellular substrates have been identified for UCHs, their highly tissue-specific expression patterns and the association of UCH-L1 mutations with human disease strongly suggest a critical role. The structure of the yeast UCH Yuh1-ubiquitin aldehyde complex identified an active site crossover loop predicted to limit the size of suitable substrates. We report the 1.45 {angstrom} resolution crystal structure of human UCH-L3 in complex with the inhibitor ubiquitin vinylmethylester, an inhibitor that forms a covalent adduct with the active site cysteine of ubiquitin-specific proteases. This structure confirms the predicted mechanism of the inhibitor and allows the direct comparison of a UCH family enzyme in the free and ligand-bound state. We also show the efficient hydrolysis by human UCH-L3 of a 13-residue peptide in isopeptide linkage with ubiquitin, consistent with considerable flexibility in UCH substrate size. We propose a model for the catalytic cycle of UCH family members which accounts for the hydrolysis of larger ubiquitin conjugates.

  13. Structure of the ubiquitin hydrolase UCH-L3 complexed with a suicide substrate.

    PubMed

    Misaghi, Shahram; Galardy, Paul J; Meester, Wim J N; Ovaa, Huib; Ploegh, Hidde L; Gaudet, Rachelle

    2005-01-14

    Ubiquitin C-terminal hydrolases (UCHs) comprise a family of small ubiquitin-specific proteases of uncertain function. Although no cellular substrates have been identified for UCHs, their highly tissue-specific expression patterns and the association of UCH-L1 mutations with human disease strongly suggest a critical role. The structure of the yeast UCH Yuh1-ubiquitin aldehyde complex identified an active site crossover loop predicted to limit the size of suitable substrates. We report the 1.45 A resolution crystal structure of human UCH-L3 in complex with the inhibitor ubiquitin vinylmethylester, an inhibitor that forms a covalent adduct with the active site cysteine of ubiquitin-specific proteases. This structure confirms the predicted mechanism of the inhibitor and allows the direct comparison of a UCH family enzyme in the free and ligand-bound state. We also show the efficient hydrolysis by human UCH-L3 of a 13-residue peptide in isopeptide linkage with ubiquitin, consistent with considerable flexibility in UCH substrate size. We propose a model for the catalytic cycle of UCH family members which accounts for the hydrolysis of larger ubiquitin conjugates.

  14. Role of phosphatase activity of soluble epoxide hydrolase in regulating simvastatin-activated endothelial nitric oxide synthase.

    PubMed

    Hou, Hsin-Han; Liao, Yi-Jen; Hsiao, Sheng-Huang; Shyue, Song-Kun; Lee, Tzong-Shyuan

    2015-08-25

    Soluble epoxide hydrolase (sEH) has C-terminal epoxide hydrolase and N-terminal lipid phosphatase activity. Its hydrolase activity is associated with endothelial nitric oxide synthase (eNOS) dysfunction. However, little is known about the role of sEH phosphatase in regulating eNOS activity. Simvastatin, a clinical lipid-lowering drug, also has a pleiotropic effect on eNOS activation. However, whether sEH phosphatase is involved in simvastatin-activated eNOS activity remains elusive. We investigated the role of sEH phosphatase activity in simvastatin-mediated activation of eNOS in endothelial cells (ECs). Simvastain increased the phosphatase activity of sEH, which was diminished by pharmacological inhibitors of sEH phosphatase. In addition, pharmacological inhibition of sEH phosphatase or overexpressing the inactive phosphatase domain of sEH enhanced simvastatin-induced NO bioavailability, tube formation and phosphorylation of eNOS, Akt, and AMP-activated protein kinase (AMPK). In contrast, overexpressing the phosphatase domain of sEH limited the simvastatin-increased NO biosynthesis and eNOS phosphorylation at Ser1179. Simvastatin evoked epidermal growth factor receptor-c-Src-increased Tyr phosphorylation of sEH and formation of an sEH-Akt-AMPK-eNOS complex, which was abolished by the c-Src kinase inhibitor PP1 or c-Src dominant-negative mutant K298M. These findings suggest that sEH phosphatase activity negatively regulates simvastatin-activated eNOS by impeding the Akt-AMPK-eNOS signaling cascade.

  15. Role of phosphatase activity of soluble epoxide hydrolase in regulating simvastatin-activated endothelial nitric oxide synthase

    PubMed Central

    Hou, Hsin-Han; Liao, Yi-Jen; Hsiao, Sheng-Huang; Shyue, Song-Kun; Lee, Tzong-Shyuan

    2015-01-01

    Soluble epoxide hydrolase (sEH) has C-terminal epoxide hydrolase and N-terminal lipid phosphatase activity. Its hydrolase activity is associated with endothelial nitric oxide synthase (eNOS) dysfunction. However, little is known about the role of sEH phosphatase in regulating eNOS activity. Simvastatin, a clinical lipid-lowering drug, also has a pleiotropic effect on eNOS activation. However, whether sEH phosphatase is involved in simvastatin-activated eNOS activity remains elusive. We investigated the role of sEH phosphatase activity in simvastatin-mediated activation of eNOS in endothelial cells (ECs). Simvastain increased the phosphatase activity of sEH, which was diminished by pharmacological inhibitors of sEH phosphatase. In addition, pharmacological inhibition of sEH phosphatase or overexpressing the inactive phosphatase domain of sEH enhanced simvastatin-induced NO bioavailability, tube formation and phosphorylation of eNOS, Akt, and AMP-activated protein kinase (AMPK). In contrast, overexpressing the phosphatase domain of sEH limited the simvastatin-increased NO biosynthesis and eNOS phosphorylation at Ser1179. Simvastatin evoked epidermal growth factor receptor–c-Src–increased Tyr phosphorylation of sEH and formation of an sEH–Akt–AMPK–eNOS complex, which was abolished by the c-Src kinase inhibitor PP1 or c-Src dominant-negative mutant K298M. These findings suggest that sEH phosphatase activity negatively regulates simvastatin-activated eNOS by impeding the Akt–AMPK–eNOS signaling cascade. PMID:26304753

  16. ENGINEERING OF PEPTIDOGLYCAN HYDROLASES FOR CONTROL OF PATHOGENIC BACTERIA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophages are viruses exclusively infecting bacteria and therefore offer suitable tools for their detection and control. At the end of their multiplication cycle, most phages lyse their hosts from within by means of an endolysin (peptidoglycan hydrolase), thereby enabling release of the phage p...

  17. Curation of characterized glycoside hydrolases of Fungal origin

    PubMed Central

    Murphy, Caitlin; Powlowski, Justin; Wu, Min; Butler, Greg; Tsang, Adrian

    2011-01-01

    Fungi produce a wide range of extracellular enzymes to break down plant cell walls, which are composed mainly of cellulose, lignin and hemicellulose. Among them are the glycoside hydrolases (GH), the largest and most diverse family of enzymes active on these substrates. To facilitate research and development of enzymes for the conversion of cell-wall polysaccharides into fermentable sugars, we have manually curated a comprehensive set of characterized fungal glycoside hydrolases. Characterized glycoside hydrolases were retrieved from protein and enzyme databases, as well as literature repositories. A total of 453 characterized glycoside hydrolases have been cataloged. They come from 131 different fungal species, most of which belong to the phylum Ascomycota. These enzymes represent 46 different GH activities and cover 44 of the 115 CAZy GH families. In addition to enzyme source and enzyme family, available biochemical properties such as temperature and pH optima, specific activity, kinetic parameters and substrate specificities were recorded. To simplify comparative studies, enzyme and species abbreviations have been standardized, Gene Ontology terms assigned and reference to supporting evidence provided. The annotated genes have been organized in a searchable, online database called mycoCLAP (Characterized Lignocellulose-Active Proteins of fungal origin). It is anticipated that this manually curated collection of biochemically characterized fungal proteins will be used to enhance functional annotation of novel GH genes. Database URL: http://mycoCLAP.fungalgenomics.ca/ PMID:21622642

  18. Recognition of corn defense chitinases by fungal polyglycine hydrolases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn (Zea mays ssp. mays). These target-specific endoproteases are unusual because they do not cut a defined pep...

  19. Bacteriophage virion-associated peptidoglycan hydrolases: potential new enzybiotics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virion-associated peptidoglycan hydrolases (VAPGH) are phage-encoded lytic enzymes that locally degrade the peptidoglycan (PG) of the bacterial cell wall during infection. Their action usually generates a small hole through which the phage tail crosses the cell envelope to inject the phage genetic m...

  20. ORGANOPHOSPHORUS HYDROLASE-BASED ASSAY FOR ORGANOPHOSPHATE PESTICIDES

    EPA Science Inventory

    We report a rapid and versatile Organophosphorus hydrolase (OPH)-based method for measurement of organophosphates. This assay is based on a substrate-dependent change in pH at the local vicinity of the enzyme. The pH change is monitored using fluorescein isothiocyanate (FITC), ...

  1. Novel microbial epoxide hydrolases for biohydrolysis of glycidyl derivatives.

    PubMed

    Kotik, Michael; Brichac, Jiri; Kyslík, Pavel

    2005-12-06

    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 degrees 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.

  2. Method for enhancing amidohydrolase activity of fatty acid amide hydrolase

    SciTech Connect

    John, George; Nagarajan, Subbiah; Chapman, Kent; Faure, Lionel; Koulen, Peter

    2016-10-25

    A method for enhancing amidohydrolase activity of Fatty Acid Amide Hydrolase (FAAH) is disclosed. The method comprising administering a phenoxyacylethanolamide that causes the enhanced activity. The enhanced activity can have numerous effects on biological organisms including, for example, enhancing the growth of certain seedlings. The subject matter disclosed herein relates to enhancers of amidohydrolase activity.

  3. The 2014 Bernard B. Brodie Award Lecture—Epoxide Hydrolases: Drug Metabolism to Therapeutics for Chronic Pain

    PubMed Central

    Kodani, Sean D.

    2015-01-01

    Dr. Bernard Brodie’s legacy is built on fundamental discoveries in pharmacology and drug metabolism that were then translated to the clinic to improve patient care. Similarly, the development of a novel class of therapeutics termed the soluble epoxide hydrolase (sEH) inhibitors was originally spurred by fundamental research exploring the biochemistry and physiology of the sEH. Here, we present an overview of the history and current state of research on epoxide hydrolases, specifically focusing on sEHs. In doing so, we start with the translational project studying the metabolism of the insect juvenile hormone mimic R-20458 [(E)-6,7-epoxy-1-(4-ethylphenoxy)-3,7-dimethyl-2-octene], which led to the identification of the mammalian sEH. Further investigation of this enzyme and its substrates, including the epoxyeicosatrienoic acids, led to insight into mechanisms of inflammation, chronic and neuropathic pain, angiogenesis, and other physiologic processes. This basic knowledge in turn led to the development of potent inhibitors of the sEH that are promising therapeutics for pain, hypertension, chronic obstructive pulmonary disorder, arthritis, and other disorders. PMID:25762541

  4. Pharmacokinetics of OpdA, an organophosphorus hydrolase, in the African Green Monkey

    PubMed Central

    Jackson, Colin J; Scott, Colin; Carville, Angela; Mansfield, Keith; Ollis, David L.

    2010-01-01

    Organophosphorus (OP) pesticides are a broad class of acetylcholinesterase inhibitors that are responsible for tremendous morbidity and mortality worldwide, contributing to an estimated 300,000 deaths annually. Current pharmacotherapy for acute OP poisoning includes the use of atropine, an oxime, and benzodiazepines. However, even with such therapy, the mortality from these agents are as high as 40%. Enzymatic hydrolysis of OPs is an attractive new potential therapy for acute OP poisoning. A number of bacterial OP hydrolases have been isolated. A promising OP hydrolase is an enzyme isolated from Agrobacterium radiobacter, named OpdA. OpdA has been shown to decrease lethality in rodent models of parathion and dichlorvos poisoning. However, pharmacokinetic data have not been obtained. In this study, we examined the pharmacokinetics of OpdA in an African Green Monkey model. At a dose of 1.2 mg/kg the half-life of OpdA was approximately 40 minutes, with a mean residence time of 57 minutes. As expected, the half-life did not change with the dose of OpdA given: at doses of 0.15 and 0.45 mg/kg, the half-life of OpdA was 43.1 and 38.9 minutes, respectively. In animals subjected to 5 daily doses of OpdA, the residual activity that was measured 24 hours after each OpdA dose increased 5-fold for the 0.45 mg/kg dose and 11-fold for the 1.2 mg/kg dose. OpdA exhibits pharmacokinetics favorable for the further development as a therapy for acute OP poisoning, particularly for hydrophilic OP pesticides. Future work to increase the half-life of OpdA may be beneficial. PMID:20599794

  5. Combined cocaine hydrolase gene transfer and anti-cocaine vaccine synergistically block cocaine-induced locomotion.

    PubMed

    Carroll, Marilyn E; Zlebnik, Natalie E; Anker, Justin J; Kosten, Thomas R; Orson, Frank M; Shen, Xiaoyun; Kinsey, Berma; Parks, Robin J; Gao, Yang; Brimijoin, Stephen

    2012-01-01

    Mice and rats were tested for reduced sensitivity to cocaine-induced hyper-locomotion after pretreatment with anti-cocaine antibody or cocaine hydrolase (CocH) derived from human butyrylcholinesterase (BChE). In Balb/c mice, direct i.p. injection of CocH protein (1 mg/kg) had no effect on spontaneous locomotion, but it suppressed responses to i.p. cocaine up to 80 mg/kg. When CocH was injected i.p. along with a murine cocaine antiserum that also did not affect spontaneous locomotion, there was no response to any cocaine dose. This suppression of locomotor activity required active enzyme, as it was lost after pretreatment with iso-OMPA, a selective BChE inhibitor. Comparable results were obtained in rats that developed high levels of CocH by gene transfer with helper-dependent adenoviral vector, and/or high levels of anti-cocaine antibody by vaccination with norcocaine hapten conjugated to keyhole limpet hemocyanin (KLH). After these treatments, rats were subjected to a locomotor sensitization paradigm involving a "training phase" with an initial i.p. saline injection on day 1 followed by 8 days of repeated cocaine injections (10 mg/kg, i.p.). A 15-day rest period then ensued, followed by a final "challenge" cocaine injection. As in mice, the individual treatment interventions reduced cocaine-stimulated hyperactivity to a modest extent, while combined treatment produced a greater reduction during all phases of testing compared to control rats (with only saline pretreatment). Overall, the present results strongly support the view that anti-cocaine vaccine and cocaine hydrolase vector treatments together provide enhanced protection against the stimulatory actions of cocaine in rodents. A similar combination therapy in human cocaine users might provide a robust therapy to help maintain abstinence.

  6. Molecular modeling studies on nucleoside hydrolase from the biological warfare agent Brucella suis.

    PubMed

    Mancini, Daiana T; Matos, Karina S; da Cunha, Elaine F F; Assis, Tamiris M; Guimarães, Ana P; França, Tanos C C; Ramalho, Teodorico C

    2012-01-01

    Brucella suis is a dangerous biological warfare agent already used for military purposes. This bacteria cause brucellosis, a zoonosis highly infective and difficult to fight. An important selective target for chemotherapy against this disease is nucleoside hydrolase (NH), an enzyme still not found in mammals. We present here the first three-dimensional structure of B. suis NH (BsNH) and propose this enzyme as a molecular target to the drug design in the fight against brucellosis. In addition, we performed molecular docking studies, aiming to analyze the three-dimensional positioning of nine known inhibitors of Chritidia fasciculata NH (CfNH) in the active sites of BsNH and CfNH. We also analyzed the main interactions of some of these compounds inside the active site of BsNH and the relevant factors to biological activity. These results, together with further molecular dynamics (MD) simulations, pointed out to the most promising compound as lead for the design of potential inhibitors of BsNH. Most of the docking and MD results corroborated to each other and the docking results also suggested a good correlation with experimental data.

  7. Synthesis of Phenoxyacyl-Ethanolamides and Their Effects on Fatty Acid Amide Hydrolase Activity*

    PubMed Central

    Faure, Lionel; Nagarajan, Subbiah; Hwang, Hyeondo; Montgomery, Christa L.; Khan, Bibi Rafeiza; John, George; Koulen, Peter; Blancaflor, Elison B.; Chapman, Kent D.

    2014-01-01

    N-Acylethanolamines (NAEs) are involved in numerous biological activities in plant and animal systems. The metabolism of these lipids by fatty acid amide hydrolase (FAAH) is a key regulatory point in NAE signaling activity. Several active site-directed inhibitors of FAAH have been identified, but few compounds have been described that enhance FAAH activity. Here we synthesized two sets of phenoxyacyl-ethanolamides from natural products, 3-n-pentadecylphenolethanolamide and cardanolethanolamide, with structural similarity to NAEs and characterized their effects on the hydrolytic activity of FAAH. Both compounds increased the apparent Vmax of recombinant FAAH proteins from both plant (Arabidopsis) and mammalian (Rattus) sources. These NAE-like compounds appeared to act by reducing the negative feedback regulation of FAAH activity by free ethanolamine. Both compounds added to seedlings relieved, in part, the negative growth effects of exogenous NAE12:0. Cardanolethanolamide reduced neuronal viability and exacerbated oxidative stress-mediated cell death in primary cultured neurons at nanomolar concentrations. This was reversed by FAAH inhibitors or exogenous NAE substrate. Collectively, our data suggest that these phenoxyacyl-ethanolamides act to enhance the activity of FAAH and may stimulate the turnover of NAEs in vivo. Hence, these compounds might be useful pharmacological tools for manipulating FAAH-mediated regulation of NAE signaling in plants or animals. PMID:24558037

  8. Role of ubiquitin C-terminal hydrolase-L1 in antipolyspermy defense of mammalian oocytes.

    PubMed

    Susor, Andrej; Liskova, Lucie; Toralova, Tereza; Pavlok, Antonin; Pivonkova, Katerina; Karabinova, Pavla; Lopatarova, Miloslava; Sutovsky, Peter; Kubelka, Michal

    2010-06-01

    The ubiquitin-proteasome system regulates many cellular processes through rapid proteasomal degradation of ubiquitin-tagged proteins. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is one of the most abundant proteins in mammalian oocytes. It has weak hydrolytic activity as a monomer and acts as a ubiquitin ligase in its dimeric or oligomeric form. Recently published data show that insufficiency in UCHL1 activity coincides with polyspermic fertilization; however, the mechanism by which UCHL1 contributes to this process remains unclear. Using UCHL1-specific inhibitors, we induced a high rate of polyspermy in bovine zygotes after in vitro fertilization. We also detected decreased levels in the monomeric ubiquitin and polyubiquitin pool. The presence of UCHL1 inhibitors in maturation medium enhanced formation of presumptive UCHL1 oligomers and subsequently increased abundance of K63-linked polyubiquitin chains in oocytes. We analyzed the dynamics of cortical granules (CGs) in UCHL1-inhibited oocytes; both migration of CGs toward the cortex during oocyte maturation and fertilization-induced extrusion of CGs were impaired. These alterations in CG dynamics coincided with high polyspermy incidence in in vitro-produced UCHL1-inhibited zygotes. These data indicate that antipolyspermy defense in bovine oocytes may rely on UCHL1-controlled functioning of CGs.

  9. Pharmacological inhibition of soluble epoxide hydrolase ameliorates diet-induced metabolic syndrome in rats.

    PubMed

    Iyer, Abishek; Kauter, Kathleen; Alam, Md Ashraful; Hwang, Sung Hee; Morisseau, Christophe; Hammock, Bruce D; Brown, Lindsay

    2012-01-01

    The signs of metabolic syndrome following chronic excessive macronutrient intake include body weight gain, excess visceral adipose deposition, hyperglycaemia, glucose and insulin intolerances, hypertension, dyslipidaemia, endothelial damage, cardiovascular hypertrophy, inflammation, ventricular contractile dysfunction, fibrosis, and fatty liver disease. Recent studies show increased activity of soluble epoxide hydrolase (sEH) during obesity and metabolic dysfunction. We have tested whether sEH inhibition has therapeutic potential in a rat model of diet-induced metabolic syndrome. In these high-carbohydrate, high-fat-fed rats, chronic oral treatment with trans-4-[4-(3-adamantan-1-ylureido)-cyclohexyloxy]-benzoic acid (t-AUCB), a potent sEH inhibitor, alleviated the signs of metabolic syndrome in vivo including glucose, insulin, and lipid abnormalities, changes in pancreatic structure, increased systolic blood pressure, cardiovascular structural and functional abnormalities, and structural and functional changes in the liver. The present study describes the pharmacological responses to this selective sEH inhibitor in rats with the signs of diet-induced metabolic syndrome.

  10. Cytosolic and microsomal epoxide hydrolases are immunologically distinguishable from each other in the rat and mouse.

    PubMed

    Guenthner, T M; Hammock, B D; Vogel, U; Oesch, F

    1981-04-10

    Antibodies raised to homogeneous rat liver microsomal epoxide hydrolase were used to distinguish microsomal epoxide hydrolase from epoxide hydrolase of cytosolic origin in mice and rats. Using double diffusion analysis in agarose gels, we show that anti-rat liver microsomal epoxide hydrolase forms a single precipitin line with solubilized microsomes from rat and mouse liver, but no reaction is seen with the corresponding cytosolic fractions. Rat or mouse microsomal epoxide hydrolase activity (using benzo[a]pyrene 4,5-oxide as substrate) can be completely precipitated out of solubilized preparations by the antibody, which is equipotent against rat and mouse microsomal epoxide hydrolase. No precipitation of cytosolic hydrolase activity (using trans-beta-ethyl styrene oxide as substrate) is seen with any concentration of the antibody tested. Thus, in the case of microsomal epoxide hydrolase, extensive immunological cross-reactivity exists between the two species, rat and mouse. In contrast, no cross-reactivity is detectable between cytosolic and microsomal epoxide hydrolase, even when enzymes from the same species are compared. We conclude that microsomal and cytosolic epoxide hydrolase activities represent distinct and immunologically non-cross-reactive protein species.

  11. Diversity and Biocatalytic Potential of Epoxide Hydrolases Identified by Genome Analysis†

    PubMed Central

    van Loo, Bert; Kingma, Jaap; Arand, Michael; Wubbolts, Marcel G.; Janssen, Dick B.

    2006-01-01

    Epoxide hydrolases play an important role in the biodegradation of organic compounds and are potentially useful in enantioselective biocatalysis. An analysis of various genomic databases revealed that about 20% of sequenced organisms contain one or more putative epoxide hydrolase genes. They were found in all domains of life, and many fungi and actinobacteria contain several putative epoxide hydrolase-encoding genes. Multiple sequence alignments of epoxide hydrolases with other known and putative α/β-hydrolase fold enzymes that possess a nucleophilic aspartate revealed that these enzymes can be classified into eight phylogenetic groups that all contain putative epoxide hydrolases. To determine their catalytic activities, 10 putative bacterial epoxide hydrolase genes and 2 known bacterial epoxide hydrolase genes were cloned and overexpressed in Escherichia coli. The production of active enzyme was strongly improved by fusion to the maltose binding protein (MalE), which prevented inclusion body formation and facilitated protein purification. Eight of the 12 fusion proteins were active toward one or more of the 21 epoxides that were tested, and they converted both terminal and nonterminal epoxides. Four of the new epoxide hydrolases showed an uncommon enantiopreference for meso-epoxides and/or terminal aromatic epoxides, which made them suitable for the production of enantiopure (S,S)-diols and (R)-epoxides. The results show that the expression of epoxide hydrolase genes that are detected by analyses of genomic databases is a useful strategy for obtaining new biocatalysts. PMID:16597997

  12. Poly(aspartic acid) (PAA) hydrolases and PAA biodegradation: current knowledge and impact on applications.

    PubMed

    Hiraishi, Tomohiro

    2016-02-01

    Thermally synthesized poly(aspartic acid) (tPAA) is a bio-based, biocompatible, biodegradable, and water-soluble polymer that has a high proportion of β-Asp units and equivalent moles of D- and L-Asp units. Poly(aspartic acid) (PAA) hydrolase-1 and hydrolase-2 are tPAA biodegradation enzymes purified from Gram-negative bacteria. PAA hydrolase-1 selectively cleaves amide bonds between β-Asp units via an endo-type process, whereas PAA hydrolase-2 catalyzes the exo-type hydrolysis of the products of tPAA hydrolysis by PAA hydrolase-1. The novel reactivity of PAA hydrolase-1 makes it a good candidate for a biocatalyst in β-peptide synthesis. This mini-review gives an overview of PAA hydrolases with emphasis on their biochemical and functional properties, in particular, PAA hydrolase-1. Functionally related enzymes, such as poly(R-3-hydroxybutyrate) depolymerases and β-aminopeptidases, are compared to PAA hydrolases. This mini-review also provides findings that offer an insight into the catalytic mechanisms of PAA hydrolase-1 from Pedobacter sp. KP-2.

  13. Purification and characterization of pranlukast hydrolase from rat liver microsomes: the hydrolase is identical to carboxylesterase pI 6.2.

    PubMed

    Luan, L; Sugiyama, T; Takai, S; Usami, Y; Adachi, T; Katagiri, Y; Hirano, K

    1997-01-01

    Two carboxylesterases with pI 6.0 and 6.2 derived from rat liver microsomes were purified. The two isozymes were remarkably different in substrate specificity, but they had equal enzymatic activity for alpha-naphthyl acetate and were inhibited equally by phenylmethylsulfonyl fluoride (PMSF) and bis-(4-nitrophenyl) phosphate (BNPP). Carboxylesterases pI 6.0 and 6.2 are identical to the enzymes referred to as hydrolase A and B, respectively, from the results of amino acid sequence analyses. Pranlukast was effectively hydrolyzed by carboxylesterase pI 6.2 but not by the pI 6.0 enzyme, and the difference in the pranlukast metabolism between the human and the rat could be explained by the substrate specificity of carboxylesterase. Furthermore, prodrugs of angiotensin converting enzyme inhibitors were found to be converted to the active drugs after hydrolysis by the carboxylesterases pI 6.0 and 6.2. Carboxylesterases generally catalyze the hydrolysis of ester-type drugs preferentially rather than amide-type drugs.

  14. pHluorin-based in vivo assay for hydrolase screening.

    PubMed

    Schuster, Sascha; Enzelberger, Markus; Trauthwein, Harald; Schmid, Rolf D; Urlacher, Vlada B

    2005-05-01

    pHluorin, a pH-sensitive mutant of green fluorescent protein (GFP), acts as a sensor for intracellular pH shifts, triggered by hydrolytic enzymes. This principle was used to develop a pHluorin-based in vivo assay for hydrolase screening. The presented assay was evaluated for Escherichia coli (E. coli) cells, producing heterologous pHluorin and an esterase from Geobacillus stearothermophilus which is considered as a model hydrolase. Subsequently, the utility of this detection system was also demonstrated with recombinantly expressed hydantoinase and amidase in E. coli. This in vivo assay also shows capability for readout with flow cytometric devices. Population shifts of pHluorin-expressing E. coli cells were easily recognized due to pH changes caused by substrate hydrolysis.

  15. Isolation and characterization of Xenopus soluble epoxide hydrolase.

    PubMed

    Purba, Endang R; Oguro, Ami; Imaoka, Susumu

    2014-07-01

    Soluble epoxide hydrolase (sEH) contributes to cell growth, but the contribution of sEH to embryonic development is not well understood. In this study, Xenopus sEH cDNA was isolated from embryos of Xenopus laevis. The Xenopus sEH was expressed in Escherichia coli and was purified. The epoxide hydrolase and phosphatase activities of purified sEH were investigated. The Xenopus sEH did not show phosphatase activity toward 4-methylumbelliferyl phosphate or several lysophosphatidic acids although it had EH activity. The amino acid sequence of Xenopus sEH was compared with that reported previously. We found amino acid substitutions of the 29th Thr to Asn and the 146th Arg to His and prepared a sEH mutant (N29T/H146R), designed as mutant 1. Neither wild-type sEH nor mutant 1 had phosphatase activity. Additional substitution of the 11th Gly with Asp was found by comparison with human sEH which has phosphatase activity, but the Xenopus sEH mutant G11D prepared as mutant 2 did not have phosphatase activity. The epoxide hydrolase activity of sEH seemed to be similar to that of human sEH, while Xenopus sEH did not have phosphatase activity toward several substrates that human sEH metabolizes.

  16. Human lung hydrolases delineate Mycobacterium tuberculosis-macrophage interactions and the capacity to control infection.

    PubMed

    Arcos, Jesús; Sasindran, Smitha J; Fujiwara, Nagatoshi; Turner, Joanne; Schlesinger, Larry S; Torrelles, Jordi B

    2011-07-01

    Pulmonary surfactant contains homeostatic and antimicrobial hydrolases. When Mycobacterium tuberculosis is initially deposited in the terminal bronchioles and alveoli, as well as following release from lysed macrophages, bacilli are in intimate contact with these lung surfactant hydrolases. We identified and measured several hydrolases in human alveolar lining fluid and lung tissue that, at their physiological concentrations, dramatically modified the M. tuberculosis cell envelope. Independent of their action time (15 min to 12 h), the effects of the hydrolases on the M. tuberculosis cell envelope resulted in a significant decrease (60-80%) in M. tuberculosis association with, and intracellular growth of the bacteria within, human macrophages. The cell envelope-modifying effects of the hydrolases also led to altered M. tuberculosis intracellular trafficking and induced a protective proinflammatory response to infection. These findings add a new concept to our understanding of M. tuberculosis-macrophage interactions (i.e., the impact of lung surfactant hydrolases on M. tuberculosis infection).

  17. Annotation and comparative analysis of the glycoside hydrolase genes in Brachypodium distachyon

    SciTech Connect

    Tyler, Ludmila; Bragg, Jennifer; Wu, Jiajie; Yang, Xiaohan; Tuskan, Gerald A; Vogel, John

    2010-01-01

    Background Glycoside hydrolases cleave the bond between a carbohydrate and another carbohydrate, a protein, lipid or other moiety. Genes encoding glycoside hydrolases are found in a wide range of organisms, from archea to animals, and are relatively abundant in plant genomes. In plants, these enzymes are involved in diverse processes, including starch metabolism, defense, and cell-wall remodeling. Glycoside hydrolase genes have been previously cataloged for Oryza sativa (rice), the model dicotyledonous plant Arabidopsis thaliana, and the fast-growing tree Populus trichocarpa (poplar). To improve our understanding of glycoside hydrolases in plants generally and in grasses specifically, we annotated the glycoside hydrolase genes in the grasses Brachypodium distachyon (an emerging monocotyledonous model) and Sorghum bicolor (sorghum). We then compared the glycoside hydrolases across species, both at the whole-genome level and at the level of individual glycoside hydrolase families. Results We identified 356 glycoside hydrolase genes in Brachypodium and 404 in sorghum. The corresponding proteins fell into the same 34 families that are represented in rice, Arabidopsis, and poplar, helping to define a glycoside hydrolase family profile which may be common to flowering plants. Examination of individual glycoside hydrolase familes (GH5, GH13, GH18, GH19, GH28, and GH51) revealed both similarities and distinctions between monocots and dicots, as well as between species. Shared evolutionary histories appear to be modified by lineage-specific expansions or deletions. Within families, the Brachypodium and sorghum proteins generally cluster with those from other monocots. Conclusions This work provides the foundation for further comparative and functional analyses of plant glycoside hydrolases. Defining the Brachypodium glycoside hydrolases sets the stage for Brachypodium to be a monocot model for investigations of these enzymes and their diverse roles in planta. Insights

  18. Contribution of hydrolase and phosphatase domains in soluble epoxide hydrolase to vascular endothelial growth factor expression and cell growth.

    PubMed

    Oguro, Ami; Sakamoto, Koichi; Suzuki, Sachiko; Imaoka, Susumu

    2009-12-01

    Soluble epoxide hydrolase (sEH) is an important pharmacological target because it metabolizes potent bioactive substrates, epoxyeicosatrienoinc acids (EETs) and other lipid epoxide. EETs have a variety of biological functions including angiogenesis and cancer metastasis. However, the regulation and physiological function of sEH is not well understood. In this study, we found that hypoxia significantly suppressed the expression of sEH in mouse liver and a human hepatoma cell line, Hep3B. Hypoxia promotes the proliferation of vascular endothelial cells or carcinoma cells. Knockdown of sEH in Hep3B cells induced vascular endothelial growth factor (VEGF) mRNA and cell growth, both of which were suppressed by overexpression of sEH. sEH has phosphatase activity as well as epoxide hydrolase (EH) activity. We prepared mutant clones which lacking EH or phosphatase activity using the amino acid change Asp335Ser or Asp9Ala, respectively. The effects of WT sEH on cell growth were lost by mutation of either the EH domain or phosphatase domain. However, mutation of the phosphatase domain but not EH domain did not influence the expression of VEGF. These results suggest that sEH plays an important role in the physiology of cells including proliferation and that the epoxide hydrolase and phosphatase domains of sEH have different biological functions.

  19. Immunohistochemical study of epoxide hydrolase induced by trichloroethylene in rat liver

    SciTech Connect

    Kawamoto, T.; Hobara, T.; Ogino, K.; Takemoto, T.; Nakamura, K.; Imamura, A.; Koshiro, A.; Kobayashi, H.; Iwamoto, S.; Sakai, T.

    1987-10-01

    Epoxide hydrolase catalyzes the hydrolation of potentially toxic, electrophilic epoxides that are often generated during cytochrome P-450 catalyzed monooxigenation, forming the corresponding transdihydrodiols. It is well-known that trichloroethylene is metabolized by cytochrome P-450 containing mixed-function oxidase systems to trichloroethylene oxide, which decomposes to other metabolites. As trichloroethylene is an epoxide, epoxide hydrolase is suspected to catalyze the hydrolation of trichloroethylene oxide. No reports have appeared about the relationship between trichloroethylene and epoxide hydrolase. In this report, the authors studied the effect of trichloroethylene on epoxide hydrolase immunohistochemically.

  20. An isofenphos-methyl hydrolase (Imh) capable of hydrolyzing the P-O-Z moiety of organophosphorus pesticides containing an aryl or heterocyclic group.

    PubMed

    Li, Rong; Liu, Yuan; Zhang, Jian; Chen, Kai; Li, Shunpeng; Jiang, Jiandong

    2012-06-01

    Organophosphorus pesticide (OP) hydrolases play key roles in the degradation and decontamination of agricultural and household OPs and in the detoxification of chemical warfare agents. In this study, an isofenphos-methyl hydrolase gene (imh) was cloned from the isocarbophos-degrading strain of Arthrobacter sp. scl-2 using the polymerase chain reaction method. Isofenphos-methyl hydrolase (Imh) showed 98% sequence identity with the isofenphos hydrolase from Arthrobacter sp. strain B-5. Imh was highly expressed in Escherichia coli BL21 (DE3), and the His(6)-tagged Imh was purified (1.7 mg/ml) with a specific activity of 14.35 U/mg for the substrate isofenphos-methyl. The molecular mass of the denatured Imh is about 44 kDa, and the isoelectric point (pI) value was estimated to be 3.4. The optimal pH and temperature for hydrolysis of isofenphos-methyl were pH 8.0 and 35 °C, respectively. The secondary structure of Imh shows that Imh is a metallo-dependent hydrolase, and it was found that Imh was completely inhibited by the metalloprotease inhibitor 1,10-phenanthroline (0.5 mM), and the catalytic activity was restored by the subsequent addition of Zn(2+). Interestingly, Imh had a relatively broader substrate specificity and was capable of hydrolyzing 12 of the tested oxon and thion OPs with the P-O-Z moiety instead of the P-S(C)-Z moiety. Furthermore, it was found that the existence of an aryl or heterocyclic group in the leaving group (Z) is also important in determining the substrate specificity. Among all the substrates hydrolyzed by Imh, it was assumed that Imh preferred P-O-Z substrates still with a phosphamide bond (P-N), such as isofenphos-methyl, isofenphos, isocarbophos, and butamifos. The newly characterized Imh has a great potential for use in the decontamination and detoxification of agricultural and household OPs and is a good candidate for the study of the catalytic mechanism and substrate specificity of OP hydrolases.

  1. The Responses of Rat Intestinal Brush Border and Cytosol Peptide Hydrolase Activities to Variation in Dietary Protein Content DIETARY REGULATION OF INTESTINAL PEPTIDE HYDROLASES

    PubMed Central

    Nicholson, J. Alex; McCarthy, Denis M.; Kim, Young S.

    1974-01-01

    The effects of variation in dietary protein content on small intestinal brush border and cytosol peptide hydrolase activities have been investigated. One group of rats was fed a high protein diet (55% casein) and another group was fed a low protein diet (10% casein). After 1 wk, brush border peptide hydrolase activity (L-leucyl-β-naphthylamide as substrate) and cytosol peptide hydrolase activity (L-prolyl-L-leucine as substrate) were determined in mucosae taken from the proximal, middle, and distal small intestine. As judged by several parameters, brush border peptide hydrolase activity was significantly greater in rats fed the high protein diet when data for corresponding segments were compared. In contrast, no significant difference was seen in cytosol peptide hydrolase activity. In a second study, brush border and cytosol peptide hydrolase activities were determined in the proximal intestine by utilizing an additional three peptide substrates: L-leucyl-L-alanine, L-phenylalanylglycine, and glycyl-L-phenylalanine. Sucrase, maltase, and alkaline phosphatase activities were also determined. As before, brush border peptide hydrolase activities were significantly greater in rats fed the high protein diet. However, activities of the nonproteolytic brush border enzymes did not vary significantly with diet. In contrast to the results obtained with L-prolyl-L-leucine as substrate for the cytosol enzymes, cytosol activity against the three additional peptide substrates was greater in rats fed the high protein diet. It is suggested that the brush border peptide hydrolase response to variation in dietary protein content represents a functional adaptation analogous to the regulation of intestinal disaccharidases by dietary carbohydrates. The implication of the differential responses of the cytosol peptide hydrolases is uncertain, since little is known of the functional role of these nonorgan-specific enzymes. PMID:4430719

  2. Design, synthesis and evaluation of polar head group containing 2-keto-oxazole inhibitors of FAAH.

    PubMed

    Rusch, Marion; Zahov, Stefan; Vetter, Ingrid R; Lehr, Matthias; Hedberg, Christian

    2012-01-15

    2-α-Keto oxazoles containing polar head groups in their C5-side chains were designed as fatty acid amide hydrolase (FAAH) inhibitors. Variation in the spacer length resulted in submicromolar α-keto-oxazole FAAH inhibitor (IC(50)=436 nM) presenting electrostatic stabilizing interactions between its polar head group contained in the C5-side chain and the hydrophilic pocket of the enzyme.

  3. Enzymatic degradation of monocrotophos by extracellular fungal OP hydrolases.

    PubMed

    Jain, Rachna; Garg, Veena

    2013-11-01

    The present study explores the potential of extracellular fungal organophosphate (OP) hydrolase for the degradation of monocrotophos. Extracellular OP hydrolases were isolated and purified from five different fungal isolates viz. Aspergillus niger (M1), Aspergillus flavus (M2), Penicillium aculeatum (M3), Fusarium pallidoroseum (M4), and Macrophomina sp. (M5) by AmSO4 precipitation, dialysis, and G-100 chromatography. M3 showed highest percentage yield of 68.81 followed by 55.41 % for M1. Each of the purified enzyme fraction constituted of two different subunits of 33- and 67-kDa molecular weight. Optimum enzyme fraction (150 μg ml(-1)) rapidly degraded monocrotophos within 120 h in phosphorus-free liquid culture medium (CZM) with K deg of 0.0368, 0.0138, 0.048, 0.016, 0.0138, and 0.048 day(-1) and half-life of 0.79, 2.11, 0.6, 1.8, and 2.11 days for M1, M2, M3, M4, and M5, respectively. The results were further confirmed by high performance thin layer chromatography and Fourier transform infrared which indicate the disappearance of monocrotophos by hydrolytic cleavage of vinyl phosphate bond. The overall order of enzymatic degradation was found to be P. aculeatum > A. niger > F. pallidoroseum > A. flavus = Macrophomina sp. Hence, the study concludes that extracellular OP hydrolases efficiently degraded monocrotophos and could be used as a potential candidate for the detoxification of this neurotoxin pesticide.

  4. Active Site and Laminarin Binding in Glycoside Hydrolase Family 55*

    PubMed Central

    Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; Udell, Hannah S.; Yik, Eric J.; Bergeman, Lai F.; Fox, Brian G.

    2015-01-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100–10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  5. Inverting hydrolases and their use in enantioconvergent biotransformations

    PubMed Central

    Schober, Markus; Faber, Kurt

    2013-01-01

    Owing to the more abundant occurrence of racemic compounds compared to prochiral or meso forms, most enantiomerically pure products are obtained via racemate resolution. This review summarizes (chemo)enzymatic enantioconvergent processes based on the use of hydrolytic enzymes, which are able to invert a stereocenter during catalysis that can overcome the 50%-yield limitation of kinetic resolution. Recent developments are presented in the fields of inverting or retaining sulfatases, epoxide hydrolases and dehalogenases, which allow the production of secondary alcohols or vicinal diols at a 100% theoretical yield from a racemate via enantioconvergent processes. PMID:23809848

  6. Abnormal Hypermethylation at Imprinting Control Regions in Patients with S-Adenosylhomocysteine Hydrolase (AHCY) Deficiency

    PubMed Central

    Motzek, Antje; Knežević, Jelena; Switzeny, Olivier J.; Cooper, Alexis; Barić, Ivo; Beluzić, Robert; Strauss, Kevin A.; Puffenberger, Erik G.; Vugrek, Oliver; Zechner, Ulrich

    2016-01-01

    S-adenosylhomocysteine hydrolase (AHCY) deficiency is a rare autosomal recessive disorder in methionine metabolism caused by mutations in the AHCY gene. Main characteristics are psychomotor delay including delayed myelination and myopathy (hypotonia, absent tendon reflexes etc.) from birth, mostly associated with hypermethioninaemia, elevated serum creatine kinase levels and increased genome wide DNA methylation. The prime function of AHCY is to hydrolyse and efficiently remove S-adenosylhomocysteine, the by-product of transmethylation reactions and one of the most potent methyltransferase inhibitors. In this study, we set out to more specifically characterize DNA methylation changes in blood samples from patients with AHCY deficiency. Global DNA methylation was increased in two of three analysed patients. In addition, we analysed the DNA methylation levels at differentially methylated regions (DMRs) of six imprinted genes (MEST, SNRPN, LIT1, H19, GTL2 and PEG3) as well as Alu and LINE1 repetitive elements in seven patients. Three patients showed a hypermethylation in up to five imprinted gene DMRs. Abnormal methylation in Alu and LINE1 repetitive elements was not observed. We conclude that DNA hypermethylation seems to be a frequent but not a constant feature associated with AHCY deficiency that affects different genomic regions to different degrees. Thus AHCY deficiency may represent an ideal model disease for studying the molecular origins and biological consequences of DNA hypermethylation due to impaired cellular methylation status. PMID:26974671

  7. Abnormal Hypermethylation at Imprinting Control Regions in Patients with S-Adenosylhomocysteine Hydrolase (AHCY) Deficiency.

    PubMed

    Motzek, Antje; Knežević, Jelena; Switzeny, Olivier J; Cooper, Alexis; Barić, Ivo; Beluzić, Robert; Strauss, Kevin A; Puffenberger, Erik G; Mudd, S Harvey; Vugrek, Oliver; Zechner, Ulrich

    2016-01-01

    S-adenosylhomocysteine hydrolase (AHCY) deficiency is a rare autosomal recessive disorder in methionine metabolism caused by mutations in the AHCY gene. Main characteristics are psychomotor delay including delayed myelination and myopathy (hypotonia, absent tendon reflexes etc.) from birth, mostly associated with hypermethioninaemia, elevated serum creatine kinase levels and increased genome wide DNA methylation. The prime function of AHCY is to hydrolyse and efficiently remove S-adenosylhomocysteine, the by-product of transmethylation reactions and one of the most potent methyltransferase inhibitors. In this study, we set out to more specifically characterize DNA methylation changes in blood samples from patients with AHCY deficiency. Global DNA methylation was increased in two of three analysed patients. In addition, we analysed the DNA methylation levels at differentially methylated regions (DMRs) of six imprinted genes (MEST, SNRPN, LIT1, H19, GTL2 and PEG3) as well as Alu and LINE1 repetitive elements in seven patients. Three patients showed a hypermethylation in up to five imprinted gene DMRs. Abnormal methylation in Alu and LINE1 repetitive elements was not observed. We conclude that DNA hypermethylation seems to be a frequent but not a constant feature associated with AHCY deficiency that affects different genomic regions to different degrees. Thus AHCY deficiency may represent an ideal model disease for studying the molecular origins and biological consequences of DNA hypermethylation due to impaired cellular methylation status.

  8. The role of fatty acid amide hydrolase inhibition in nicotine reward and dependence

    PubMed Central

    Muldoon, Pretal P.; Lichtman, Aron H.; Parsons, Loren H.; Damaj, M. Imad

    2012-01-01

    The endogenous cannabinoid anandamide (AEA) exerts the majority of its effects at CB1 and CB2 receptors and is degraded by fatty acid amide hydrolase (FAAH). FAAH KO mice and animals treated with FAAH inhibitors are impaired in their ability to hydrolyze AEA and other non-cannabinoid lipid signaling molecules, such as oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). AEA and these other substrates activate non- cannabinoid receptor systems, including TRPV1 and PPAR-α receptors. In this mini review, we describe the functional consequences of FAAH inhibition on nicotine reward and dependence as well as the underlying endocannabinoid and non-cannabinoid receptor systems mediating these effects. Interestingly, FAAH inhibition seems to mediate nicotine dependence differently in mice and rats. Indeed, pharmacological and genetic FAAH disruption in mice enhances nicotine reward and withdrawal. However, in rats, pharmacological blockade of FAAH significantly inhibits nicotine reward and has no effect in nicotine withdrawal. Studies suggest that non-cannabinoid mechanisms may play a role in these species differences. PMID:22705310

  9. The Molecular Basis for Dual Fatty Acid Amide Hydrolase (FAAH)/Cyclooxygenase (COX) Inhibition.

    PubMed

    Palermo, Giulia; Favia, Angelo D; Convertino, Marino; De Vivo, Marco

    2016-06-20

    The design of multitarget-directed ligands is a promising strategy for discovering innovative drugs. Here, we report a mechanistic study that clarifies key aspects of the dual inhibition of the fatty acid amide hydrolase (FAAH) and the cyclooxygenase (COX) enzymes by a new multitarget-directed ligand named ARN2508 (2-[3-fluoro-4-[3-(hexylcarbamoyloxy)phenyl]phenyl]propanoic acid). This potent dual inhibitor combines, in a single scaffold, the pharmacophoric elements often needed to block FAAH and COX, that is, a carbamate moiety and the 2-arylpropionic acid functionality, respectively. Molecular modeling and molecular dynamics simulations suggest that ARN2508 uses a noncovalent mechanism of inhibition to block COXs, while inhibiting FAAH via the acetylation of the catalytic Ser241, in line with previous experimental evidence for covalent FAAH inhibition. This study proposes the molecular basis for the dual FAAH/COX inhibition by this novel hybrid scaffold, stimulating further experimental studies and offering new insights for the rational design of novel anti-inflammatory agents that simultaneously act on FAAH and COX.

  10. Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein.

    PubMed Central

    Mulbry, W W; Karns, J S

    1989-01-01

    The sequence of a 1,693-base-pair plasmid DNA fragment from Flavobacterium sp. strain ATCC 27551 containing the parathion hydrolase gene (opd) was determined. Within this sequence, there is only one open reading frame large enough to encode the 35,000-dalton membrane-associated hydrolase protein purified from Flavobacterium extracts. Amino-terminal sequence analysis of the purified Flavobacterium hydrolase demonstrated that serine is the amino-terminal residue of the hydrolase protein. The amino-terminal serine corresponds to a TCG codon located 87 base pairs downstream of the presumptive ATG initiation codon in the nucleotide sequence. The amino acid composition of the purified protein agrees well with that predicted from the nucleotide sequence, using serine as the amino-terminal residue. These data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp. Construction in Escherichia coli of a lacZ-opd gene fusion in which the first 33 amino-terminal residues of opd were replaced by the first 5 residues of lacZ resulted in the production of an active hydrolase identical in molecular mass to the hydrolase isolated from Flavobacterium sp. E. coli cells containing the lacZ-opd fusion showed higher levels of hydrolase activity than did cells containing the parent plasmid. Images PMID:2556372

  11. Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein.

    PubMed

    Mulbry, W W; Karns, J S

    1989-12-01

    The sequence of a 1,693-base-pair plasmid DNA fragment from Flavobacterium sp. strain ATCC 27551 containing the parathion hydrolase gene (opd) was determined. Within this sequence, there is only one open reading frame large enough to encode the 35,000-dalton membrane-associated hydrolase protein purified from Flavobacterium extracts. Amino-terminal sequence analysis of the purified Flavobacterium hydrolase demonstrated that serine is the amino-terminal residue of the hydrolase protein. The amino-terminal serine corresponds to a TCG codon located 87 base pairs downstream of the presumptive ATG initiation codon in the nucleotide sequence. The amino acid composition of the purified protein agrees well with that predicted from the nucleotide sequence, using serine as the amino-terminal residue. These data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp. Construction in Escherichia coli of a lacZ-opd gene fusion in which the first 33 amino-terminal residues of opd were replaced by the first 5 residues of lacZ resulted in the production of an active hydrolase identical in molecular mass to the hydrolase isolated from Flavobacterium sp. E. coli cells containing the lacZ-opd fusion showed higher levels of hydrolase activity than did cells containing the parent plasmid.

  12. Annotation and comparative analysis of the glycoside hydrolase genes in Brachypodium distachyon

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glycoside hydrolase genes have been previously cataloged for Oryza sativa (rice), the model dicotyledonous plant Arabidopsis thaliana, and the fast-growing tree Populus trichocarpa (poplar). To improve our understanding of glycoside hydrolases in plants generally and in grasses specifically, we ann...

  13. Hydration of vinyl ether groups by unsaturated glycoside hydrolases and their role in bacterial pathogenesis.

    PubMed

    Hashimoto, Wataru; Itoh, Takafumi; Maruyama, Yukie; Mikami, Bunzo; Murata, Kousaku

    2007-12-01

    Many pathogenic microorganisms invade mammalian and/or plant cells by producing polysaccharide-degrading enzymes (lyases and hydrolases). Mammalian glycosaminoglycans and plant pectins that form part of the cell surface matrix are typical targets for these microbial enzymes. Unsaturated glycoside hydrolase catalyzes the hydrolytic release of an unsaturated uronic acid from oligosaccharides, which are produced through the reaction of matrix-degrading polysaccharide lyase. This enzymatic ability suggests that unsaturated glycoside hydrolases function as virulence factors in microbial infection. This review focuses on the molecular identification, bacterial distribution, and structure/function relationships of these enzymes. In contrast to general glycoside hydrolases, in which the catalytic mechanism involves the retention or inversion of an anomeric configuration, unsaturated glycoside hydrolases uniquely trigger the hydrolysis of vinyl ether groups in unsaturated saccharides but not of their glycosidic bonds.

  14. Gene deficiency and pharmacological inhibition of soluble epoxide hydrolase confers resilience to repeated social defeat stress

    PubMed Central

    Ren, Qian; Ma, Min; Ishima, Tamaki; Morisseau, Christophe; Yang, Jun; Wagner, Karen M.; Zhang, Ji-chun; Yang, Chun; Yao, Wei; Dong, Chao; Han, Mei; Hammock, Bruce D.; Hashimoto, Kenji

    2016-01-01

    Depression is a severe and chronic psychiatric disease, affecting 350 million subjects worldwide. Although multiple antidepressants have been used in the treatment of depressive symptoms, their beneficial effects are limited. The soluble epoxide hydrolase (sEH) plays a key role in the inflammation that is involved in depression. Thus, we examined here the role of sEH in depression. In both inflammation and social defeat stress models of depression, a potent sEH inhibitor, TPPU, displayed rapid antidepressant effects. Expression of sEH protein in the brain from chronically stressed (susceptible) mice was higher than of control mice. Furthermore, expression of sEH protein in postmortem brain samples of patients with psychiatric diseases, including depression, bipolar disorder, and schizophrenia, was higher than controls. This finding suggests that increased sEH levels might be involved in the pathogenesis of certain psychiatric diseases. In support of this hypothesis, pretreatment with TPPU prevented the onset of depression-like behaviors after inflammation or repeated social defeat stress. Moreover, sEH KO mice did not show depression-like behavior after repeated social defeat stress, suggesting stress resilience. The sEH KO mice showed increased brain-derived neurotrophic factor (BDNF) and phosphorylation of its receptor TrkB in the prefrontal cortex, hippocampus, but not nucleus accumbens, suggesting that increased BDNF-TrkB signaling in the prefrontal cortex and hippocampus confer stress resilience. All of these findings suggest that sEH plays a key role in the pathophysiology of depression, and that epoxy fatty acids, their mimics, as well as sEH inhibitors could be potential therapeutic or prophylactic drugs for depression. PMID:26976569

  15. Inhibition of soluble epoxide hydrolase in mice promotes reverse cholesterol transport and regression of atherosclerosis.

    PubMed

    Shen, Li; Peng, Hongchun; Peng, Ran; Fan, Qingsong; Zhao, Shuiping; Xu, Danyan; Morisseau, Christophe; Chiamvimonvat, Nipavan; Hammock, Bruce D

    2015-04-01

    Adipose tissue is the body largest free cholesterol reservoir and abundantly expresses ATP binding cassette transporter A1 (ABCA1), which maintains plasma high-density lipoprotein (HDL) levels. HDLs have a protective role in atherosclerosis by mediating reverse cholesterol transport (RCT). Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose inhibition has various beneficial effects on cardiovascular disease. The sEH is highly expressed in adipocytes, and it converts epoxyeicosatrienoic acids (EETs) into less bioactive dihydroxyeicosatrienoic acids. We previously showed that increasing EETs levels with a sEH inhibitor (sEHI) (t-AUCB) resulted in elevated ABCA1 expression and promoted ABCA1-mediated cholesterol efflux from 3T3-L1 adipocytes. The present study investigates the impacts of t-AUCB in mice deficient for the low density lipoprotein (LDL) receptor (Ldlr(-/-) mice) with established atherosclerotic plaques. The sEH inhibitor delivered in vivo for 4 weeks decreased the activity of sEH in adipose tissue, enhanced ABCA1 expression and cholesterol efflux from adipose depots, and consequently increased HDL levels. Furthermore, t-AUCB enhanced RCT to the plasma, liver, bile and feces. It also showed the reduction of plasma LDL-C levels. Consistently, t-AUCB-treated mice showed reductions in the size of atherosclerotic plaques. These studies establish that raising adipose ABCA1 expression, cholesterol efflux, and plasma HDL levels with t-AUCB treatment promotes RCT, decreasing LDL-C and atherosclerosis regression, suggesting that sEH inhibition may be a promising strategy to treat atherosclerotic vascular disease.

  16. Metabolic products of soluble epoxide hydrolase are essential for monocyte chemotaxis to MCP-1 in vitro and in vivo.

    PubMed

    Kundu, Suman; Roome, Talat; Bhattacharjee, Ashish; Carnevale, Kevin A; Yakubenko, Valentin P; Zhang, Renliang; Hwang, Sung Hee; Hammock, Bruce D; Cathcart, Martha K

    2013-02-01

    Monocyte chemoattractant protein-1 (MCP-1)-induced monocyte chemotaxis is a major event in inflammatory disease. Our prior studies have demonstrated that MCP-1-dependent chemotaxis requires release of arachidonic acid (AA) by activated cytosolic phospholipase A(2) (cPLA(2)). Here we investigated the involvement of AA metabolites in chemotaxis. Neither cyclooxygenase nor lipoxygenase pathways were required, whereas pharmacologic inhibitors of both the cytochrome-P450 (CYP) and the soluble epoxide hydrolase (sEH) pathways blocked monocyte chemotaxis to MCP-1. To verify specificity, we demonstrated that the CYP and sEH products epoxyeiscosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs), respectively, restored chemotaxis in the presence of the inhibitors, indicating that sEH-derived products are essential for MCP-1-driven chemotaxis. Importantly, DHETs also rescued chemotaxis in cPLA(2)-deficient monocytes and monocytes with blocked Erk1/2 activity, because Erk controls cPLA(2) activation. The in vitro findings regarding the involvement of CYP/sEH pathways were further validated in vivo using two complementary approaches measuring MCP-1-dependent chemotaxis in mice. These observations reveal the importance of sEH in MCP-1-regulated monocyte chemotaxis and may explain the observed therapeutic value of sEH inhibitors in treatment of inflammatory diseases, cardiovascular diseases, pain, and even carcinogenesis. Their effectiveness, often attributed to increasing EET levels, is probably influenced by the impairment of DHET formation and inhibition of chemotaxis.

  17. Inhibition of fatty acid amide hydrolase (FAAH) reduces spinal nociceptive responses and expression of spinal long-term potentiation (LTP).

    PubMed

    Eriksen, Guro S; Jacobsen, Line Melå; Mahmood, Aqsa; Pedersen, Linda M; Gjerstad, Johannes

    2012-02-10

    Fatty acid amide hydrolase (FAAH) is an enzyme that metabolizes endocannabinoids and fatty acid amides possibly linked to activation of the opioid system. To examine how this enzyme affects spinal signalling, electrophysiological recordings in the dorsal horn and qPCR on dorsal horn tissue following systemic administration of the FAAH inhibitor URB597 (0.3 and 1.0mg/kg i.v.) and spinal administration of the opioid receptor antagonist naloxone (0.1 μg/μl i.th.), were performed. The present data showed that the suppressive effect of the FAAH inhibitor URB597 (1.0mg/kg i.v.) on the spinal nociceptive responses was prevented by spinal administration of the opioid receptor antagonist naloxone (0.1 μg/μl i.th.). Moreover, the present findings demonstrated that the FAAH inhibitor URB597 (1.0mg/kg i.v.) partly reversed expression of spinal long-term potentiation (LTP) and also attenuated the LTP-associated increased Zif expression. We conclude that pharmacological inactivation of FAAH may be a promising strategy to inhibit the development of central hyperalgesia; thereby reinforcing the role of FAAH as a potential therapeutic target.

  18. Development of surface plasmon resonance imaging biosensors for detection of ubiquitin carboxyl-terminal hydrolase L1.

    PubMed

    Sankiewicz, Anna; Laudanski, Piotr; Romanowicz, Lech; Hermanowicz, Adam; Roszkowska-Jakimiec, Wiesława; Debek, Wojciech; Gorodkiewicz, Ewa

    2015-01-15

    We have developed a new method for highly selective determination of the ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) concentration using a surface plasmon resonance imaging (SPRI) technique and two different biosensors. UCH-L1 was captured from a solution by immobilized specific rabbit monoclonal antibody or specific LDN-57444 inhibitor due to formation of receptor-UCH-L1 complex on the biosensor surface. The analytically useful dynamic response range of both biosensors is between 0.1 and 2.5ng/ml. The detection limit is 0.06ng/ml for the biosensor with antibody and 0.08ng/ml for the biosensor with inhibitor. Biosensors based on both antibody and inhibitor were found to be suitable for quantitative determination of the UCH-L1 and exhibit good tolerance to the potential interferents. Both biosensors gave comparable results in the range of 0 to 0.20ng/ml for plasma samples and 0.30 to 0.49ng/ml for cerebrospinal fluid samples. To validate the new methods, comparative determination of UCH-L1 by the commercial enzyme-linked immunosorbent assay (ELISA) kit was performed. In general, in terms of UCH-L1 concentration, a good correlation between SPRI and ELISA was found. The developed biosensors can be used successfully for the determination of UCH-L1 in body fluids.

  19. Marine extremophiles: a source of hydrolases for biotechnological applications.

    PubMed

    Dalmaso, Gabriel Zamith Leal; Ferreira, Davis; Vermelho, Alane Beatriz

    2015-04-03

    The marine environment covers almost three quarters of the planet and is where evolution took its first steps. Extremophile microorganisms are found in several extreme marine environments, such as hydrothermal vents, hot springs, salty lakes and deep-sea floors. The ability of these microorganisms to support extremes of temperature, salinity and pressure demonstrates their great potential for biotechnological processes. Hydrolases including amylases, cellulases, peptidases and lipases from hyperthermophiles, psychrophiles, halophiles and piezophiles have been investigated for these reasons. Extremozymes are adapted to work in harsh physical-chemical conditions and their use in various industrial applications such as the biofuel, pharmaceutical, fine chemicals and food industries has increased. The understanding of the specific factors that confer the ability to withstand extreme habitats on such enzymes has become a priority for their biotechnological use. The most studied marine extremophiles are prokaryotes and in this review, we present the most studied archaea and bacteria extremophiles and their hydrolases, and discuss their use for industrial applications.

  20. Marine Extremophiles: A Source of Hydrolases for Biotechnological Applications

    PubMed Central

    Dalmaso, Gabriel Zamith Leal; Ferreira, Davis; Vermelho, Alane Beatriz

    2015-01-01

    The marine environment covers almost three quarters of the planet and is where evolution took its first steps. Extremophile microorganisms are found in several extreme marine environments, such as hydrothermal vents, hot springs, salty lakes and deep-sea floors. The ability of these microorganisms to support extremes of temperature, salinity and pressure demonstrates their great potential for biotechnological processes. Hydrolases including amylases, cellulases, peptidases and lipases from hyperthermophiles, psychrophiles, halophiles and piezophiles have been investigated for these reasons. Extremozymes are adapted to work in harsh physical-chemical conditions and their use in various industrial applications such as the biofuel, pharmaceutical, fine chemicals and food industries has increased. The understanding of the specific factors that confer the ability to withstand extreme habitats on such enzymes has become a priority for their biotechnological use. The most studied marine extremophiles are prokaryotes and in this review, we present the most studied archaea and bacteria extremophiles and their hydrolases, and discuss their use for industrial applications. PMID:25854643

  1. Acetylcarnitine hydrolase activity in bovine caudal epididymal spermatozoa

    SciTech Connect

    Bruns, K.; Foster, R.A.; Casillas, E.R.

    1986-05-01

    Recently, the authors identified mM concentrations of acetylcarnitine in epidiymal fluids and have investigated the metabolism of acetylcarnitine by bovine and hamster caudal epididymal spermatozoa. (1-/sup 14/C)acetyl-L-carnitine is oxidized to /sup 14/CO/sub 2/ by washed, intact hamster and bovine sperm at maximal rates of 8.4 and 15.2 nmol/hr/10/sup 7/ cells respectively. Conversely, the carnitine moiety of acetyl-L-(/sup 3/H-methyl)carnitine is not accumulated by sperm under similar conditions. Hydrolysis of (/sup 3/H)acetyl-L-carnitine and competition of uptake of (/sup 3/H)acetate by unlabeled acetate was demonstrated in incubations of intact cells of both species. The amount of (/sup 3/H)acetate accumulated in the incubation medium is time-dependent and also depends on the concentration of unlabeled acetate. A partial solubilization of acetylcarnitine hydrolase activity from washed, intact bovine caudal epididymal spermatozoa in buffer or 0.01% Triton X-100 is observed. There is an enrichment of acetylcarnitine hydrolase activity in purified plasma membranes from bovine caudal epididymal spermatozoa when compared to the activity present in broken cell preparations or other cellular fractions. The results suggest that acetylcarnitine is a substrate for spermatozoa as they traverse the epididymis.

  2. Recovering glycoside hydrolase genes from active tundra cellulolytic bacteria.

    PubMed

    Pinnell, Lee J; Dunford, Eric; Ronan, Patrick; Hausner, Martina; Neufeld, Josh D

    2014-07-01

    Bacteria responsible for cellulose hydrolysis in situ are poorly understood, largely because of the relatively recent development of cultivation-independent methods for their detection and characterization. This study combined DNA stable-isotope probing (DNA-SIP) and metagenomics for identifying active bacterial communities that assimilated carbon from glucose and cellulose in Arctic tundra microcosms. Following DNA-SIP, bacterial fingerprint analysis of gradient fractions confirmed isotopic enrichment. Sequenced fingerprint bands and clone library analysis of 16S rRNA genes identified active bacterial taxa associated with cellulose-associated labelled DNA, including Bacteroidetes (Sphingobacteriales), Betaproteobacteria (Burkholderiales), Alphaproteobacteria (Caulobacteraceae), and Chloroflexi (Anaerolineaceae). We also compared glycoside hydrolase metagenomic profiles from bulk soil and heavy DNA recovered from DNA-SIP incubations. Active populations consuming [(13)C]glucose and [(13)C]cellulose were distinct, based on ordinations of light and heavy DNA. Metagenomic analysis demonstrated a ∼3-fold increase in the relative abundance of glycoside hydrolases in DNA-SIP libraries over bulk-soil libraries. The data also indicate that multiple displacement amplification introduced bias into the resulting metagenomic analysis. This research identified DNA-SIP incubation conditions for glucose and cellulose that were suitable for Arctic tundra soil and confirmed that DNA-SIP enrichment can increase target gene frequencies in metagenomic libraries.

  3. Mechanistic Investigations of Unsaturated Glucuronyl Hydrolase from Clostridium perfringens*

    PubMed Central

    Jongkees, Seino A. K.; Yoo, Hayoung; Withers, Stephen G.

    2014-01-01

    Experiments were carried out to probe the details of the hydration-initiated hydrolysis catalyzed by the Clostridium perfringens unsaturated glucuronyl hydrolase of glycoside hydrolase family 88 in the CAZy classification system. Direct 1H NMR monitoring of the enzymatic reaction detected no accumulated reaction intermediates in solution, suggesting that rearrangement of the initial hydration product occurs on-enzyme. An attempt at mechanism-based trapping of on-enzyme intermediates using a 1,1-difluoro-substrate was unsuccessful because the probe was too deactivated to be turned over by the enzyme. Kinetic isotope effects arising from deuterium-for-hydrogen substitution at carbons 1 and 4 provide evidence for separate first-irreversible and overall rate-determining steps in the hydration reaction, with two potential mechanisms proposed to explain these results. Based on the positioning of catalytic residues in the enzyme active site, the lack of efficient turnover of a 2-deoxy-2-fluoro-substrate, and several unsuccessful attempts at confirmation of a simpler mechanism involving a covalent glycosyl-enzyme intermediate, the most plausible mechanism is one involving an intermediate bearing an epoxide on carbons 1 and 2. PMID:24573682

  4. Structure of unsaturated rhamnogalacturonyl hydrolase complexed with substrate

    SciTech Connect

    Itoh, Takafumi; Ochiai, Akihito; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku . E-mail: kmurata@kais.kyoto-u.ac.jp

    2006-09-08

    Bacillus subtilis strain 168 YteR has been identified as a novel enzyme 'unsaturated rhamnogalacturonyl hydrolase' classified in glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) produced from plant cell wall RG type-I treated with RG lyases, releasing unsaturated galacturonic acid ({delta}GalA) from the substrate. The most likely candidate catalytic residue is Asp-143. Here, we show the structure of D143N in complex with unsaturated RG disaccharide (substrate) determined at 1.9 A resolution by X-ray crystallography. This structural feature directly contributes to the postulation of the enzyme reaction mechanism. YteR triggers the hydration of vinyl ether group in {delta}GalA, but not of glycoside bond, by using Asp-143 as a general acid and base catalyst. Asp-143 donates proton to the double bond of {delta}GalA as an acid catalyst and also deprotonates a water molecule as a base catalyst. Deprotonated water molecule attacks the C5 atom of {delta}GalA.

  5. POTENT UREA AND CARBAMATE INHIBITORS OF SOLUBLE EPOXIDE HYDROLASES. (R825433)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  6. Sequence of the bphD gene encoding 2-hydroxy-6-oxo-(phenyl/chlorophenyl)hexa-2,4-dienoic acid (HOP/cPDA) hydrolase involved in the biphenyl/polychlorinated biphenyl degradation pathway in Comamonas testosteroni: evidence suggesting involvement of Ser112 in catalytic activity.

    PubMed

    Ahmad, D; Fraser, J; Sylvestre, M; Larose, A; Khan, A; Bergeron, J; Juteau, J M; Sondossi, M

    1995-04-14

    The nucleotide sequence of bphD, encoding 2-hydroxy-6-oxo-(phenyl/chlorophenyl)hexa-2,4-dienoic acid hydrolase involved in the biphenyl/polychlorinated biphenyl degradation pathway of Comamonas testosteroni strain B-356, was determined. Comparison of the deduced amino-acid sequence with published sequences led to the identification of a 'lipase box', containing a consensus pentapeptide sequence GlyXaaSerXaaGly. This suggested that the mechanism of action of this enzyme may involve an Asp-Ser-His catalytic triad similar to that of classical lipases and serine hydrolases. Further biochemical and genetic evidence for the active-site involvement of Ser112 was obtained by showing that a semipurified enzyme was inhibited by PMSF, a classic inhibitor of serine hydrolases, and by site-directed Ser112-->Ala mutagenesis.

  7. A Proton Wire and Water Channel Revealed in the Crystal Structure of Isatin Hydrolase

    PubMed Central

    Bjerregaard-Andersen, Kaare; Sommer, Theis; Jensen, Jan K.; Jochimsen, Bjarne; Etzerodt, Michael; Morth, J. Preben

    2014-01-01

    The high resolution crystal structures of isatin hydrolase from Labrenzia aggregata in the apo and the product state are described. These are the first structures of a functionally characterized metal-dependent hydrolase of this fold. Isatin hydrolase converts isatin to isatinate and belongs to a novel family of metalloenzymes that include the bacterial kynurenine formamidase. The product state, mimicked by bound thioisatinate, reveals a water molecule that bridges the thioisatinate to a proton wire in an adjacent water channel and thus allows the proton released by the reaction to escape only when the product is formed. The functional proton wire present in isatin hydrolase isoform b represents a unique catalytic feature common to all hydrolases is here trapped and visualized for the first time. The local molecular environment required to coordinate thioisatinate allows stronger and more confident identification of orthologous genes encoding isatin hydrolases within the prokaryotic kingdom. The isatin hydrolase orthologues found in human gut bacteria raise the question as to whether the indole-3-acetic acid degradation pathway is present in human gut flora. PMID:24917679

  8. Identification and characterization of a new epoxide hydrolase from mouse liver microsomes.

    PubMed

    Guenthner, T M; Oesch, F

    1983-12-25

    A new microsomal epoxide hydrolase (mEH2) has been identified and characterized. This enzyme has properties which distinguish it from previously described cytosolic (cEH) or membrane-bound (mEH1) epoxide hydrolases. The enzyme is an integral microsomal protein which is not dissociated from the membrane by repeated washing, high ionic strength salt, or chaotropic agent solutions, or by sonication. It is very different from the normally described microsomal epoxide hydrolase (mEH1) as shown by its different substrate specificity and kinetic properties and by immunological criteria. In contrast to the hitherto described microsomal epoxide hydrolase, mEH1, the new enzyme effectively catalyzes the hydration of transdisubstituted oxiranes such as trans-stilbene oxide and trans-beta-ethyl styrene oxide and has no appreciable activity toward benzo(a)pyrene 4,5-oxide. It is also structurally distinct, in that it does not cross-react with antibodies raised against the normally described microsomal epoxide hydrolase mEH1. This newly described microsomal epoxide hydrolase probably represents an important factor in the control of reactive epoxides; its location in the membrane ensures access to lipophilic epoxides generated by membrane-bound monooxygenases, and its substrate specificity is such that it can hydrolyze epoxides poorly metabolized by the previously described microsomal epoxide hydrolase.

  9. Refined homology model of monoacylglycerol lipase: toward a selective inhibitor

    NASA Astrophysics Data System (ADS)

    Bowman, Anna L.; Makriyannis, Alexandros

    2009-11-01

    Monoacylglycerol lipase (MGL) is primarily responsible for the hydrolysis of 2-arachidonoylglycerol (2-AG), an endocannabinoid with full agonist activity at both cannabinoid receptors. Increased tissue 2-AG levels consequent to MGL inhibition are considered therapeutic against pain, inflammation, and neurodegenerative disorders. However, the lack of MGL structural information has hindered the development of MGL-selective inhibitors. Here, we detail a fully refined homology model of MGL which preferentially identifies MGL inhibitors over druglike noninhibitors. We include for the first time insight into the active-site geometry and potential hydrogen-bonding interactions along with molecular dynamics simulations describing the opening and closing of the MGL helical-domain lid. Docked poses of both the natural substrate and known inhibitors are detailed. A comparison of the MGL active-site to that of the other principal endocannabinoid metabolizing enzyme, fatty acid amide hydrolase, demonstrates key differences which provide crucial insight toward the design of selective MGL inhibitors as potential drugs.

  10. Inhibition of fatty acid amide hydrolase (FAAH) as a novel therapeutic strategy in the treatment of pain and inflammatory diseases in the gastrointestinal tract.

    PubMed

    Sałaga, Maciej; Sobczak, Marta; Fichna, Jakub

    2014-02-14

    Fatty acid amide hydrolase (FAAH) is the enzyme crucially involved in the modulation of physiological processes mediated by anandamide (AEA), as well as other endocannabinoids and non-cannabinoid biolipids in the gastrointestinal (GI) tract. FAAH also plays a major role in the etiology and the course of GI diseases and the inhibition of the enzyme has recently become a potential target for their therapy. In this review we look at the pharmacology of FAAH and possible clinical application of FAAH inhibitors in the treatment of GI disorders. In particular, we focus on inflammatory bowel diseases (IBD) and irritable bowel syndrome (IBS), whose symptoms include abdominal pain and motility disturbances. We also discuss why the inhibitor-based drugs may replace in future conventional therapies for IBD and IBS.

  11. Soluble epoxide hydrolase: Gene structure, expression and deletion

    PubMed Central

    Harris, Todd R.; Hammock, Bruce D.

    2013-01-01

    Mammalian soluble epoxide hydrolase (sEH) converts epoxides to their corresponding diols through the addition of a water molecule. sEH readily hydrolyzes lipid signaling molecules, including the epoxyeicosatrienoic acids (EETs), epoxidized lipids produced from arachidonic acid by the action of cytochrome p450s. Through its metabolism of the EETs and other lipid mediators, sEH contributes to the regulation of vascular tone, nociception, angiogenesis and the inflammatory response. Because of its central physiological role in disease states such as cardiac hypertrophy, diabetes, hypertension, and pain sEH is being investigated as a therapeutic target. This review begins with a brief introduction to sEH protein structure and function. sEH evolution and gene structure are then discussed before human small nucleotide polymorphisms and mammalian gene expression are described in the context of several disease models. The review ends with an overview of studies that have employed the sEH knockout mouse model. PMID:23701967

  12. Ubiquitin carboxyl hydrolase L1 significance for human diseases.

    PubMed

    Suong, Dang Ngoc Anh; Thao, Dang Thi Phuong; Masamitsu, Yamaguchi; Thuoc, Tran Linh

    2014-07-01

    Ubiquitin carboxyl hydrolase L1 (UCH-L1) is an abundant multifunctional neuron protein. It plays an important role in maintaining the ubiquitin proteasome system (UPS), vital for recognizing and degrading dysfunctional proteins in organisms. In recent decades, UCH-L1 has been implicated in the pathogenesis of many diseases, including neurodegenerative disorders, cancer and diabetes. However, the mechanisms of UCH-L1 involvement have yet to be revealed in detail. Since UCH-L1 contributes many different functions to cell metabolism, its role and regulation might be more complex than previously thought and it has become a research target in many laboratories. In this review, we summarize recent findings related to the actions of UCH-L1 in several human diseases.

  13. Retinyl ester hydrolases and their roles in vitamin A homeostasis.

    PubMed

    Schreiber, Renate; Taschler, Ulrike; Preiss-Landl, Karina; Wongsiriroj, Nuttaporn; Zimmermann, Robert; Lass, Achim

    2012-01-01

    In mammals, dietary vitamin A intake is essential for the maintenance of adequate retinoid (vitamin A and metabolites) supply of tissues and organs. Retinoids are taken up from animal or plant sources and subsequently stored in form of hydrophobic, biologically inactive retinyl esters (REs). Accessibility of these REs in the intestine, the circulation, and their mobilization from intracellular lipid droplets depends on the hydrolytic action of RE hydrolases (REHs). In particular, the mobilization of hepatic RE stores requires REHs to maintain steady plasma retinol levels thereby assuring constant vitamin A supply in times of food deprivation or inadequate vitamin A intake. In this review, we focus on the roles of extracellular and intracellular REHs in vitamin A metabolism. Furthermore, we will discuss the tissue-specific function of REHs and highlight major gaps in the understanding of RE catabolism. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.

  14. Thermodynamics of Enzyme-Catalyzed Reactions. Part 3. Hydrolases

    NASA Astrophysics Data System (ADS)

    Goldberg, Robert N.; Tewari, Yadu B.

    1994-11-01

    Equilibrium constants and enthalpy changes for reactions catalyzed by the hydrolase class of enzymes have been compiled. For each reaction the following information is given: The reference for the data; the reaction studied; the name of the enzyme used and its Enzyme Commission number; the method of measurement; the conditions of measurement [temperature, pH, ionic strength, and the buffer(s) and cofactor(s) used]; the data and an evaluation of it; and, sometimes, commentary on the data and on any corrections which have been applied to it or any calculations for which the data have been used. The data from 145 references have been examined and evaluated. Chemical Abstract Service registry numbers are given for the substances involved in these various reactions. There is a cross reference between the substances and the Enzyme Commission numbers of the enzymes used to catalyze the reactions in which the substances participate.

  15. Soluble epoxide hydrolase contamination of specific catalase preparations inhibits epoxyeicosatrienoic acid vasodilation of rat renal arterioles

    PubMed Central

    Olson, Lauren; Harder, Adam; Isbell, Marilyn; Imig, John D.; Gutterman, David D.; Falck, J. R.; Campbell, William B.

    2011-01-01

    Cytochrome P-450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H2O2), are important signaling molecules in the kidney. In renal arteries, EETs cause vasodilation whereas H2O2 causes vasoconstriction. To determine the physiological contribution of H2O2, catalase is used to inactivate H2O2. However, the consequence of catalase action on EET vascular activity has not been determined. In rat renal afferent arterioles, 14,15-EET caused concentration-related dilations that were inhibited by Sigma bovine liver (SBL) catalase (1,000 U/ml) but not Calbiochem bovine liver (CBL) catalase (1,000 U/ml). SBL catalase inhibition was reversed by the soluble epoxide hydrolase (sEH) inhibitor tAUCB (1 μM). In 14,15-EET incubations, SBL catalase caused a concentration-related increase in a polar metabolite. Using mass spectrometry, the metabolite was identified as 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), the inactive sEH metabolite. 14,15-EET hydrolysis was not altered by the catalase inhibitor 3-amino-1,2,4-triazole (3-ATZ; 10–50 mM), but was abolished by the sEH inhibitor BIRD-0826 (1–10 μM). SBL catalase EET hydrolysis showed a regioisomer preference with greatest hydrolysis of 14,15-EET followed by 11,12-, 8,9- and 5,6-EET (Vmax = 0.54 ± 0.07, 0.23 ± 0.06, 0.18 ± 0.01 and 0.08 ± 0.02 ng DHET·U catalase−1·min−1, respectively). Of five different catalase preparations assayed, EET hydrolysis was observed with two Sigma liver catalases. These preparations had low specific catalase activity and positive sEH expression. Mass spectrometric analysis of the SBL catalase identified peptide fragments matching bovine sEH. Collectively, these data indicate that catalase does not affect EET-mediated dilation of renal arterioles. However, some commercial catalase preparations are contaminated with sEH, and these contaminated preparations diminish the biological activity of H2O2 and EETs. PMID:21753077

  16. Characterization of intracellular pteroylpolyglutamate hydrolase (PPH) from human intestinal mucosa

    SciTech Connect

    Wang, T.T.Y.; Chandler, C.J.; Halsted, C.H.

    1986-03-01

    There are two forms of pteroylpolyglutamate hydrolase (PPH) in the human intestinal mucosa, one in the brush border membrane and the other intracellular; brush border PPH is an exopeptidase with optimal activity at pH 6.5 and a requirement for zinc. The presence study characterized human intracellular PPH and compared its properties to those of brush border PPH. Intracellular PPH was purified 30-fold. The enzyme had a MW of 75,000 by gel filtration, was optimally active at pH 4.5, and had an isoelectric point at pH 8.0. In contrast to brush border PPH, intracellular PPH was unstable at increasing temperatures, was unaffected by dialysis against chelating agents and showed no requirement for Zn/sup 2 +/. Using PteGlu/sub 2/(/sup 14/C)Glu as substrate, they demonstrated a K/sub m/ of 1.2 ..mu..M and increasing affinity for folates with longer glutamate chains. Intracellular PPH required the complete folic acid (PteGlu) moiety and a ..gamma..-glutamyl linkage for activity. Using ion exchange chromatography and an HPLC method to determine the hydrolytic products of the reaction, they found intracellular PPH could cleave both internal and terminal ..gamma..-glutamyl linkages, with PteGlu as an end product. After subcellular fractionation of the mucosa, PPH was found in the lysosomes. In summary, the distinct characteristics of brush border and intracellular PPH suggest that the two hydrolases serve different roles in folate metabolism.

  17. Oxidative inactivation of an extramitochondrial acetyl-CoA hydrolase by autoxidation of L-ascorbic acid.

    PubMed

    Nakanishi, Y; Isohashi, F; Matsunaga, T; Sakamoto, Y

    1985-10-15

    The activity of acetyl-CoA hydrolase (dimeric form) purified from the supernatant fraction of rat liver was shown to have a half-life (t1/2) of 3 min at 0 degree C, but to stable at 37 degrees C (t1/2 = 34 h) [Isohashi, F., Nakanishi, Y. & Sakamoto, Y. (1983) Biochemistry 22, 584-590]. Incubation of the purified enzyme with L-ascorbic acid (AsA) at 37 degrees C resulted in inactivation of the enzyme (t1/2 = 90 min at 2 mM AsA). The extent of inactivation was greatly enhanced by addition of transition metal ions (Cu2+, Fe2+, and Fe3+). Thiol reducing agents, such as reduced glutathione and DL-dithiothreitol, protected the hydrolase from inactivation by AsA. However, these materials did not restore the catalytic activity of the enzyme inactivated by AsA. When AsA solution containing Cu2+ was preincubated under aerobic conditions at 37 degrees C for various times in the absence of enzyme, and then aliquots were incubated with the enzyme solution for 20 min, remaining activity was found to decrease with increase in the preincubation time, reaching a minimum at 60 min. However, further preincubation reduced the potential for inactivation. Catalase, a hydrogen peroxide (H2O2) scavenger, almost completely prevented inactivation of the enzyme by AsA plus Cu2+. Superoxide dismutase and tiron, which are both superoxide (O2-) scavengers, also prevented inactivation of the enzyme. A high concentration of mannitol, a hydroxyl radical (OH) scavenger, partially protected the enzyme from inactivation. These results suggest that inactivation of the enzyme by AsA in the presence of Cu2+ was due to the effect of active oxygen species (H2O2, O2-, OH) that are known to be autoxidation products of AsA. Valeryl-CoA, a competitive inhibitor of acetyl-CoA hydrolase, greatly protected the enzyme from inactivation by AsA plus Cu2+, but ATP and ADP, which are both effectors of this enzyme, had only slight protective effects. These results suggest that inactivation of this enzyme by addition

  18. Synthesis and evaluation of potential inhibitors of human and Escherichia coli histidine triad nucleotide binding proteins.

    PubMed

    Bardaweel, Sanaa K; Ghosh, Brahma; Wagner, Carston R

    2012-01-01

    Based on recent substrate specificity studies, a series of ribonucleotide based esters and carbamates were synthesized and screened as inhibitors of the phosphoramidases and acyl-AMP hydrolases, Escherichia coli Histidine Triad Nucleotide Binding Protein (ecHinT) and human Histidine Triad Nucleotide Binding Protein 1 (hHint1). Using our established phosphoramidase assay, K(i) values were determined. All compounds exhibited non-competitive inhibition profiles. The carbamate based inhibitors were shown to successfully suppress the Hint1-associated phenotype in E. coli, suggesting that they are permeable intracellular inhibitors of ecHinT.

  19. Structural Analysis of a Family 101 Glycoside Hydrolase in Complex with Carbohydrates Reveals Insights into Its Mechanism*

    PubMed Central

    Gregg, Katie J.; Suits, Michael D. L.; Deng, Lehua; Vocadlo, David J.; Boraston, Alisdair B.

    2015-01-01

    O-Linked glycosylation is one of the most abundant post-translational modifications of proteins. Within the secretory pathway of higher eukaryotes, the core of these glycans is frequently an N-acetylgalactosamine residue that is α-linked to serine or threonine residues. Glycoside hydrolases in family 101 are presently the only known enzymes to be able to hydrolyze this glycosidic linkage. Here we determine the high-resolution structures of the catalytic domain comprising a fragment of GH101 from Streptococcus pneumoniae TIGR4, SpGH101, in the absence of carbohydrate, and in complex with reaction products, inhibitor, and substrate analogues. Upon substrate binding, a tryptophan lid (residues 724-WNW-726) closes on the substrate. The closing of this lid fully engages the substrate in the active site with Asp-764 positioned directly beneath C1 of the sugar residue bound within the −1 subsite, consistent with its proposed role as the catalytic nucleophile. In all of the bound forms of the enzyme, however, the proposed catalytic acid/base residue was found to be too distant from the glycosidic oxygen (>4.3 Å) to serve directly as a general catalytic acid/base residue and thereby facilitate cleavage of the glycosidic bond. These same complexes, however, revealed a structurally conserved water molecule positioned between the catalytic acid/base and the glycosidic oxygen. On the basis of these structural observations we propose a new variation of the retaining glycoside hydrolase mechanism wherein the intervening water molecule enables a Grotthuss proton shuttle between Glu-796 and the glycosidic oxygen, permitting this residue to serve as the general acid/base catalytic residue. PMID:26304114

  20. Structural Analysis of a Family 101 Glycoside Hydrolase in Complex with Carbohydrates Reveals Insights into Its Mechanism.

    PubMed

    Gregg, Katie J; Suits, Michael D L; Deng, Lehua; Vocadlo, David J; Boraston, Alisdair B

    2015-10-16

    O-Linked glycosylation is one of the most abundant post-translational modifications of proteins. Within the secretory pathway of higher eukaryotes, the core of these glycans is frequently an N-acetylgalactosamine residue that is α-linked to serine or threonine residues. Glycoside hydrolases in family 101 are presently the only known enzymes to be able to hydrolyze this glycosidic linkage. Here we determine the high-resolution structures of the catalytic domain comprising a fragment of GH101 from Streptococcus pneumoniae TIGR4, SpGH101, in the absence of carbohydrate, and in complex with reaction products, inhibitor, and substrate analogues. Upon substrate binding, a tryptophan lid (residues 724-WNW-726) closes on the substrate. The closing of this lid fully engages the substrate in the active site with Asp-764 positioned directly beneath C1 of the sugar residue bound within the -1 subsite, consistent with its proposed role as the catalytic nucleophile. In all of the bound forms of the enzyme, however, the proposed catalytic acid/base residue was found to be too distant from the glycosidic oxygen (>4.3 Å) to serve directly as a general catalytic acid/base residue and thereby facilitate cleavage of the glycosidic bond. These same complexes, however, revealed a structurally conserved water molecule positioned between the catalytic acid/base and the glycosidic oxygen. On the basis of these structural observations we propose a new variation of the retaining glycoside hydrolase mechanism wherein the intervening water molecule enables a Grotthuss proton shuttle between Glu-796 and the glycosidic oxygen, permitting this residue to serve as the general acid/base catalytic residue.

  1. Soluble epoxide hydrolase inhibition does not prevent cardiac remodeling and dysfunction after aortic constriction in rats and mice.

    PubMed

    Morgan, Lisa A; Olzinski, Alan R; Upson, John J; Zhao, Shufang; Wang, Tao; Eisennagel, Stephen H; Hoang, Bao; Tunstead, James R; Marino, Joseph P; Willette, Robert N; Jucker, Beat M; Behm, David J

    2013-04-01

    Epoxyeicosatrienoic acids, substrates for soluble epoxide hydrolase (sEH), exhibit vasodilatory and antihypertrophic activities. Inhibitors of sEH might therefore hold promise as heart failure therapeutics. We examined the ability of sEH inhibitors GSK2188931 and GSK2256294 to modulate cardiac hypertrophy, fibrosis, and function after transverse aortic constriction (TAC) in rats and mice. GSK2188931 administration was initiated in rats 1 day before TAC, whereas GSK2256294 treatment was initiated in mice 2 weeks after TAC. Four weeks later, cardiovascular function was assessed, plasma was collected for drug and sEH biomarker concentrations, and left ventricle was isolated for messenger RNA and histological analyses. In rats, although GSK2188931 prevented TAC-mediated increases in certain genes associated with hypertrophy and fibrosis (α-skeletal actin and connective tissue growth factor), the compound failed to attenuate TAC-induced increases in left ventricle mass, posterior wall thickness, end-diastolic volume and pressure, and perivascular fibrosis. Similarly, in mice, GSK2256294 did not reverse cardiac remodeling or systolic dysfunction induced by TAC. Both compounds increased the sEH substrate/product (leukotoxin/leukotoxin diol) ratio, indicating sEH inhibition. In summary, sEH inhibition does not prevent cardiac remodeling or dysfunction after TAC. Thus, targeting sEH seems to be insufficient for reducing pressure overload hypertrophy.

  2. Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis

    PubMed Central

    2016-01-01

    Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb “Hydrolase important for pathogenesis 1” (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections. PMID:27739665

  3. High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC).

    PubMed

    Camilo, Cesar M; Polikarpov, Igor

    2014-07-01

    Recent advances in DNA sequencing techniques have led to an explosion in the amount of available genome sequencing data and this provided an inexhaustible source of uncharacterized glycoside hydrolases (GH) to be studied both structurally and enzymatically. Ligation-Independent Cloning (LIC), an interesting alternative to traditional, restriction enzyme-based cloning, and commercial recombinatorial cloning, was adopted and optimized successfully for a high throughput cloning, expression and purification pipeline. Using this platform, 130 genes encoding mainly uncharacterized glycoside hydrolases from 13 different organisms were cloned and submitted to a semi-automated protein expression and solubility screening in Escherichia coli, resulting in 73 soluble targets. The high throughput approach proved to be a powerful tool for production of recombinant glycoside hydrolases for further structural and biochemical characterization and confirmed that thioredoxin fusion tag (TRX) is a better choice to increase solubility of recombinant glycoside hydrolases expressed in E. coli, when compared to His-tag alone.

  4. DETOXIFICATION OF ORGANOPHOSPHATE PESTICIDES BY IMMOBILIZED ESCHERICHIA COLI EXPRESSING ORGANOPHOSPHORUS HYDROLASE ON CELL SURFACE. (R823663)

    EPA Science Inventory

    An improved whole-cell technology for detoxifying organophosphate nerve agents was recently developed based on genetically engineered Escherichia coli with organophosphorus hydrolase anchored on the surface. This article reports the immobilization of these novel biocatalys...

  5. Pharmacological inhibition of soluble epoxide hydrolase prevents renal interstitial fibrogenesis in obstructive nephropathy

    PubMed Central

    Kim, Jinu; Yoon, Sang Pil; Toews, Myron L.; Imig, John D.; Hwang, Sung Hee; Hammock, Bruce D.

    2014-01-01

    Treating chronic kidney disease (CKD) has been challenging because of its pathogenic complexity. Epoxyeicosatrienoic acids (EETs) are cytochrome P-450-dependent derivatives of arachidonic acid with antihypertensive, anti-inflammatory, and profibrinolytic functions. We recently reported that genetic ablation of soluble epoxide hydrolase (sEH), an enzyme that converts EETs to less active dihydroxyeicosatrienoic acids, prevents renal tubulointerstitial fibrosis and inflammation in experimental mouse models of CKD. Here, we tested the hypothesis that pharmacological inhibition of sEH after unilateral ureteral obstruction (UUO) would attenuate tubulointerstitial fibrosis and inflammation in mouse kidneys and may provide a novel approach to manage the progression of CKD. Inhibition of sEH enhanced levels of EET regioisomers and abolished tubulointerstitial fibrosis, as demonstrated by reduced collagen deposition and myofibroblast formation after UUO. The inflammatory response was also attenuated, as demonstrated by decreased influx of neutrophils and macrophages and decreased expression of inflammatory cytokines keratinocyte chemoattractant, macrophage inflammatory protein-2, monocyte chemotactic protein-1, TNF-α, and ICAM-1 in kidneys after UUO. UUO upregulated transforming growth factor-β1/Smad3 signaling and induced NF-κB activation, oxidative stress, tubular injury, and apoptosis; in contrast, it downregulated antifibrotic factors, including peroxisome proliferator-activated receptor (PPAR) isoforms, especially PPAR-γ. sEH inhibition mitigated the aforementioned malevolent effects in UUO kidneys. These data demonstrate that pharmacological inhibition of sEH promotes anti-inflammatory and fibroprotective effects in UUO kidneys by preventing tubular injury, downregulation of NF-κB, transforming growth factor-β1/Smad3, and inflammatory signaling pathways, and activation of PPAR isoforms. Our data suggest the potential use of sEH inhibitors in treating fibrogenesis

  6. Soluble Epoxide Hydrolase Inhibition and Epoxyeicosatrienoic Acid Treatment Improve Vascularization of Engineered Skin Substitutes

    PubMed Central

    Hahn, Jennifer M.; McFarland, Kevin L.; Combs, Kelly A.; Lee, Kin Sing Stephen; Inceoglu, Bora; Wan, Debin; Boyce, Steven T.; Hammock, Bruce D.

    2016-01-01

    Background: Autologous engineered skin substitutes comprised of keratinocytes, fibroblasts, and biopolymers can serve as an adjunctive treatment for excised burns. However, engineered skin lacks a vascular plexus at the time of grafting, leading to slower vascularization and reduced rates of engraftment compared with autograft. Hypothetically, vascularization of engineered skin grafts can be improved by treatment with proangiogenic agents at the time of grafting. Epoxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid that are inactivated by soluble epoxide hydrolase (sEH). EETs have multiple biological activities and have been shown to promote angiogenesis. Inhibitors of sEH (sEHIs) represent attractive therapeutic agents because they increase endogenous EET levels. We investigated sEHI administration, alone or combined with EET treatment, for improved vascularization of engineered skin after grafting to mice. Methods: Engineered skin substitutes, prepared using primary human fibroblasts and keratinocytes, were grafted to full-thickness surgical wounds in immunodeficient mice. Mice were treated with the sEHI 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), which was administered in drinking water throughout the study period, with or without topical EET treatment, and were compared with vehicle-treated controls. Vascularization was quantified by image analysis of CD31-positive areas in tissue sections. Results: At 2 weeks after grafting, significantly increased vascularization was observed in the TPPU and TPPU + EET groups compared with controls, with no evidence of toxicity. Conclusions: The results suggest that sEH inhibition can increase vascularization of engineered skin grafts after transplantation, which may contribute to enhanced engraftment and improved treatment of full-thickness wounds. PMID:28293507

  7. Peptidase E, a Peptidase Specific for N-Terminal Aspartic Dipeptides, Is a Serine Hydrolase

    PubMed Central

    Lassy, Rachel A. L.; Miller, Charles G.

    2000-01-01

    Salmonella enterica serovar Typhimurium peptidase E (PepE) is an N-terminal Asp-specific dipeptidase. PepE is not inhibited by any of the classical peptidase inhibitors, and its amino acid sequence does not place it in any of the known peptidase structural classes. A comparison of the amino acid sequence of PepE with a number of related sequences has allowed us to define the amino acid residues that are strongly conserved in this family. To ensure the validity of this comparison, we have expressed one of the most distantly related relatives (Xenopus) in Escherichia coli and have shown that it is indeed an Asp-specific dipeptidase with properties very similar to those of serovar Typhimurium PepE. The sequence comparison suggests that PepE is a serine hydrolase. We have used site-directed mutagenesis to change all of the conserved Ser, His, and Asp residues and have found that Ser120, His157, and Asp135 are all required for activity. Conversion of Ser120 to Cys leads to severely reduced (104-fold) but still detectable activity, and this activity but not that of the parent is inhibited by thiol reagents; these results confirm that this residue is likely to be the catalytic nucleophile. These results suggest that PepE is the prototype of a new family of serine peptidases. The phylogenetic distribution of the family is unusual, since representatives are found in eubacteria, an insect (Drosophila), and a vertebrate (Xenopus) but not in the Archaea or in any of the other eukaryotes for which genome sequences are available. PMID:10762256

  8. Oligomeric structure of proclavaminic acid amidino hydrolase: evolution of a hydrolytic enzyme in clavulanic acid biosynthesis.

    PubMed Central

    Elkins, Jonathan M; Clifton, Ian J; Hernández, Helena; Doan, Linh X; Robinson, Carol V; Schofield, Christopher J; Hewitson, Kirsty S

    2002-01-01

    During biosynthesis of the clinically used beta-lactamase inhibitor clavulanic acid, one of the three steps catalysed by clavaminic acid synthase is separated from the other two by a step catalysed by proclavaminic acid amidino hydrolase (PAH), in which the guanidino group of an intermediate is hydrolysed to give proclavaminic acid and urea. PAH shows considerable sequence homology with the primary metabolic arginases, which hydrolyse arginine to ornithine and urea, but does not accept arginine as a substrate. Like other members of the bacterial sub-family of arginases, PAH is hexameric in solution and requires Mn2+ ions for activity. Other metal ions, including Co2+, can substitute for Mn2+. Two new substrates for PAH were identified, N-acetyl-(L)-arginine and (3R)-hydroxy-N-acetyl-(L)-arginine. Crystal structures of PAH from Streptomyces clavuligerus (at 1.75 A and 2.45 A resolution, where 1 A=0.1 nm) imply how it binds beta-lactams rather than the amino acid substrate of the arginases from which it evolved. The structures also suggest how PAH selects for a particular alcohol intermediate in the clavam biosynthesis pathway. As observed for the arginases, each PAH monomer consists of a core of beta-strands surrounded by alpha-helices, and its active site contains a di-Mn2+ centre with a bridging water molecule responsible for hydrolytic attack on to the guanidino group of the substrate. Comparison of structures obtained under different conditions reveals different conformations of a flexible loop, which must move to allow substrate binding. PMID:12020346

  9. Compositional profile of α/β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

    PubMed Central

    Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    2015-01-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; < 5%). Detailed analysis of the genes predicted to encode proteins of the abH08 superfamily revealed a high proportion related to epoxide hydrolases and haloalkane dehalogenases in polluted mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. PMID:25171437

  10. Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites.

    PubMed

    Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    2015-05-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; < 5%). Detailed analysis of the genes predicted to encode proteins of the abH08 superfamily revealed a high proportion related to epoxide hydrolases and haloalkane dehalogenases in polluted mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes.

  11. Human Lung Hydrolases Delineate Mycobacterium tuberculosis–Macrophage Interactions and the Capacity To Control Infection

    PubMed Central

    Arcos, Jesus; Sasindran, Smitha J.; Fujiwara, Nagatoshi; Turner, Joanne; Schlesinger, Larry S.; Torrelles, Jordi B.

    2014-01-01

    Pulmonary surfactant contains homeostatic and antimicrobial hydrolases. When Mycobacterium tuberculosis is initially deposited in the terminal bronchioles and alveoli, as well as following release from lysed macrophages, bacilli are in intimate contact with these lung surfactant hydrolases. We identified and measured several hydrolases in human alveolar lining fluid and lung tissue that, at their physiological concentrations, dramatically modified the M. tuberculosis cell envelope. Independent of their action time (15 min to 12 h), the effects of the hydrolases on the M. tuberculosis cell envelope resulted in a significant decrease (60–80%) in M. tuberculosis association with, and intracellular growth of the bacteria within, human macrophages. The cell envelope-modifying effects of the hydrolases also led to altered M. tuberculosis intracellular trafficking and induced a protective proin-flammatory response to infection. These findings add a new concept to our understanding of M. tuberculosis–macrophage inter-actions (i.e., the impact of lung surfactant hydrolases on M. tuberculosis infection). PMID:21602490

  12. A Phylogenetically Informed Comparison of GH1 Hydrolases between Arabidopsis and Rice Response to Stressors

    PubMed Central

    Cao, Yun-Ying; Yang, Jing-Fang; Liu, Tie-Yuan; Su, Zhen-Feng; Zhu, Fu-Yuan; Chen, Mo-Xian; Fan, Tao; Ye, Neng-Hui; Feng, Zhen; Wang, Ling-Juan; Hao, Ge-Fei; Zhang, Jianhua; Liu, Ying-Gao

    2017-01-01

    Glycoside hydrolases Family 1 (GH1) comprises enzymes that can hydrolyze β-O-glycosidic bond from a carbohydrate moiety. The plant GH1 hydrolases participate in a number of developmental processes and stress responses, including cell wall modification, plant hormone activation or deactivation and herbivore resistance. A large number of members has been observed in this family, suggesting their potential redundant functions in various biological processes. In this study, we have used 304 sequences of plant GH1 hydrolases to study the evolution of this gene family in plant lineage. Gene duplication was found to be a common phenomenon in this gene family. Although many members of GH1 hydrolases showed a high degree of similarity in Arabidopsis and rice, they showed substantial tissue specificity and differential responses to various stress treatments. This differential regulation implies each enzyme may play a distinct role in plants. Furthermore, some of salt-responsive Arabidopsis GH1 hydrolases were selected to test their genetic involvement in salt responses. The knockout mutants of AtBGLU1 and AtBGLU19 were observed to be less-sensitive during NaCl treatment in comparison to the wild type seedlings, indicating their participation in salt stress response. In summary, Arabidopsis and rice GH1 glycoside hydrolases showed distinct features in their evolutionary path, transcriptional regulation and genetic functions. PMID:28392792

  13. Purification and characterization of three parathion hydrolases from gram-negative bacterial strains.

    PubMed

    Mulbry, W W; Karns, J S

    1989-02-01

    Three unique parathion hydrolases were purified from gram-negative bacterial isolates and characterized. All three purified enzymes had roughly comparable affinities for ethyl parathion and had broad temperature optima at ca. 40 degrees C. The membrane-bound hydrolase of Flavobacterium sp. strain ATCC 27551 was composed of a single subunit of approximately 35,000 daltons (Da) and was inhibited by sulfhydryl reagents such as dithiothreitol (DTT) and by metal salts such as CuCl2. The cytosolic hydrolase of strain B-1 was composed of a single subunit of approximately 43,000 Da and was stimulated by DTT and inhibited by CuCl2. The membrane-bound hydrolase of strain SC was composed of four identical subunits of 67,000 Da and was inhibited by DTT and stimulated by CuCl2. The substrate ranges of the three enzymes also differed, as evidenced by their relative affinities for parathion and the related organophosphate insecticide O-ethyl-O-4-nitrophenyl phenylphosphonothioate (EPN). The B-1 hydrolase displayed equal affinity for both compounds, the Flavobacterium enzyme showed twofold-lower affinity for EPN than for parathion, and the SC hydrolase displayed no activity toward EPN. The range in characteristics of these three enzymes can be exploited in different waste disposal strategies.

  14. Purification and characterization of three parathion hydrolases from gram-negative bacterial strains.

    PubMed Central

    Mulbry, W W; Karns, J S

    1989-01-01

    Three unique parathion hydrolases were purified from gram-negative bacterial isolates and characterized. All three purified enzymes had roughly comparable affinities for ethyl parathion and had broad temperature optima at ca. 40 degrees C. The membrane-bound hydrolase of Flavobacterium sp. strain ATCC 27551 was composed of a single subunit of approximately 35,000 daltons (Da) and was inhibited by sulfhydryl reagents such as dithiothreitol (DTT) and by metal salts such as CuCl2. The cytosolic hydrolase of strain B-1 was composed of a single subunit of approximately 43,000 Da and was stimulated by DTT and inhibited by CuCl2. The membrane-bound hydrolase of strain SC was composed of four identical subunits of 67,000 Da and was inhibited by DTT and stimulated by CuCl2. The substrate ranges of the three enzymes also differed, as evidenced by their relative affinities for parathion and the related organophosphate insecticide O-ethyl-O-4-nitrophenyl phenylphosphonothioate (EPN). The B-1 hydrolase displayed equal affinity for both compounds, the Flavobacterium enzyme showed twofold-lower affinity for EPN than for parathion, and the SC hydrolase displayed no activity toward EPN. The range in characteristics of these three enzymes can be exploited in different waste disposal strategies. Images PMID:2541658

  15. A New Family of Biuret Hydrolases Involved in S-Triazine Ring Metabolism

    PubMed Central

    Cameron, Stephan M.; Durchschein, Katharina; Richman, Jack E.; Sadowsky, Michael J.; Wackett, Lawrence P.

    2011-01-01

    Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain 3841 encoding biuret hydrolase was synthesized, transformed into Escherichia coli, and expressed. The enzyme was purified and found to be stable. Biuret hydrolase catalyzed the hydrolysis of biuret to allophanate and ammonia. The kcat/KM of 1.7 × 105 M−1s−1 and the relatively low KM of 23 ± 4 μM together suggested that this enzyme acts uniquely on biuret physiologically. This is supported by the fact that of the 34 substrate analogs of biuret tested, only two demonstrated reactivity, both at less than 5% of the rate determined for biuret. Biuret hydrolase does not react with carboxybiuret, the product of the enzyme immediately preceding biuret hydrolase in the metabolic pathway for cyanuric acid. This suggests an unusual metabolic strategy of an enzymatically-produced intermediate undergoing non-enzymatic decarboxylation to produce the substrate for the next enzyme in the pathway. PMID:21897878

  16. Expression of Nudix hydrolase genes in barley under UV irradiation

    NASA Astrophysics Data System (ADS)

    Tanaka, Sayuri; Sugimoto, Manabu; Kihara, Makoto

    Seed storage and cultivation should be necessary to self-supply foods when astronauts would stay and investigate during long-term space travel and habitation in the bases on the Moon and Mars. Thought the sunlight is the most importance to plants, both as the ultimate energy source and as an environmental signal regulating growth and development, UV presenting the sunlight can damage many aspects of plant processes at the physiological and DNA level. Especially UV-C, which is eliminated by the stratospheric ozone layer, is suspected to be extremely harmful and give a deadly injury to plants in space. However, the defense mechanism against UV-C irradiation damage in plant cells has not been clear. In this study, we investigated the expression of Nudix hydrolases, which defense plants from biotic / abiotic stress, in barley under UV irradiation. The genes encoding the amino acid sequences, which show homology to those of 28 kinds of Nudix hydrolases in Arabidopsis thaliana, were identified in the barley full-length cDNA library. BLAST analysis showed 14 kinds of barley genes (HvNUDX1-14), which encode the Nudix motif sequence. A phylogenetic tree showed that HvNUDX1, HvNUDX7, HvNUDX9 and HvNUDX11 belonged to the ADP-ribose pyrophosphohydrolase, ADP-sugar pyrophosphohydrolase, NAD(P)H pyrophosphohydrolase and FAD pyrophosphohydrolase subfamilies, respectively, HvNUDX3, HvNUDX6, and HvNUDX8 belonged to the Ap _{n}A pyrophosphohydrolase subfamilies, HvNUDX5 and HvNUDX14 belonged to the coenzyme A pyrophosphohydrolase subfamilies, HvNUDX12 and HvNUDX13 belonged to the Ap _{4}A pyrophosphohydrolase subfamilies. Induction of HvNUDX genes by UV-A (340nm), UV-B (312nm), and UV-C (260nm) were analyzed by quantitative RT-PCR. The results showed that HvNUDX4 was induced by UV-A and UV-B, HvNUDX6 was induced by UV-B and UV-C, and HvNUDX7 and HvNUDX14 were induced by UV-C, significantly. Our results suggest that the response of HvNUDXs to UV irradiation is different by UV

  17. Piperidinyl thiazole isoxazolines: A new series of highly potent, slowly reversible FAAH inhibitors with analgesic properties.

    PubMed

    Pember, Stephen O; Mejia, Galo L; Price, Theodore J; Pasteris, Robert J

    2016-06-15

    Fatty acid amide hydrolase (FAAH) is a membrane anchored serine hydrolase that has a principle role in the metabolism of the endogenous cannabinoid anandamide. Docking studies using representative FAAH crystal structures revealed that compounds containing a novel piperidinyl thiazole isoxazoline core fit within the ligand binding domains. New potential FAAH inhibitors were designed and synthesized incorporating urea, carbamate, alkyldione and thiourea reactive centers as potential pharmacophores. A small library of candidate compounds (75) was then screened against human FAAH leading to the identification of new carbamate and urea based inhibitors (Ki=pM and nM, respectively). Representative carbamate and urea based chemotypes displayed slow, time dependent inhibition kinetics leading to enzyme inactivation which was slowly reversible. However, evidence indicated that features of the mechanism of inactivation differ between the two pharmacophore types. Selected compounds were also evaluated for analgesic activity in the mouse-tail flick test.

  18. Identification and characterization of a bile salt hydrolase from Lactobacillus salivarius for development of novel alternatives to antibiotic growth promoters.

    PubMed

    Wang, Zhong; Zeng, Ximin; Mo, Yiming; Smith, Katie; Guo, Yuming; Lin, Jun

    2012-12-01

    Antibiotic growth promoters (AGPs) have been used as feed additives to improve average body weight gain and feed efficiency in food animals for more than 5 decades. However, there is a worldwide trend to limit AGP use to protect food safety and public health, which raises an urgent need to discover effective alternatives to AGPs. The growth-promoting effect of AGPs has been shown to be highly correlated with the decreased activity of intestinal bile salt hydrolase (BSH), an enzyme that is produced by various gut microflora and involved in host lipid metabolism. Thus, BSH inhibitors are likely promising feed additives to AGPs to improve animal growth performance. In this study, the genome of Lactobacillus salivarius NRRL B-30514, a BSH-producing strain isolated from chicken, was sequenced by a 454 GS FLX sequencer. A BSH gene identified by genome analysis was cloned and expressed in an Escherichia coli expression system for enzymatic analyses. The BSH displayed efficient hydrolysis activity for both glycoconjugated and tauroconjugated bile salts, with slightly higher catalytic efficiencies (k(cat)/K(m)) on glycoconjugated bile salts. The optimal pH and temperature for the BSH activity were 5.5 and 41°C, respectively. Examination of a panel of dietary compounds using the purified BSH identified some potent BSH inhibitors, in which copper and zinc have been recently demonstrated to promote feed digestion and body weight gain in different food animals. In sum, this study identified and characterized a BSH with broad substrate specificity from a chicken L. salivarius strain and established a solid platform for us to discover novel BSH inhibitors, the promising feed additives to replace AGPs for enhancing the productivity and sustainability of food animals.

  19. A Personal Retrospective: Elevating Anandamide (AEA) by Targeting Fatty Acid Amide Hydrolase (FAAH) and the Fatty Acid Binding Proteins (FABPs)

    PubMed Central

    Deutsch, Dale G.

    2016-01-01

    This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that “solubilize” anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions. At the International Cannabis Research Society Symposium in 1992, Raphe Mechoulam revealed that his laboratory isolated an endogenous lipid molecule that binds to the CB1 receptor (cannabinoid receptor type 1) and this became the milestone paper published in December of that year describing anandamide (AEA, Devane et al., 1992

  20. A Personal Retrospective: Elevating Anandamide (AEA) by Targeting Fatty Acid Amide Hydrolase (FAAH) and the Fatty Acid Binding Proteins (FABPs).

    PubMed

    Deutsch, Dale G

    2016-01-01

    This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that "solubilize" anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions. At the International Cannabis Research Society Symposium in 1992, Raphe Mechoulam revealed that his laboratory isolated an endogenous lipid molecule that binds to the CB1 receptor (cannabinoid receptor type 1) and this became the milestone paper published in December of that year describing anandamide (AEA, Devane et al., 1992). As to

  1. Reaction Pathway for Cocaine Hydrolase-Catalyzed Hydrolysis of (+)-Cocaine

    PubMed Central

    Yao, Yuan; Liu, Junjun; Zheng, Fang; Zhan, Chang-Guo

    2017-01-01

    A recently designed and discovered cocaine hydrolase (CocH), engineered from human butyrylcholinesterase (BChE), has been proven promising as a novel enzyme therapy for treatment of cocaine overdose and addiction because it is highly efficient in catalyzing hydrolysis of naturally occurring (−)-cocaine. It has been known that the CocH also has a high catalytic efficiency against (+)-cocaine, a synthetic enantiomer of cocaine. Reaction pathway and the corresponding free energy profile for the CocH-catalyzed hydrolysis of (+)-cocaine have been determined, in the present study, by performing first-principles pseudobond quantum mechanical/molecular mechanical (QM/MM)-free energy (FE) calculations. Acordingt to the QM/MM-FE results, the catalytic hydrolysis process is initiated by the nucleophilic attack on carbonyl carbon of (−)-cocaine benzoyl ester via hydroxyl oxygen of S198 side chain, and the second reaction step (i.e. dissociation of benzoyl ester) is rate-determining. This finding for CocH-catalyzed hydrolysis of (+)-cocaine is remarkably different from that for the (+)-cocaine hydrolysis catalyzed by bacterial cocaine esterase in which the first reaction step of the deacylation is associated with the highest free energy barrier (~17.9 kcal/mol). The overall free energy barrier (~16.0 kcal/mol) calculated for the acylation stage of CocH-catalyzed hydrolysis of (+)-cocaine is in good agreement with the experimental free energy barrier of ~14.5 kcal/mol derivated from the experimental kinetic data.

  2. Genetic regulation of expression of leukotriene A4 hydrolase

    PubMed Central

    Castaldi, Peter; Cho, Michael H.; Blalock, J. Edwin; Gaggar, Amit

    2016-01-01

    In chronic inflammatory lung disorders such as chronic obstructive pulmonary disease (COPD), the concurrent organ-specific and systemic inflammatory responses lead to airway remodelling and vascular dysfunction. Although a major common risk factor for COPD, cigarette smoke alone cannot explain the progression of this disease; there is increasing evidence that genetic predisposition also plays a role in COPD susceptibility and progression. A key enzyme in chronic lung inflammation is leukotriene A4 hydrolase (LTA4H). With its aminopeptidase activity, LTA4H degrades the neutrophil chemoattractant tripeptide PGP. In this study, we used the luciferase reporter gene analysis system and quantitative trait locus analysis to explore the impact of single-nucleotide polymorphisms (SNPs) in the putative promoter region of LTA4H on LTA4H expression. We show that not only is the putative promoter of LTA4H larger than previously reported but also that SNPs in the expanded promoter region regulate expression of LTA4H both in cell-based systems and in peripheral blood samples from human subjects. These findings provide significant evidence for an active region upstream of the previously reported LTA4H promoter, which may have implications related to ongoing inflammatory processes in chronic lung disease. PMID:27730172

  3. Regulation of S-Adenosylhomocysteine Hydrolase by Lysine Acetylation*

    PubMed Central

    Wang, Yun; Kavran, Jennifer M.; Chen, Zan; Karukurichi, Kannan R.; Leahy, Daniel J.; Cole, Philip A.

    2014-01-01

    S-Adenosylhomocysteine hydrolase (SAHH) is an NAD+-dependent tetrameric enzyme that catalyzes the breakdown of S-adenosylhomocysteine to adenosine and homocysteine and is important in cell growth and the regulation of gene expression. Loss of SAHH function can result in global inhibition of cellular methyltransferase enzymes because of high levels of S-adenosylhomocysteine. Prior proteomics studies have identified two SAHH acetylation sites at Lys401 and Lys408 but the impact of these post-translational modifications has not yet been determined. Here we use expressed protein ligation to produce semisynthetic SAHH acetylated at Lys401 and Lys408 and show that modification of either position negatively impacts the catalytic activity of SAHH. X-ray crystal structures of 408-acetylated SAHH and dually acetylated SAHH have been determined and reveal perturbations in the C-terminal hydrogen bonding patterns, a region of the protein important for NAD+ binding. These crystal structures along with mutagenesis data suggest that such hydrogen bond perturbations are responsible for SAHH catalytic inhibition by acetylation. These results suggest how increased acetylation of SAHH may globally influence cellular methylation patterns. PMID:25248746

  4. Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization.

    PubMed

    de Oliveira, Gabriel Stephani; Adriani, Patricia Pereira; Borges, Flavia Garcia; Lopes, Adriana Rios; Campana, Patricia T; Chambergo, Felipe S

    2016-08-01

    Epoxide hydrolases (EHs) are enzymes that are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EHs have biotechnological potential in chiral chemistry. We report the cloning, purification, enzymatic activity, and conformational analysis of the TrEH gene from Trichoderma reesei strain QM9414 using circular dichroism spectroscopy. The EH gene has an open reading frame encoding a protein of 343 amino acid residues, resulting in a molecular mass of 38.2kDa. The enzyme presents an optimum pH of 7.2, and it is highly active at temperatures ranging from 23 to 50°C and thermally inactivated at 70°C (t1/2=7.4min). The Michaelis constants (Km) were 4.6mM for racemic substrate, 21.7mM for (R)-(+)-styrene oxide and 3.0mM for (S)-(-)-styrene oxide. The kcat/Km analysis indicated that TrEH is enantioselective and preferentially hydrolyzes (S)-(-)-styrene oxide. The conformational stability studies suggested that, despite the extreme conditions (high temperatures and extremely acid and basic pHs), TrEH is able to maintain a considerable part of its regular structures, including the preservation of the native cores in some cases. The recombinant protein showed enantioselectivity that was distinct from other fungus EHs, making this protein a potential biotechnological tool.

  5. Molecular Basis of Arabinobio-hydrolase Activity in Phytopathogenic Fungi

    PubMed Central

    Carapito, Raphaël; Imberty, Anne; Jeltsch, Jean-Marc; Byrns, Simon C.; Tam, Pui-Hang; Lowary, Todd L.; Varrot, Annabelle; Phalip, Vincent

    2009-01-01

    The phytopathogenic fungus Fusarium graminearum secretes a very diverse pool of glycoside hydrolases (GHs) aimed at degrading plant cell walls. α-l-Arabinanases are essential GHs participating in the complete hydrolysis of hemicellulose, a natural resource for various industrial processes, such as bioethanol or pharmaceuticals production. Arb93A, the exo-1,5-α-l-arabinanase of F. graminearum encoded by the gene fg03054.1, belongs to the GH93 family, for which no structural data exists. The enzyme is highly active (1065 units/mg) and displays a strict substrate specificity for linear α-1,5-l-arabinan. Biochemical assays and NMR experiments demonstrated that the enzyme releases α-1,5-l-arabinobiose from the nonreducing end of the polysaccharide. We determined the crystal structure of the native enzyme and its complex with α-1,5-l-arabinobiose, a degradation product of α-Me-1,5-l-arabinotetraose, at 1.85 and 2.05Å resolution, respectively. Arb93A is a monomeric enzyme, which presents the six-bladed β-propeller fold characteristic of sialidases of clan GHE. The configuration of the bound arabinobiose is consistent with the retaining mechanism proposed for the GH93 family. Catalytic residues were proposed from the structural analysis, and site-directed mutagenesis was used to validate their role. They are significantly different from those observed for GHE sialidases. PMID:19269961

  6. Expression of organophosphate hydrolase in the filamentous fungus Gliocladium virens.

    PubMed

    Dave, K I; Lauriano, C; Xu, B; Wild, J R; Kenerley, C M

    1994-05-01

    The broad-spectrum organophosphate hydrolase (OPH; EC 3.1.8.1) encoded by the organophosphate-degrading gene (opd) from Pseudomonas diminuta MG and Flavobacterium sp. ATCC 27551 possesses capabilities of both P-O bond hydrolysis (e.g. paraoxon) and P-F bond hydrolysis [e.g. sarin and diisopropylfluorophosphate (DFP)]. In the present study a 9.4-kb plasmid, pCL1, was used to transform the saprophytic fungus Gliocladium virens. pCL1 was derived from pJS294 by placing the fungal promoter (prom1) from Cochliobolus heterostrophus upstream and the trpC terminator from Aspergillus nidulans down-stream of the opd gene. Southern analysis of restricted genomic DNA from various transformants indicated that integration occurred non-specifically at multiple sites. Western blot analysis of mycelial extracts from transformants confirmed the production of a processed form of the enzyme in the fungus. Maximal levels of OPH activity (rate of p-nitrophenol production from paraoxon) were observed after 168 h of culture and activity levels correlated with biomass production in mature vegetative growth.

  7. Investigation of the Bacillus cereus phosphonoacetaldehyde hydrolase catalytic mechanism

    SciTech Connect

    Hepburn, T.W.

    1988-01-01

    The enzyme phosphonoacetaldehyde hydrolase (phosphonatase) from Bacillus cereus catalyzes the conversion of phosphonoacetaldehyde and phosphate. We have demonstrated that phosphonatase is inactivated when incubated with either acetaldehyde or phosphonoacetaldehyde for short time periods at low temperature in the presence of NaBH{sup 4}. This result suggests that the Schiff base mechanism is operative since such treatment might be expected to inactivate the enzyme by reducing an iminium cation mechanistic intermediate. The inactivation process was shown to be highly specific for a single lysine residue. Incubation of phosphonatase with ({sup 3}H)-NaBH{sub 4} and phosphonacetaldehyde ({sup 14}C)-acetaldehyde and NaBH{sub 4} or (C{sub 2}-{sup 3}H)- phosphonoacetaldehyde and NaBH{sup 4} resulted in radiolabeled inactivated enzyme. Tryptic hydrolysis and reverse phase HPLC chromatography of the resulting digests demonstrated that the (C{sub 2} - {sup 3}H)- phosphonoacetaldehyde/NaBH{sub 4} methodology afforded the most specifically tritium labeled, inactivated phosphonatase. The radiolabeled, active site peptide was purified to homogeneity and its amino acid sequence was determined.

  8. Mapping human brain fatty acid amide hydrolase activity with PET

    PubMed Central

    Rusjan, Pablo M; Wilson, Alan A; Mizrahi, Romina; Boileau, Isabelle; Chavez, Sofia E; Lobaugh, Nancy J; Kish, Stephen J; Houle, Sylvain; Tong, Junchao

    2013-01-01

    Endocannabinoid tone has recently been implicated in a number of prevalent neuropsychiatric conditions. [11C]CURB is the first available positron emission tomography (PET) radiotracer for imaging fatty acid amide hydrolase (FAAH), the enzyme which metabolizes the prominent endocannabinoid anandamide. Here, we sought to determine the most suitable kinetic modeling approach for quantifying [11C]CURB that binds selectively to FAAH. Six healthy volunteers were scanned with arterial blood sampling for 90 minutes. Kinetic parameters were estimated regionally using a one-tissue compartment model (TCM), a 2-TCM with and without irreversible trapping, and an irreversible 3-TCM. The 2-TCM with irreversible trapping provided the best identifiability of PET outcome measures among the approaches studied (coefficient of variation (COV) of the net influx constant Ki and the composite parameter λk3 (λ=K1/k2) <5%, and COV(k3)<10%). Reducing scan time to 60 minutes did not compromise the identifiability of rate constants. Arterial spin labeling measures of regional cerebral blood flow were only slightly correlated with Ki, but not with k3 or λk3. Our data suggest that λk3 is sensitive to changes in FAAH activity, therefore, optimal for PET quantification of FAAH activities with [11C]CURB. Simulations showed that [11C]CURB binding in healthy subjects is far from a flow-limited uptake. PMID:23211960

  9. Ubiquitin C-Terminal Hydrolase L1 in Tumorigenesis

    PubMed Central

    Hurst-Kennedy, Jennifer; Chin, Lih-Shen; Li, Lian

    2012-01-01

    Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1, aka PGP9.5) is an abundant, neuronal deubiquitinating enzyme that has also been suggested to possess E3 ubiquitin-protein ligase activity and/or stabilize ubiquitin monomers in vivo. Recent evidence implicates dysregulation of UCH-L1 in the pathogenesis and progression of human cancers. Although typically only expressed in neurons, high levels of UCH-L1 have been found in many nonneuronal tumors, including breast, colorectal, and pancreatic carcinomas. UCH-L1 has also been implicated in the regulation of metastasis and cell growth during the progression of nonsmall cell lung carcinoma, colorectal cancer, and lymphoma. Together these studies suggest UCH-L1 has a potent oncogenic role and drives tumor development. Conversely, others have observed promoter methylation-mediated silencing of UCH-L1 in certain tumor subtypes, suggesting a potential tumor suppressor role for UCH-L1. In this paper, we provide an overview of the evidence supporting the involvement of UCH-L1 in tumor development and discuss the potential mechanisms of action of UCH-L1 in oncogenesis. PMID:22811913

  10. Thermus thermophilus Glycoside Hydrolase Family 57 Branching Enzyme

    PubMed Central

    Palomo, Marta; Pijning, Tjaard; Booiman, Thijs; Dobruchowska, Justyna M.; van der Vlist, Jeroen; Kralj, Slavko; Planas, Antoni; Loos, Katja; Kamerling, Johannis P.; Dijkstra, Bauke W.; van der Maarel, Marc J. E. C.; Dijkhuizen, Lubbert; Leemhuis, Hans

    2011-01-01

    Branching enzyme (EC 2.4.1.18; glycogen branching enzyme; GBE) catalyzes the formation of α1,6-branching points in glycogen. Until recently it was believed that all GBEs belong to glycoside hydrolase family 13 (GH13). Here we describe the cloning and expression of the Thermus thermophilus family GH57-type GBE and report its biochemical properties and crystal structure at 1.35-Å resolution. The enzyme has a central (β/α)7-fold catalytic domain A with an inserted domain B between β2 and α5 and an α-helix-rich C-terminal domain, which is shown to be essential for substrate binding and catalysis. A maltotriose was modeled in the active site of the enzyme which suggests that there is insufficient space for simultaneously binding of donor and acceptor substrates, and that the donor substrate must be cleaved before acceptor substrate can bind. The biochemical assessment showed that the GH57 GBE possesses about 4% hydrolytic activity with amylose and in vitro forms a glucan product with a novel fine structure, demonstrating that the GH57 GBE is clearly different from the GH13 GBEs characterized to date. PMID:21097495

  11. Soluble epoxide hydrolase activity regulates inflammatory responses and seizure generation in two mouse models of temporal lobe epilepsy.

    PubMed

    Hung, Yu-Wen; Hung, Shao-Wen; Wu, Yi-Chen; Wong, Lin-King; Lai, Ming-Tsong; Shih, Yang-Hsin; Lee, Tzong-Shyuan; Lin, Yung-Yang

    2015-01-01

    Neuroinflammation is known to be involved in epileptogenesis with unclear mechanisms. Inhibition of soluble epoxide hydrolase (sEH) seems to offer anti-inflammatory protection to ischemic brain injury in rodents. Thus, it is hypothesized that sEH inhibition might also affect the neuroinflammatory responses caused by epileptic seizures. In the present study, we investigated the involvement of sEH in neuroinflammation, seizure generation and subsequent epileptogenesis using two mouse models of temporal lobe epilepsy. Experimental epileptic seizures were induced by either pilocarpine or electrical amygdala kindling in both wild-type (WT) C57BL/6 mice and sEH knockout (sEH KO) mice. The sEH expression in the hippocampus was detected by immunohistochemistry and Western blot analysis. The effects of the sEH hydrolase inhibitors, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) and N-[1-(1-oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy) phenyl)-urea (TPPU), and of the genetic deletion of sEH on seizure-induced neuroinflammatory responses and the development of epilepsy were evaluated. In the hippocampus of WT mice, sEH was mainly expressed in astrocytes (GFAP(+)), neurons (NeuN(+)) and scattered microglia (Iba-1(+)) in the regions of CA1, CA3 and dentate gyrus. Expression of sEH was significantly increased on day 7, 14, 21 and 28 after pilocarpine-induced status epilepticus (SE). Administration with sEH inhibitors attenuated the SE-induced up-regulation of interleukin-1β (IL-1β) and interleukin-6 (IL-6), the degradation of EETs, as well as IκB phosphorylation. Following treatment with AUDA, the frequency and duration of spontaneous motor seizures in the pilocarpine-SE mice were decreased and the seizure-induction threshold of the fully kindled mice was increased. Up-regulation of hippocampal IL-1β and IL-6 was found in both WT and sEH KO mice after successful induction of SE. Notably, sEH KO mice were more susceptible to seizures than WT mice. Seizure related

  12. Detoxification of acetylcholinesterase inhibitors. Final report, 1 June 1984-30 November 1986

    SciTech Connect

    Wild, J.R.; O'Donovan, G.A.; Chang, T.

    1987-02-19

    The research support by ARO contract 21288-LS entitled Detoxification of acetylcholinesterase inhibitors resulted in the cloning and partial sequence of two opd genes from Pseudomonas diminuta and Flavobacterium species. It has been possible to isolate the enzyme in association with a small membrane fraction and initiate an evaluation of the organophosphate hydrolase. Collaborative interactions with research scientists at the Chemical Defense Research Command have suggested that the Pseudomonas species is competent to degrade selected types of biological neurotoxins.

  13. Angiogenesis Inhibitors

    MedlinePlus

    ... inhibitors: current strategies and future prospects. CA: A Cancer Journal for Clinicians 2010; 60(4):222–243. [PubMed Abstract] Chen HX, Cleck JN. Adverse effects of anticancer agents that target the VEGF pathway. Nature Reviews Clinical Oncology 2009; 6(8):465– ...

  14. Carboxylesterase inhibitors

    PubMed Central

    Hatfield, M. Jason; Potter, Philip M.

    2011-01-01

    Introduction Carboxylesterases play major roles in the hydrolysis of numerous therapeutically active compounds. This is, in part, due to the prevalence of the ester moiety in these small molecules. However, the impact these enzymes may play on drug stability and pharmacokinetics is rarely considered prior to molecule development. Therefore, the application of selective inhibitors of this class of proteins may have utility in modulating the metabolism, distribution and toxicity of agents that are subjected to enzyme hydrolysis. Areas covered This review details the development of all such compounds dating back to 1986, but principally focuses on the very recent identification of selective human carboxylesterases inhibitors. Expert opinion The implementation of carboxylesterase inhibitors may significantly revolutionize drug discovery. Such molecules may allow for improved efficacy of compounds inactivated by this class of enzymes and/or reduce the toxicity of agents that are activated by these proteins. Furthermore, since lack of carboxylesterase activity appears to have no obvious biological consequence, these compounds could be applied in combination with virtually any esterified drug. Therefore, inhibitors of these proteins may have utility in altering drug hydrolysis and distribution in vivo. The characteristics, chemical and biological properties, and potential uses of such agents, are discussed here. PMID:21609191

  15. Therapeutic potential of monoacylglycerol lipase inhibitors.

    PubMed

    Mulvihill, Melinda M; Nomura, Daniel K

    2013-03-19

    Marijuana and aspirin have been used for millennia to treat a wide range of maladies including pain and inflammation. Both cannabinoids, like marijuana, that exert anti-inflammatory action through stimulating cannabinoid receptors, and cyclooxygenase (COX) inhibitors, like aspirin, that suppress pro-inflammatory eicosanoid production have shown beneficial outcomes in mouse models of neurodegenerative diseases and cancer. Both cannabinoids and COX inhibitors, however, have untoward effects that discourage their chronic usage, including cognitive deficits and gastrointestinal toxicity, respectively. Recent studies have uncovered that the serine hydrolase monoacylglycerol lipase (MAGL) links the endocannabinoid and eicosanoid systems together through hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG) to provide the major arachidonic acid (AA) precursor pools for pro-inflammatory eicosanoid synthesis in specific tissues. Studies in recent years have shown that MAGL inhibitors elicit anti-nociceptive, anxiolytic, and anti-emetic responses and attenuate precipitated withdrawal symptoms in addiction paradigms through enhancing endocannabinoid signaling. MAGL inhibitors have also been shown to exert anti-inflammatory action in the brain and protect against neurodegeneration through lowering eicosanoid production. In cancer, MAGL inhibitors have been shown to have anti-cancer properties not only through modulating the endocannabinoid-eicosanoid network, but also by controlling fatty acid release for the synthesis of protumorigenic signaling lipids. Thus, MAGL serves as a critical node in simultaneously coordinating multiple lipid signaling pathways in both physiological and disease contexts. This review will discuss the diverse (patho)physiological roles of MAGL and the therapeutic potential of MAGL inhibitors in treating a vast array of complex human diseases.

  16. Development of a differential medium for bile salt hydrolase-active Lactobacillus spp.

    PubMed Central

    Dashkevicz, M P; Feighner, S D

    1989-01-01

    An agar plate assay was developed to detect bile salt hydrolase activity in lactobacilli. On Lactobacillus-selective MRS or Rogosa SL medium supplemented with taurodeoxycholic, taurocholic, or taurochenodeoxycholic acids, bile salt hydrolysis was manifested at two intensities: (i) the formation of precipitate halos around colonies or (ii) the formation of opaque granular white colonies. Sixty-six lactobacilli were tested for bile salt hydrolase activity by both the plate assay and a sensitive radiochemical assay. No false-positive or false-negative results were detected by the plate assay. Based on results of experiments with Eubacterium lentum and Bacteroides species, the plate assay was dependent on two factors: (i) the presence of bile salt hydrolytic activity and (ii) the ability of the organism to sufficiently acidify the medium to protonate free bile acids. The availability of a differential medium for determination of bile salt hydrolase activity will provide a rapid method for determining shifts in a specific functional activity of intestinal Lactobacillus species and provide a rapid screening capability for identifying bile salt hydrolase-deficient mutants. The latter application should allow bile salt hydrolase activity to be used as a marker enzyme in genetic experiments. Images PMID:2705765

  17. Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice

    SciTech Connect

    Harris, Todd R.; Bettaieb, Ahmed; Kodani, Sean; Dong, Hua; Myers, Richard; Chiamvimonvat, Nipavan; Haj, Fawaz G.; Hammock, Bruce D.

    2015-07-15

    Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl{sub 4})-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in the liver was increased five-fold in the CCl{sub 4}-treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl{sub 4}-treated group relative to the Control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl{sub 4}-treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-(4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy)-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl{sub 4}, presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress. - Highlights: • We administer an inhibitor of sEH in a CCl4 murine model. • sEH inhibition reduces liver collagen deposition and pro-fibrotic gene expression. • sEH inhibition induces MMP-1a activity.

  18. Prunasin hydrolases during fruit development in sweet and bitter almonds.

    PubMed

    Sánchez-Pérez, Raquel; Belmonte, Fara Sáez; Borch, Jonas; Dicenta, Federico; Møller, Birger Lindberg; Jørgensen, Kirsten

    2012-04-01

    Amygdalin is a cyanogenic diglucoside and constitutes the bitter component in bitter almond (Prunus dulcis). Amygdalin concentration increases in the course of fruit formation. The monoglucoside prunasin is the precursor of amygdalin. Prunasin may be degraded to hydrogen cyanide, glucose, and benzaldehyde by the action of the β-glucosidase prunasin hydrolase (PH) and mandelonitirile lyase or be glucosylated to form amygdalin. The tissue and cellular localization of PHs was determined during fruit development in two sweet and two bitter almond cultivars using a specific antibody toward PHs. Confocal studies on sections of tegument, nucellus, endosperm, and embryo showed that the localization of the PH proteins is dependent on the stage of fruit development, shifting between apoplast and symplast in opposite patterns in sweet and bitter cultivars. Two different PH genes, Ph691 and Ph692, have been identified in a sweet and a bitter almond cultivar. Both cDNAs are 86% identical on the nucleotide level, and their encoded proteins are 79% identical to each other. In addition, Ph691 and Ph692 display 92% and 86% nucleotide identity to Ph1 from black cherry (Prunus serotina). Both proteins were predicted to contain an amino-terminal signal peptide, with the size of 26 amino acid residues for PH691 and 22 residues for PH692. The PH activity and the localization of the respective proteins in vivo differ between cultivars. This implies that there might be different concentrations of prunasin available in the seed for amygdalin synthesis and that these differences may determine whether the mature almond develops into bitter or sweet.

  19. Long-acting cocaine hydrolase for addiction therapy

    PubMed Central

    Chen, Xiabin; Xue, Liu; Hou, Shurong; Jin, Zhenyu; Zhang, Ting; Zheng, Fang; Zhan, Chang-Guo

    2016-01-01

    Cocaine abuse is a world-wide public health and social problem without a US Food and Drug Administration-approved medication. An ideal anticocaine medication would accelerate cocaine metabolism, producing biologically inactive metabolites by administration of an efficient cocaine-specific exogenous enzyme. Our recent studies have led to the discovery of the desirable, highly efficient cocaine hydrolases (CocHs) that can efficiently detoxify and inactivate cocaine without affecting normal functions of the CNS. Preclinical and clinical data have demonstrated that these CocHs are safe for use in humans and are effective for accelerating cocaine metabolism. However, the actual therapeutic use of a CocH in cocaine addiction treatment is limited by its short biological half-life (e.g., 8 h or shorter in rats). Here we demonstrate a novel CocH form, a catalytic antibody analog, which is a fragment crystallizable (Fc)-fused CocH dimer (CocH-Fc) constructed by using CocH to replace the Fab region of human IgG1. The CocH-Fc not only has a high catalytic efficiency against cocaine but also, like an antibody, has a considerably longer biological half-life (e.g., ∼107 h in rats). A single dose of CocH-Fc was able to accelerate cocaine metabolism in rats even after 20 d and thus block cocaine-induced hyperactivity and toxicity for a long period. Given the general observation that the biological half-life of a protein drug is significantly longer in humans than in rodents, the CocH-Fc reported in this study could allow dosing once every 2–4 wk, or longer, for treatment of cocaine addiction in humans. PMID:26712009

  20. Soluble epoxide hydrolase limits mechanical hyperalgesia during inflammation

    PubMed Central

    2011-01-01

    Background Cytochrome-P450 (CYP450) epoxygenases metabolise arachidonic acid (AA) into four different biologically active epoxyeicosatrienoic acid (EET) regioisomers. Three of the EETs (i.e., 8,9-, 11,12- and 14,15-EET) are rapidly hydrolysed by the enzyme soluble epoxide hydrolase (sEH). Here, we investigated the role of sEH in nociceptive processing during peripheral inflammation. Results In dorsal root ganglia (DRG), we found that sEH is expressed in medium and large diameter neurofilament 200-positive neurons. Isolated DRG-neurons from sEH-/- mice showed higher EET and lower DHET levels. Upon AA stimulation, the largest changes in EET levels occurred in culture media, indicating both that cell associated EET concentrations quickly reach saturation and EET-hydrolyzing activity mostly effects extracellular EET signaling. In vivo, DRGs from sEH-deficient mice exhibited elevated 8,9-, 11,12- and 14,15-EET-levels. Interestingly, EET levels did not increase at the site of zymosan-induced inflammation. Cellular imaging experiments revealed direct calcium flux responses to 8,9-EET in a subpopulation of nociceptors. In addition, 8,9-EET sensitized AITC-induced calcium increases in DRG neurons and AITC-induced calcitonin gene related peptide (CGRP) release from sciatic nerve axons, indicating that 8,9-EET sensitizes TRPA1-expressing neurons, which are known to contribute to mechanical hyperalgesia. Supporting this, sEH-/- mice showed increased nociceptive responses to mechanical stimulation during zymosan-induced inflammation and 8,9-EET injection reduced mechanical thresholds in naive mice. Conclusion Our results show that the sEH can regulate mechanical hyperalgesia during inflammation by inactivating 8,9-EET, which sensitizes TRPA1-expressing nociceptors. Therefore we suggest that influencing the CYP450 pathway, which is actually highly considered to treat cardiovascular diseases, may cause pain side effects. PMID:21970373

  1. Mechanistic studies of ubiquitin C-terminal hydrolase L1.

    PubMed

    Case, April; Stein, Ross L

    2006-02-21

    Ubiquitin C-terminal hydrolases (UCHs) cleave Ub-X bonds (Ub is ubiquitin and X an alcohol, an amine, or a protein) through a thioester intermediate that is produced by nucleophilic attack of the Cys residue of a Cys-SH/His-Im catalytic diad. We are studying the mechanism of UCH-L1, a UCH that is implicated in Parkinson's disease, and now wish to report our initial findings. (i) Pre-steady-state kinetic studies for UCH-L1-catalyzed hydrolysis of Ub-AMC (AMC, 7-amido-4-methylcoumarin) indicate that k(cat) is rate-limited by acyl-enzyme formation. Thus, K(m) = K(s), the dissociation constant for the Michaelis complex, and k(cat) = k(2), the rate constant for acyl-enzyme formation. (ii) For K(assoc) (=K(s)(-)(1)), DeltaC(p) = -0.8 kcal mol(-)(1) deg(-)(1) and is consistent with coupling between substrate association and a conformational change of the enzyme. For k(2), DeltaS(++) = 0 and suggests that in the E-S, substrate and active site residues are precisely aligned for reaction. (iii) Solvent isotope effects are (D)K(assoc) = 0.5 and (D)k(2) = 0.9, suggesting that the substrate binds to a form of free enzyme in which the active site Cys exists as the thiol. In the resultant Michaelis complex, the diad has tautomerized to ion pair Cys-S(-)/His-ImH(+). Subsequent attack of thiolate produces the acyl-enzyme species. In contrast, isotope effects for association of UCH-L1 with transition-state analogue ubiquitin aldehyde suggest that an alternative mechanistic pathway can sometimes be available to UCH-L1 involving general base-catalyzed attack of Cys-SH by His-Im.

  2. Bioprospecting metagenomics of decaying wood: mining for new glycoside hydrolases

    SciTech Connect

    Li L. L.; van der Lelie D.; Taghavi, S.; McCorkle, S. M.; Zhang, Y.-B.; Blewitt, M. G.; Brunecky, R.; Adney, W. S.; Himmel, M. E.; Brumm, P.; Drinkwater, C.; Mead, D. A.; Tringe, S. G.

    2011-08-01

    To efficiently deconstruct recalcitrant plant biomass to fermentable sugars in industrial processes, biocatalysts of higher performance and lower cost are required. The genetic diversity found in the metagenomes of natural microbial biomass decay communities may harbor such enzymes. Our goal was to discover and characterize new glycoside hydrolases (GHases) from microbial biomass decay communities, especially those from unknown or never previously cultivated microorganisms. From the metagenome sequences of an anaerobic microbial community actively decaying poplar biomass, we identified approximately 4,000 GHase homologs. Based on homology to GHase families/activities of interest and the quality of the sequences, candidates were selected for full-length cloning and subsequent expression. As an alternative strategy, a metagenome expression library was constructed and screened for GHase activities. These combined efforts resulted in the cloning of four novel GHases that could be successfully expressed in Escherichia coli. Further characterization showed that two enzymes showed significant activity on p-nitrophenyl-{alpha}-L-arabinofuranoside, one enzyme had significant activity against p-nitrophenyl-{beta}-D-glucopyranoside, and one enzyme showed significant activity against p-nitrophenyl-{beta}-D-xylopyranoside. Enzymes were also tested in the presence of ionic liquids. Metagenomics provides a good resource for mining novel biomass degrading enzymes and for screening of cellulolytic enzyme activities. The four GHases that were cloned may have potential application for deconstruction of biomass pretreated with ionic liquids, as they remain active in the presence of up to 20% ionic liquid (except for 1-ethyl-3-methylimidazolium diethyl phosphate). Alternatively, ionic liquids might be used to immobilize or stabilize these enzymes for minimal solvent processing of biomass.

  3. Reaction Mechanisms in Carbohydrate-Active Enzymes: Glycoside Hydrolases and Glycosyltransferases. Insights from ab Initio Quantum Mechanics/Molecular Mechanics Dynamic Simulations.

    PubMed

    Ardèvol, Albert; Rovira, Carme

    2015-06-24

    Carbohydrate-active enzymes such as glycoside hydrolases (GHs) and glycosyltransferases (GTs) are of growing importance as drug targets. The development of efficient competitive inhibitors and chaperones to treat diseases related to these enzymes requires a detailed knowledge of their mechanisms of action. In recent years, sophisticated first-principles modeling approaches have significantly advanced in our understanding of the catalytic mechanisms of GHs and GTs, not only the molecular details of chemical reactions but also the significant implications that just the conformational dynamics of a sugar ring can have on these mechanisms. Here we provide an overview of the progress that has been made in the past decade, combining molecular dynamics simulations with density functional theory to solve these sweet mysteries of nature.

  4. Strategies to reduce end-product inhibition in family 48 glycoside hydrolases.

    PubMed

    Chen, Mo; Bu, Lintao; Alahuhta, Markus; Brunecky, Roman; Xu, Qi; Lunin, Vladimir V; Brady, John W; Crowley, Michael F; Himmel, Michael E; Bomble, Yannick J

    2016-03-01

    Family 48 cellobiohydrolases are some of the most abundant glycoside hydrolases in nature. They are able to degrade cellulosic biomass and therefore serve as good enzyme candidates for biofuel production. Family 48 cellulases hydrolyze cellulose chains via a processive mechanism, and produce end products composed primarily of cellobiose as well as other cellooligomers (dp ≤ 4). The challenge of utilizing cellulases in biofuel production lies in their extremely slow turnover rate. A factor contributing to the low enzyme activity is suggested to be product binding to enzyme and the resulting performance inhibition. In this study, we quantitatively evaluated the product inhibitory effect of four family 48 glycoside hydrolases using molecular dynamics simulations and product expulsion free-energy calculations. We also suggested a series of single mutants of the four family 48 glycoside hydrolases with theoretically reduced level of product inhibition. The theoretical calculations provide a guide for future experimental studies designed to produce mutant cellulases with enhanced activity.

  5. Structure-Guided Engineering of Molinate Hydrolase for the Degradation of Thiocarbamate Pesticides

    PubMed Central

    Paiva, Ana M.; Ferreira-da-Silva, Frederico; Matias, Pedro M.; Nunes, Olga C.; Gales, Luís

    2015-01-01

    Molinate is a recalcitrant thiocarbamate used to control grass weeds in rice fields. The recently described molinate hydrolase, from Gulosibacter molinativorax ON4T, plays a key role in the only known molinate degradation pathway ending in the formation of innocuous compounds. Here we report the crystal structure of recombinant molinate hydrolase at 2.27 Å. The structure reveals a homotetramer with a single mononuclear metal-dependent active site per monomer. The active site architecture shows similarities with other amidohydrolases and enables us to propose a general acid-base catalysis mechanism for molinate hydrolysis. Molinate hydrolase is unable to degrade bulkier thiocarbamate pesticides such as thiobencarb which is used mostly in rice crops. Using a structural-based approach, we were able to generate a mutant (Arg187Ala) that efficiently degrades thiobencarb. The engineered enzyme is suitable for the development of a broader thiocarbamate bioremediation system. PMID:25905461

  6. Murein hydrolase activity of surface layer proteins from Lactobacillus acidophilus against Escherichia coli.

    PubMed

    Meng, Jun; Gao, Shu-Ming; Zhang, Qiu-Xiang; Lu, Rong-Rong

    2015-08-01

    The aim of this study was to investigate the murein hydrolase activities of the surface layer proteins (SLPs) from two strains of Lactobacillus acidophilus using zymography. The influence of these hydrolase activities on Escherichia coli ATCC 43893 was also evaluated by analysing their growth curve, cell morphology and physiological state. After the incubation of E. coli with SLPs, growth was inhibited, the number of viable cells was significantly reduced, examination by transmission electron microscopy showed that the cell wall was damaged and flow cytometry results indicated that the majority of the cells were sublethally injured. All of these results suggested that the SLPs of both L. acidophilus strains possessed murein hydrolase activities that were sublethal to E. coli cells.

  7. Strategies to reduce end-product inhibition in family 48 glycoside hydrolases

    SciTech Connect

    Chen, Mo; Bu, Lintao; Alahuhta, Markus; Brunecky, Roman; Xu, Qi; Lunin, Vladimir V.; Brady, John W.; Crowley, Michael F.; Himmel, Michael E.; Bomble, Yannick J.

    2016-02-01

    Family 48 cellobiohydrolases are some of the most abundant glycoside hydrolases in nature. They are able to degrade cellulosic biomass and therefore serve as good enzyme candidates for biofuel production. Family 48 cellulases hydrolyze cellulose chains via a processive mechanism, and produce end products composed primarily of cellobiose as well as other cellooligomers (dp ≤ 4). The challenge of utilizing cellulases in biofuel production lies in their extremely slow turnover rate. A factor contributing to the low enzyme activity is suggested to be product binding to enzyme and the resulting performance inhibition. In this study, we quantitatively evaluated the product inhibitory effect of four family 48 glycoside hydrolases using molecular dynamics simulations and product expulsion free-energy calculations. We also suggested a series of single mutants of the four family 48 glycoside hydrolases with theoretically reduced level of product inhibition. As a result, the theoretical calculations provide a guide for future experimental studies designed to produce mutant cellulases with enhanced activity.

  8. Structure-guided engineering of molinate hydrolase for the degradation of thiocarbamate pesticides.

    PubMed

    Leite, José P; Duarte, Márcia; Paiva, Ana M; Ferreira-da-Silva, Frederico; Matias, Pedro M; Nunes, Olga C; Gales, Luís

    2015-01-01

    Molinate is a recalcitrant thiocarbamate used to control grass weeds in rice fields. The recently described molinate hydrolase, from Gulosibacter molinativorax ON4T, plays a key role in the only known molinate degradation pathway ending in the formation of innocuous compounds. Here we report the crystal structure of recombinant molinate hydrolase at 2.27 Å. The structure reveals a homotetramer with a single mononuclear metal-dependent active site per monomer. The active site architecture shows similarities with other amidohydrolases and enables us to propose a general acid-base catalysis mechanism for molinate hydrolysis. Molinate hydrolase is unable to degrade bulkier thiocarbamate pesticides such as thiobencarb which is used mostly in rice crops. Using a structural-based approach, we were able to generate a mutant (Arg187Ala) that efficiently degrades thiobencarb. The engineered enzyme is suitable for the development of a broader thiocarbamate bioremediation system.

  9. Phosphonoacetic acid utilization by fungal isolates: occurrence and properties of a phosphonoacetate hydrolase in some penicillia.

    PubMed

    Forlani, Giuseppe; Klimek-Ochab, Magdalena; Jaworski, Jakub; Lejczak, Barbara; Picco, Anna M

    2006-12-01

    Among a collection of 18 fungal strains representing eight genera, only two strains (Penicillium oxalicum and P. minioluteum) were capable of growth on phosphonoacetic acid as sole phosphorous source. Enrichment liquid cultures in minimal medium with the compound as the only P-source selected four isolates, that were also identified as Penicillium spp. Phosphonoacetate metabolism did not lead to extracellular release of inorganic phosphate. In all cases phosphonoacetate hydrolase activity was detected in partially purified extracts, and a protein of the expected molecular mass reacted with polyclonal antibodies raised against the enzyme from P. oxalicum. There was no relation between phosphonoacetate hydrolase specific activity and growth rate or yield. Phosphonoacetic acid was the inducer of the hydrolase, independently of the concurrent availability of inorganic phosphate. Notwithstanding this, the utilization of the phosphonate was significantly inhibited in the presence of phosphate, suggesting an interference of the latter with phosphonoacetic acid uptake.

  10. Soluble epoxide hydrolase deficiency alters pancreatic islet size and improves glucose homeostasis in a model of insulin resistance.

    PubMed

    Luria, Ayala; Bettaieb, Ahmed; Xi, Yannan; Shieh, Guang-Jong; Liu, Hsin-Chen; Inoue, Hiromi; Tsai, Hsing-Ju; Imig, John D; Haj, Fawaz G; Hammock, Bruce D

    2011-05-31

    Visceral obesity has been defined as an important element of the metabolic syndrome and contributes to the development of insulin resistance and cardiovascular disease. Increasing endogenous levels of epoxyeicosatrienoic acids (EETs) are known for their analgesic, antihypertensive, and antiinflammatory effects. The availability of EETs is limited primarily by the soluble epoxide hydrolase (sEH, EPHX2), which metabolizes EETs to their less active diols. In this study, we tested the hypothesis that EETs are involved in glucose regulation and in retarding the development of insulin resistance. To address the role of EETs in regulating glucose homeostasis and insulin signaling, we used mice with targeted gene deletion of sEH (Ephx2-null mice) and a subsequent study with a selective sEH inhibitor. When wild-type mice are fed a high fat diet, insulin resistance develops. However, knockout or inhibition of sEH activity resulted in a significant decrease in plasma glucose. These findings are characterized by enhancement of tyrosyl phosphorylation of the insulin receptor, insulin receptor substrate 1, and their downstream cascade. In addition, pancreatic islets were larger when sEH was disrupted. This effect was associated with an increase in vasculature. These observations were supported by pharmacological inhibition of sEH. These data suggest that an increase in EETs due to sEH-gene knockout leads to an increase in the size of islets and improved insulin signaling and sensitivity.

  11. Parabens inhibit fatty acid amide hydrolase: A potential role in paraben-enhanced 3T3-L1 adipocyte differentiation.

    PubMed

    Kodani, Sean D; Overby, Haley B; Morisseau, Christophe; Chen, Jiangang; Zhao, Ling; Hammock, Bruce D

    2016-11-16

    Parabens are a class of small molecules that are regularly used as preservatives in a variety of personal care products. Several parabens, including butylparaben and benzylparaben, have been found to interfere with endocrine signaling and to stimulate adipocyte differentiation. We hypothesized these biological effects could be due to interference with the endocannabinoid system and identified fatty acid amide hydrolase (FAAH) as the direct molecular target of parabens. FAAH inhibition by parabens yields mixed-type and time-independent kinetics. Additionally, structure activity relationships indicate FAAH inhibition is selective for the paraben class of compounds and the more hydrophobic parabens have higher potency. Parabens enhanced 3T3-L1 adipocyte differentiation in a dose dependent fashion, different from two other FAAH inhibitors URB597 and PF622. Moreover, parabens, URB597 and PF622 all failed to enhance AEA-induced differentiation. Furthermore, rimonabant, a cannabinoid receptor 1 (CB1)-selective antagonist, did not attenuate paraben-induced adipocyte differentiation. Thus, adipogenesis mediated by parabens likely occurs through modulation of endocannabinoids, but cell differentiation is independent of direct activation of CB1 by endocannabinoids.

  12. Recombinant production, crystallization and X-ray crystallographic structure determination of the peptidyl-tRNA hydrolase of Pseudomonas aeruginosa

    SciTech Connect

    Hughes, Ronny C.; McFeeters, Hana; Coates, Leighton; McFeeters, Robert L.

    2014-10-15

    The peptidyl-tRNA hydrolase enzyme from the pathogenic bacterium Pseudomonas aeruginosa (Pth; EC 3.1.1.29) has been cloned, expressed in Escherichia coli and crystallized for X-ray structural analysis. Suitable crystals were grown using the sitting-drop vapour-diffusion method after one week of incubation against a reservoir solution consisting of 20% polyethylene glycol 4000, 100 mM Tris pH 7.5, 10%(v/v) isopropyl alcohol. The crystals were used to obtain the three-dimensional structure of the native protein at 1.77 Å resolution. The structure was determined by molecular replacement of the crystallographic data processed in space group P6122 with unit-cell parameters a = b = 63.62,c = 155.20 Å, α = β = 90, γ = 120°. The asymmetric unit of the crystallographic lattice was composed of a single copy of the enzyme molecule with a 43% solvent fraction, corresponding to a Matthews coefficient of 2.43 Å3 Da-1. The crystallographic structure reported here will serve as the foundation for future structure-guided efforts towards the development of novel small-molecule inhibitors specific to bacterial Pths.

  13. Fragment Screening of Soluble Epoxide Hydrolase for Lead Generation-Structure-Based Hit Evaluation and Chemistry Exploration.

    PubMed

    Xue, Yafeng; Olsson, Thomas; Johansson, Carina A; Öster, Linda; Beisel, Hans-Georg; Rohman, Mattias; Karis, David; Bäckström, Stefan

    2016-03-04

    Soluble epoxide hydrolase (sEH) is involved in the regulation of many biological processes by metabolizing the key bioactive lipid mediator, epoxyeicosatrienoic acids. For the development of sEH inhibitors with improved physicochemical properties, we performed both a fragment screening and a high-throughput screening aiming at an integrated hit evaluation and lead generation. Followed by a joint dose-response analysis to confirm the hits, the identified actives were then effectively triaged by a structure-based hit-classification approach to three prioritized series. Two distinct scaffolds were identified as tractable starting points for potential lead chemistry work. The oxoindoline series bind at the right-hand side of the active-site pocket with hydrogen bonds to the protein. The 2-phenylbenzimidazole-4-sulfonamide series bind at the central channel with significant induced fit, which has not been previously reported. On the basis of the encouraging initial results, we envision that a new lead series with improved properties could be generated if a vector is found that could merge the cyclohexyl functionality of the oxoindoline series with the trifluoromethyl moiety of the 2-phenylbenzimidazole-4-sulfonamide series.

  14. High-resolution structures of complexes of plant S-­adenosyl-l-homocysteine hydrolase (Lupinus luteus)

    PubMed Central

    Brzezinski, Krzysztof; Dauter, Zbigniew; Jaskolski, Mariusz

    2012-01-01

    S-Adenosyl-l-homocysteine hydrolase (SAHase) catalyzes the reversible breakdown of S-adenosyl-l-homocysteine (SAH) to adenosine and homocysteine. SAH is formed in methylation reactions that utilize S-adenosyl-l-methionine (SAM) as a methyl donor. By removing the SAH byproduct, SAHase serves as a major regulator of SAM-dependent biological methylation reactions. Here, the first crystal structure of SAHase of plant origin, that from the legume yellow lupin (LlSAHase), is presented. Structures have been determined at high resolution for three complexes of the enzyme: those with a reaction byproduct/substrate (adenosine), with its nonoxidizable analog (cordycepin) and with a product of inhibitor cleavage (adenine). In all three cases the enzyme has a closed conformation. A sodium cation is found near the active site, coordinated by residues from a conserved loop that hinges domain movement upon reactant binding. An insertion segment that is present in all plant SAHases is located near a substrate-pocket access channel and participates in its formation. In contrast to mammalian and bacterial SAHases, the channel is open when adenosine or cordycepin is bound and is closed in the adenine complex. In contrast to SAHases from other organisms, which are active as tetramers, the plant enzyme functions as a homodimer in solution. PMID:22349223

  15. Polyglycine hydrolases: fungal b-lactamase-like endoproteases that cleave polyglycine regions within plant class IV chitinases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyglycine hydrolases are secreted fungal proteases that cleave glycine-glycine peptide bonds in the inter-domain linker region of specific plant defense chitinases. Previously, we reported the catalytic activity of polyglycine hydrolases from the phytopathogens Epicoccum sorghi (Es-cmp) and Cochli...

  16. Cloning and expression of diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from Lupinus angustifolius L.

    PubMed Central

    Maksel, D; Guranowski, A; Ilgoutz, S C; Moir, A; Blackburn, M G; Gayler, K R

    1998-01-01

    The first isolation, cloning and expression of cDNA encoding an asymmetric diadenosine 5',5'''P1,P4-tetraphosphate pyrophosphohydrolase (Ap4A hydrolase) from a higher plant is described. Ap4A hydrolase protein was purified from seeds of both Lupinus luteus and Lupinus angustifolius and partially sequenced. The Ap4A hydrolase cDNA was cloned from L. angustifolius cotyledonary polyadenylated RNA using reverse transcription and PCR with primers based on the amino acid sequence. The cDNA encoded a protein of 199 amino acids, molecular mass 22982Da. When expressed in Escherichia coli fused to a maltose-binding protein, the enzyme catalysed asymmetric cleavage of Ap4A to AMP and ATP which was inhibited at concentrations of F- as low as 3 microM. These are properties characteristic of Ap4A hydrolase (asymmetrical) (EC 3.6.1. 17). Comparison of the Ap4A hydrolase sequences derived from the four known cDNAs from pig, human, lupin and fission yeast showed that, like the mammalian hydrolase, the lupin enzyme possesses a Mut T motif but no other significant similarities. No sequence similarity to the human fragile histidine triad protein, as found in the Ap4A hydrolase from Schizosaccharomyces pombe, was detected in the Ap4A hydrolase from lupin. PMID:9425114

  17. Congenital hypothyroidism mutations affect common folding and trafficking in the α/β-hydrolase fold proteins.

    PubMed

    De Jaco, Antonella; Dubi, Noga; Camp, Shelley; Taylor, Palmer

    2012-12-01

    The α/β-hydrolase fold superfamily of proteins is composed of structurally related members that, despite great diversity in their catalytic, recognition, adhesion and chaperone functions, share a common fold governed by homologous residues and conserved disulfide bridges. Non-synonymous single nucleotide polymorphisms within the α/β-hydrolase fold domain in various family members have been found for congenital endocrine, metabolic and nervous system disorders. By examining the amino acid sequence from the various proteins, mutations were found to be prevalent in conserved residues within the α/β-hydrolase fold of the homologous proteins. This is the case for the thyroglobulin mutations linked to congenital hypothyroidism. To address whether correct folding of the common domain is required for protein export, we inserted the thyroglobulin mutations at homologous positions in two correlated but simpler α/β-hydrolase fold proteins known to be exported to the cell surface: neuroligin3 and acetylcholinesterase. Here we show that these mutations in the cholinesterase homologous region alter the folding properties of the α/β-hydrolase fold domain, which are reflected in defects in protein trafficking, folding and function, and ultimately result in retention of the partially processed proteins in the endoplasmic reticulum. Accordingly, mutations at conserved residues may be transferred amongst homologous proteins to produce common processing defects despite disparate functions, protein complexity and tissue-specific expression of the homologous proteins. More importantly, a similar assembly of the α/β-hydrolase fold domain tertiary structure among homologous members of the superfamily is required for correct trafficking of the proteins to their final destination.

  18. Purification and characterization of allophanate hydrolase (AtzF) from Pseudomonas sp. strain ADP.

    PubMed

    Shapir, Nir; Sadowsky, Michael J; Wackett, Lawrence P

    2005-06-01

    AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a k(cat)/K(m) of 1.1 x 10(4) s(-1) M(-1), and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced k(cat)/K(m) of 21 s(-1) M(-1). Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of

  19. A pro-nociceptive phenotype unmasked in mice lacking fatty-acid amide hydrolase

    PubMed Central

    Carey, Lawrence M; Slivicki, Richard A; Leishman, Emma; Cornett, Ben; Mackie, Ken; Bradshaw, Heather

    2016-01-01

    Fatty-acid amide hydrolase (FAAH) is the major enzyme responsible for degradation of anandamide, an endocannabinoid. Pharmacological inhibition or genetic deletion of FAAH (FAAH KO) produces antinociception in preclinical pain models that is largely attributed to anandamide-induced activation of cannabinoid receptors. However, FAAH metabolizes a wide range of structurally related, biologically active lipid signaling molecules whose functions remain largely unknown. Some of these endogenous lipids, including anandamide itself, may exert pro-nociceptive effects under certain conditions. In our study, FAAH KO mice exhibited a characteristic analgesic phenotype in the tail flick test and in both formalin and carrageenan models of inflammatory nociception. Nonetheless, intradermal injection of the transient receptor potential channel V1 (TRPV1) agonist capsaicin increased nocifensive behavior as well as mechanical and heat hypersensitivity in FAAH KO relative to wild-type mice. This pro-nociceptive phenotype was accompanied by increases in capsaicin-evoked Fos-like immunoreactive (FLI) cells in spinal dorsal horn regions implicated in nociceptive processing and was attenuated by CB1 (AM251) and TRPV1 (AMG9810) antagonists. When central sensitization was established, FAAH KO mice displayed elevated levels of anandamide, other fatty-acid amides, and endogenous TRPV1 agonists in both paw skin and lumbar spinal cord relative to wild-type mice. Capsaicin decreased spinal cord 2-AG levels and increased arachidonic acid and prostaglandin E2 levels in both spinal cord and paw skin irrespective of genotype. Our studies identify a previously unrecognized pro-nociceptive phenotype in FAAH KO mice that was unmasked by capsaicin challenge. The heightened nociceptive response was mediated by CB1 and TRPV1 receptors and accompanied by enhanced spinal neuronal activation. Moreover, genetic deletion of FAAH has a profound impact on the peripheral and central lipidome. Thus, genetic

  20. A pro-nociceptive phenotype unmasked in mice lacking fatty-acid amide hydrolase.

    PubMed

    Carey, Lawrence M; Slivicki, Richard A; Leishman, Emma; Cornett, Ben; Mackie, Ken; Bradshaw, Heather; Hohmann, Andrea G

    2016-01-01

    Fatty-acid amide hydrolase (FAAH) is the major enzyme responsible for degradation of anandamide, an endocannabinoid. Pharmacological inhibition or genetic deletion of FAAH (FAAH KO) produces antinociception in preclinical pain models that is largely attributed to anandamide-induced activation of cannabinoid receptors. However, FAAH metabolizes a wide range of structurally related, biologically active lipid signaling molecules whose functions remain largely unknown. Some of these endogenous lipids, including anandamide itself, may exert pro-nociceptive effects under certain conditions. In our study, FAAH KO mice exhibited a characteristic analgesic phenotype in the tail flick test and in both formalin and carrageenan models of inflammatory nociception. Nonetheless, intradermal injection of the transient receptor potential channel V1 (TRPV1) agonist capsaicin increased nocifensive behavior as well as mechanical and heat hypersensitivity in FAAH KO relative to wild-type mice. This pro-nociceptive phenotype was accompanied by increases in capsaicin-evoked Fos-like immunoreactive (FLI) cells in spinal dorsal horn regions implicated in nociceptive processing and was attenuated by CB1 (AM251) and TRPV1 (AMG9810) antagonists. When central sensitization was established, FAAH KO mice displayed elevated levels of anandamide, other fatty-acid amides, and endogenous TRPV1 agonists in both paw skin and lumbar spinal cord relative to wild-type mice. Capsaicin decreased spinal cord 2-AG levels and increased arachidonic acid and prostaglandin E2 levels in both spinal cord and paw skin irrespective of genotype. Our studies identify a previously unrecognized pro-nociceptive phenotype in FAAH KO mice that was unmasked by capsaicin challenge. The heightened nociceptive response was mediated by CB1 and TRPV1 receptors and accompanied by enhanced spinal neuronal activation. Moreover, genetic deletion of FAAH has a profound impact on the peripheral and central lipidome. Thus, genetic

  1. Purification and Characterization of Allophanate Hydrolase (AtzF) from Pseudomonas sp. Strain ADP

    PubMed Central

    Shapir, Nir; Sadowsky, Michael J.; Wackett, Lawrence P.

    2005-01-01

    AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a kcat/Km of 1.1 × 104 s−1 M−1, and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced kcat/Km of 21 s−1 M−1. Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of enzymes

  2. Diversity of glycosyl hydrolase enzymes from metagenome and their application in food industry.

    PubMed

    Sathya, T A; Khan, Mahejibin

    2014-11-01

    Traditional use of enzymes for food processing and production of food ingredients resulted in fast-growing enzyme industries world over. The advances in technologies gave rise to exploring newer enzymes and/or modified enzymes for specific application. Search for novel enzymes that can augment catalytic efficiency and advances in molecular biology techniques including sequencing has targeted microbial diversity through metagenomic approaches for sourcing enzymes from difficult to culture organisms. Such mining studies have received more attention in characterizing hydrolases, their prevalence, broad substrate specificities, stability, and independence of cofactors. The focus on glycosyl hydrolases from metagenome for their application in food sector is reviewed.

  3. Data set of optimal parameters for colorimetric red assay of epoxide hydrolase activity.

    PubMed

    de Oliveira, Gabriel Stephani; Adriani, Patricia Pereira; Borges, Flavia Garcia; Lopes, Adriana Rios; Campana, Patricia T; Chambergo, Felipe S

    2016-09-01

    The data presented in this article are related to the research article entitled "Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization" [1]. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. This article describes the optimal parameters for the colorimetric red assay to determine the enzymatic activity, with an emphasis on the characterization of the kinetic parameters, pH optimum and thermal stability of this enzyme. The effects of reagents that are not resistant to oxidation by sodium periodate on the reactions can generate false positives and interfere with the final results of the red assay.

  4. Inhibition of soluble epoxide hydrolase contributes to the anti-inflammatory effect of antimicrobial triclocarban in a murine model

    SciTech Connect

    Liu Junyan; Qiu Hong; Morisseau, Christophe; Hwang, Sung Hee; Tsai, Hsing-Ju; Ulu, Arzu; Chiamvimonvat, Nipavan; Hammock, Bruce D.

    2011-09-01

    The increasing use of the antimicrobial triclocarban (TCC) in personal care products (PCPs) has resulted in concern regarding environmental pollution. TCC is a potent inhibitor of soluble epoxide hydrolase (sEH). Inhibitors of sEH (sEHIs) are anti-inflammatory, anti-hypertensive and cardio-protective in multiple animal models. However, the in vivo effects anticipated from a sEHI have not been reported for TCC. Here we demonstrated the anti-inflammatory effects in vivo of TCC in a murine model. TCC was employed in a lipopolysaccharide (LPS)-challenged murine model. Systolic blood pressure, plasma levels of several inflammatory cytokines and chemokine, and metabolomic profile of plasma oxylipins were determined. TCC significantly reversed LPS-induced morbid hypotension in a time-dependent manner. TCC significantly repressed the increased release of inflammatory cytokines and chemokine caused by LPS. Furthermore, TCC significantly shifted the oxylipin profile in vivo in a time-dependent manner towards resolution of inflammation as expected from a sEHI. These results demonstrated that at the doses used TCC is anti-inflammatory in the murine model. This study suggests that TCC may provide some benefits in humans in addition to its antimicrobial activities due to its potent inhibition of sEH. It may be a promising starting point for developing new low volume high value applications of TCC. However these biological effects also caution against the general over use of TCC in PCPs. - Graphical abstract: Display Omitted Research Highlights: > Anti-microbial triclocarban (TCC) is anti-inflammatory in a murine model. > TCC significantly shifted the oxylipin profile in vivo as expected from a sEHI. > TCC significantly reversed LPS-induced morbid hypotension in a time-dependent manner. > TCC significantly repressed LPS-induced increased release of inflammatory cytokines.

  5. Streptococcus pneumoniae Endohexosaminidase D, Structural and Mechanistic Insight into Substrate-Assisted Catalysis in Family 85 Glycoside Hydrolases

    SciTech Connect

    Abbott, D.; Macauley, M; Vocadlo, D; Boraston, A

    2009-01-01

    Endo-?-d-glucosaminidases from family 85 of glycoside hydrolases (GH85 endohexosaminidases) act to cleave the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. Endohexosaminidase D (Endo-D), produced by Streptococcus pneumoniae, is believed to contribute to the virulence of this organism by playing a role in the deglycosylation of IgG antibodies. Endohexosaminidases have received significant attention for this reason and, moreover, because they are powerful tools for chemoenzymatic synthesis of proteins having defined glycoforms. Here we describe mechanistic and structural studies of the catalytic domain (SpGH85) of Endo-D that provide compelling support for GH85 enzymes using a catalytic mechanism involving substrate-assisted catalysis. Furthermore, the structure of SpGH85 in complex with the mechanism-based competitive inhibitor NAG-thiazoline (Kd = 28 ?m) provides a coherent rationale for previous mutagenesis studies of Endo-D and other related GH85 enzymes. We also find GH85, GH56, and GH18 enzymes have a similar configuration of catalytic residues. Notably, GH85 enzymes have an asparagine in place of the aspartate residue found in these other families of glycosidases. We propose that this residue, as the imidic acid tautomer, acts analogously to the key catalytic aspartate of GH56 and GH18 enzymes. This topographically conserved arrangement of the asparagine residue and a conserved glutamic acid, coupled with previous kinetic studies, suggests these enzymes may use an unusual proton shuttle to coordinate effective general acid and base catalysis to aid cleavage of the glycosidic bond. These results collectively provide a blueprint that may be used to facilitate protein engineering of these enzymes to improve their function as biocatalysts for synthesizing glycoproteins having defined glycoforms and also may serve as a guide for generating inhibitors of GH85 enzymes.

  6. Fatty acid amide hydrolase (FAAH) blockade ameliorates experimental colitis by altering microRNA expression and suppressing inflammation.

    PubMed

    Shamran, Haidar; Singh, Narendra P; Zumbrun, Elizabeth E; Murphy, Angela; Taub, Dennis D; Mishra, Manoj K; Price, Robert L; Chatterjee, Saurabh; Nagarkatti, Mitzi; Nagarkatti, Prakash S; Singh, Udai P

    2017-01-01

    Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), which is thought to result from immune-mediated inflammatory disorders, leads to high morbidity and health care cost. Fatty acid amide hydrolase (FAAH) is an enzyme crucially involved in the modulation of intestinal physiology through anandamide (AEA) and other endocannabinoids. Here we examined the effects of an FAAH inhibitor (FAAH-II), on dextran sodium sulphate (DSS)-induced experimental colitis in mice. Treatments with FAAH-II improved overall clinical scores by reversing weight loss and colitis-associated pathogenesis. The frequencies of activated CD4(+) T cells in spleens, mesenteric lymph nodes (MLNs), Peyer's patches (PPs), and colon lamina propiria (LP) were reduced by FAAH inhibition. Similarly, the frequencies of macrophages, neutrophils, natural killer (NK), and NKT cells in the PPs and LP of mice with colitis declined after FAAH blockade, as did concentrations of systemic and colon inflammatory cytokines. Microarray analysis showed that 26 miRNAs from MLNs and 217 from PPs had a 1.5-fold greater difference in expression after FAAH inhibition. Among them, 8 miRNAs were determined by reverse-transcription polymerase chain reaction (RT-PCR) analysis to have anti-inflammatory properties. Pathway analysis demonstrated that differentially regulated miRNAs target mRNA associated with inflammation. Thus, FAAH-II ameliorates experimental colitis by reducing not only the number of activated T cells but also the frequency of macrophages, neutrophils, and NK/NKT cell, as well as inflammatory miRNAs and cytokine at effector sites in the colon. These studies demonstrate for the first time that FAAH-II inhibitor may suppress colitis through regulation of pro-inflammatory miRNAs expression.

  7. Inhibition of fatty-acid amide hydrolase enhances cannabinoid stress-induced analgesia: sites of action in the dorsolateral periaqueductal gray and rostral ventromedial medulla.

    PubMed

    Suplita, Richard L; Farthing, Jesse N; Gutierrez, Tannia; Hohmann, Andrea G

    2005-12-01

    Recent research in our laboratory has demonstrated that stress activates an endogenous cannabinoid mechanism that suppresses sensitivity to pain [Nature 435 (2005) 1108]. In this work, CB(1) antagonists administered systemically blocked stress-induced analgesia induced by brief, continuous foot-shock. The present studies were conducted to examine the role of cannabinoid CB(1) receptors in the brainstem rostral ventromedial medulla (RVM) and midbrain dorsolateral periaqueductal gray (PAG) in cannabinoid stress-induced analgesia (SIA). Pharmacological blockade of vanilloid TRPV1 receptors with capsazepine, administered systemically, did not alter cannabinoid SIA, suggesting that cannabinoid SIA was not dependent upon TRPV1. Microinjection of the competitive CB(1) antagonist rimonabant (SR141716A) into either the RVM or dorsolateral PAG suppressed stress antinociception in this model. Rimonabant was maximally effective following microinjection into the dorsolateral PAG. The fatty-acid amide hydrolase (FAAH) inhibitor arachidonoyl serotonin (AA-5-HT) was subsequently used to block hydrolysis of endocannabinoids and enhance SIA. Systemic and site-specific injections of AA-5-HT into either the dorsolateral PAG or RVM induced CB(1)-mediated enhancements of SIA. Palmitoyltrifluoromethylketone, a potent inhibitor of FAAH and phospholipase A2 activity, administered systemically, exerted similar effects. In all conditions, the antinociceptive effects of each FAAH inhibitor were completely blocked by coadministration of the CB(1) antagonist rimonabant. The present results provide evidence that a descending cannabinergic neural system is activated by environmental stressors to modulate pain sensitivity in a CB(1)-dependent manner.

  8. Phenotypic and genotypic characterization of peptidoglycan hydrolases of Lactobacillus sakei

    PubMed Central

    Najjari, Afef; Amairi, Houda; Chaillou, Stéphane; Mora, Diego; Boudabous, Abdellatif; Zagorec, Monique; Ouzari, Hadda

    2015-01-01

    Lactobacillus sakei, a lactic acid bacterium naturally found in fresh meat and sea products, is considered to be one of the most important bacterial species involved in meat fermentation and bio-preservation. Several enzymes of Lb. sakei species contributing to microbial safeguarding and organoleptic properties of fermented-meat were studied. However, the specific autolytic mechanisms and associated enzymes involved in Lb. sakei are not well understood. The autolytic phenotype of 22 Lb. sakei strains isolated from Tunisian meat and seafood products was evaluated under starvation conditions, at pH 6.5 and 8.5, and in the presence of different carbon sources. A higher autolytic rate was observed when cells were grown in the presence of glucose and incubated at pH 6.5. Almost all strains showed high resistance to mutanolysin, indicating a minor role of muramidases in Lb. sakei cell lysis. Using Micrococcus lysodeikticus cells as a substrate in activity gels zymogram, peptidoglycan hydrolase (PGH) patterns for all strains was characterized by two lytic bands of ∼80 (B1) and ∼70 kDa (B2), except for strain BMG.167 which harbored two activity signals at a lower MW. Lytic activity was retained in high salt and in acid/basic conditions and was active toward cells of Lb. sakei, Listeria monocytogenes, Listeria ivanovii and Listeria innocua. Analysis of five putative PGH genes found in the Lb. sakei 23 K model strain genome, indicated that one gene, lsa1437, could encode a PGH (N-acetylmuramoyl-L-alanine amidase) containing B1 and B2 as isoforms. According to this hypothesis, strain BMG.167 showed an allelic version of lsa1437 gene deleted of one of the five LysM domains, leading to a reduction in the MW of lytic bands and the high autolytic rate of this strain. Characterization of autolytic phenotype of Lb. sakei should expand the knowledge of their role in fermentation processes where they represent the dominant species. PMID:26843981

  9. Conformational Variability of Organophosphorus Hydrolase upon Soman and Paraoxon Binding

    SciTech Connect

    Gomes, Diego Eb; Lins, Roberto D.; Pascutti, Pedro G.; Lei, Chenghong; Soares, Thereza A.

    2011-12-31

    The bacterial enzyme organophosphorus hydrolase (OPH) exhibits both catalytic and substrate promiscuity. It hydrolyzes bonds in a variety of phosphotriester (P-O), phosphonothioate (P-S), phosphofluoridate (P-F) and phosphonocyanate (F-CN) compounds. However, its catalytic efficiency varies markedly for different substrates, limiting the broad-range application of OPH as catalyst in the bioremediation of pesticides and chemical war agents. In the present study, pK{sub a} calculations and multiple explicit-solvent molecular dynamics (MD) simulations were performed to characterize and contrast the structural dynamics of OPH bound to two substrates hydrolyzed with very distinct catalytic efficiencies: the nerve agent soman (O-pinacolyl-methyl-phosphonofluoridate) and the pesticide paraoxon (diethyl p-nitrophenyl phosphate). pK{sub a} calculations for the substrate-bound and unbound enzyme showed a significant pK{sub a} shift from standard values ({Delta}pK{sub a} = {+-} 3 units) for residues 254His and 275Arg. MD simulations of the doubly protonated 254His revealed a dynamic hydrogen bond network connecting the catalytic residue 301Asp via 254His to 232Asp, 233Asp, 275Arg and 235Asp, and is consistent with a previously postulated proton relay mechanism to ferry protons away from the active site with substrates that do not require activation of the leaving group. Hydrogen bonds between 301Asp and 254His were persistent in the OPH-paraoxon complex but not in the OPH-soman one, suggesting a potential role for such interaction in the more efficient hydrolysis of paraoxon over soman by OPH. These results are in line with previous mutational studies of residue 254His, which led to an increase of the catalytic efficiency of OPH over soman yet decreased its efficiency for paraoxon. In addition, comparative analysis of the molecular trajectories for OPH bound to soman and paraoxon suggests that binding of the latter facilitates the conformational transition of OPH from the

  10. Phenotypic and genotypic characterization of peptidoglycan hydrolases of Lactobacillus sakei.

    PubMed

    Najjari, Afef; Amairi, Houda; Chaillou, Stéphane; Mora, Diego; Boudabous, Abdellatif; Zagorec, Monique; Ouzari, Hadda

    2016-01-01

    Lactobacillus sakei, a lactic acid bacterium naturally found in fresh meat and sea products, is considered to be one of the most important bacterial species involved in meat fermentation and bio-preservation. Several enzymes of Lb. sakei species contributing to microbial safeguarding and organoleptic properties of fermented-meat were studied. However, the specific autolytic mechanisms and associated enzymes involved in Lb. sakei are not well understood. The autolytic phenotype of 22 Lb. sakei strains isolated from Tunisian meat and seafood products was evaluated under starvation conditions, at pH 6.5 and 8.5, and in the presence of different carbon sources. A higher autolytic rate was observed when cells were grown in the presence of glucose and incubated at pH 6.5. Almost all strains showed high resistance to mutanolysin, indicating a minor role of muramidases in Lb. sakei cell lysis. Using Micrococcus lysodeikticus cells as a substrate in activity gels zymogram, peptidoglycan hydrolase (PGH) patterns for all strains was characterized by two lytic bands of ∼80 (B1) and ∼70 kDa (B2), except for strain BMG.167 which harbored two activity signals at a lower MW. Lytic activity was retained in high salt and in acid/basic conditions and was active toward cells of Lb. sakei, Listeria monocytogenes, Listeria ivanovii and Listeria innocua. Analysis of five putative PGH genes found in the Lb. sakei 23 K model strain genome, indicated that one gene, lsa1437, could encode a PGH (N-acetylmuramoyl-L-alanine amidase) containing B1 and B2 as isoforms. According to this hypothesis, strain BMG.167 showed an allelic version of lsa1437 gene deleted of one of the five LysM domains, leading to a reduction in the MW of lytic bands and the high autolytic rate of this strain. Characterization of autolytic phenotype of Lb. sakei should expand the knowledge of their role in fermentation processes where they represent the dominant species.

  11. Hypermethioninemias of genetic and non-genetic origin: A review.

    PubMed

    Mudd, S Harvey

    2011-02-15

    This review covers briefly the major conditions, genetic and non-genetic, sometimes leading to abnormally elevated methionine, with emphasis on recent developments. A major aim is to assist in the differential diagnosis of hypermethioninemia. The genetic conditions are: (1) Homocystinuria due to cystathionine β-synthase (CBS) deficiency. At least 150 different mutations in the CBS gene have been identified since this deficiency was established in 1964. Hypermethioninemia is due chiefly to remethylation of the accumulated homocysteine. (2) Deficient activity of methionine adenosyltransferases I and III (MAT I/III), the isoenzymes the catalytic subunit of which are encoded by MAT1A. Methionine accumulates because its conversion to S-adenosylmethionine (AdoMet) is impaired. (3) Glycine N-methyltrasferase (GNMT) deficiency. Disruption of a quantitatively major pathway for AdoMet disposal leads to AdoMet accumulation with secondary down-regulation of methionine flux into AdoMet. (4) S-adenosylhomocysteine (AdoHcy) hydrolase (AHCY) deficiency. Not being catabolized normally, AdoHcy accumulates and inhibits many AdoMet-dependent methyltransferases, producing accumulation of AdoMet and, thereby, hypermethioninemia. (5) Citrin deficiency, found chiefly in Asian countries. Lack of this mitochondrial aspartate-glutamate transporter may produce (usually transient) hypermethioninemia, the immediate cause of which remains uncertain. (6) Fumarylacetoacetate hydrolase (FAH) deficiency (tyrosinemia type I) may lead to hypermethioninemia secondary either to liver damage and/or to accumulation of fumarylacetoacetate, an inhibitor of the high K(m) MAT. Additional possible genetic causes of hypermethioninemia accompanied by elevations of plasma AdoMet include mitochondrial disorders (the specificity and frequency of which remain to be elucidated). Non-genetic conditions include: (a) Liver disease, which may cause hypermethioninemia, mild, or severe. (b) Low-birth-weight and

  12. Autophagy inhibitors.

    PubMed

    Pasquier, Benoit

    2016-03-01

    Autophagy is a lysosome-dependent mechanism of intracellular degradation. The cellular and molecular mechanisms underlying this process are highly complex and involve multiple proteins, including the kinases ULK1 and Vps34. The main function of autophagy is the maintenance of cell survival when modifications occur in the cellular environment. During the past decade, extensive studies have greatly improved our knowledge and autophagy has exploded as a research field. This process is now widely implicated in pathophysiological processes such as cancer, metabolic, and neurodegenerative disorders, making it an attractive target for drug discovery. In this review, we will summarize the different types of inhibitors that affect the autophagy machinery and provide some potential therapeutic perspectives.

  13. Rehabilitation of faulty kinetic determinations and misassigned glycoside hydrolase family of retaining mechanism ß-xylosidases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We obtained Cx1 from a commercial supplier, whose catalog listed it as a ß-xylosidase of glycoside hydrolase family 43. NMR experiments indicate retention of anomeric configuration in its reaction stereochemistry, opposing the assignment of GH43, which follows an inverting mechanism. Partial protein...

  14. Functional characterization and structural modeling of synthetic polyester-degrading hydrolases from Thermomonospora curvata

    PubMed Central

    2014-01-01

    Thermomonospora curvata is a thermophilic actinomycete phylogenetically related to Thermobifida fusca that produces extracellular hydrolases capable of degrading synthetic polyesters. Analysis of the genome of T. curvata DSM43183 revealed two genes coding for putative polyester hydrolases Tcur1278 and Tcur0390 sharing 61% sequence identity with the T. fusca enzymes. Mature proteins of Tcur1278 and Tcur0390 were cloned and expressed in Escherichia coli TOP10. Tcur1278 and Tcur0390 exhibited an optimal reaction temperature against p-nitrophenyl butyrate at 60°C and 55°C, respectively. The optimal pH for both enzymes was determined at pH 8.5. Tcur1278 retained more than 80% and Tcur0390 less than 10% of their initial activity following incubation for 60 min at 55°C. Tcur0390 showed a higher hydrolytic activity against poly(ε-caprolactone) and polyethylene terephthalate (PET) nanoparticles compared to Tcur1278 at reaction temperatures up to 50°C. At 55°C and 60°C, hydrolytic activity against PET nanoparticles was only detected with Tcur1278. In silico modeling of the polyester hydrolases and docking with a model substrate composed of two repeating units of PET revealed the typical fold of α/β serine hydrolases with an exposed catalytic triad. Molecular dynamics simulations confirmed the superior thermal stability of Tcur1278 considered as the main reason for its higher hydrolytic activity on PET. PMID:25405080

  15. Proteomic Analysis of a Novel Bacillus Jumbo Phage Revealing Glycoside Hydrolase As Structural Component

    PubMed Central

    Yuan, Yihui; Gao, Meiying

    2016-01-01

    Tailed phages with genomes of larger than 200 kbp are classified as Jumbo phages and exhibited extremely high uncharted diversity. The genomic annotation of Jumbo phage is often disappointing because most of the predicted proteins, including structural proteins, failed to make good hits to the sequences in the databases. In this study, 23 proteins of a novel Bacillus Jumbo phage, vB_BpuM_BpSp, were identified as phage structural proteins by the structural proteome analysis, including 14 proteins of unknown function, 5 proteins with predicted function as structural proteins, a glycoside hydrolase, a Holliday junction resolvase, a RNA-polymerase β-subunit, and a host-coding portal protein, which might be hijacked from the host strain during phage virion assembly. The glycoside hydrolase (Gp255) was identified as phage virion component and was found to interact with the phage baseplate protein. Gp255 shows specific lytic activity against the phage host strain GR8 and has high temperature tolerance. In situ peptidoglycan-hydrolyzing activities analysis revealed that the expressed Gp255 and phage structural proteome exhibited glycoside hydrolysis activity against the tested GR8 cell extracts. This study identified the first functional individual structural glycoside hydrolase in phage virion. The presence of activated glycoside hydrolase in phage virions might facilitate the injection of the phage genome during infection by forming pores on the bacterial cell wall. PMID:27242758

  16. Expression of recombinant organophosphorus hydrolase in the original producer of the enzyme, Sphingobium fuliginis ATCC 27551.

    PubMed

    Nakayama, Kosuke; Ohmori, Takeshi; Ishikawa, Satoshi; Iwata, Natsumi; Seto, Yasuo; Kawahara, Kazuyoshi

    2016-05-01

    The plasmid encoding His-tagged organophosphorus hydrolase (OPH) cloned from Sphingobium fuliginis was modified to be transferred back to this bacterium. The replication function of S. amiense plasmid was inserted at downstream of OPH gene, and S. fuliginis was transformed with this plasmid. The transformant produced larger amount of active OPH with His-tag than E. coli.

  17. ORGANOPHOSPHORUS HYDROLASE-BASED AMPEROMETRIC SENSOR: MODULATION OF SENSITIVITY AND SUBSTRATE SELECTIVITY

    EPA Science Inventory

    The detection of organophosphate (OP) insecticides with nitrophenyl substituents is reported using an enzyme electrode composed of Organophosphorus Hydrolase (OPH) and albumin co-immobilized to a nylon net and attached to a carbon paste electrode. The mechanism for this biosen...

  18. BIODEGRADATION OF ORGANOPHOSPHORUS PESTICIDES BY SURFACE-EXPRESSED ORGANOPHOSPHORUS HYDROLASE. (R823663)

    EPA Science Inventory

    Organophosphorus hydrolase (OPH) was displayed and anchored onto the surface of
    Escherichia coli using an Lpp-OmpA fusion system. Production of the fusion proteins in membrane
    fractions was verified by immunoblotting with OmpA antisera. inclusion of the organophosphorus
    ...

  19. Assessment of rat liver microsomal epoxide hydrolase as a marker of hepatocarcinogenesis.

    PubMed

    Kizer, D E; Clouse, J A; Ringer, D P; Hanson-Painton, O; Vaz, A D; Palakodety, R B; Griffin, M J

    1985-05-15

    The influence of eleven xenobiotics on the activity and amount of hepatic microsomal epoxide hydrolase was determined. Activity was assayed using three different substrates after rats were fed, throughout 3 weeks, diets containing one of six hepatocarcinogens, viz. 2-acetylaminofluorene, 3'-methyl-4-dimethylaminoazobenzene, 4'-fluoro-4-dimethylaminoazobenzene, thioacetamide, aflatoxin B1 and ethionine. Five hepatocarcinogens induced activity 4- to 10-fold; ethionine was relatively ineffective as an inducer. Two non-carcinogenic analogues of hepatocarcinogens, viz. fluorene and p-aminoazobenzene, caused no appreciable increase in enzyme activity, but phenobarbital, barbital and 1-naphthylisothiocyanate induced activity 2- to 3-fold. All eleven xenobiotics increased the amount of microsomal epoxide hydrolase 2- to 9-fold when examined immunochemically using either a radial diffusion assay or an enzyme-linked immunosorbent assay (ELISA). Serum glutamic oxaloacetic acid transaminase activity was not appreciably elevated by feeding ten of the xenobiotics, suggesting that inductions were not owing to toxicity. Using ELISA, microsomal epoxide hydrolase was detected in post-microsomal (PM) supernatant fractions from control rat liver, thus confirming an earlier report by Gill et al. [Carcinogenesis 3, 1307 (1982)]. The eleven xenobiotics induced the amount of ELISA-detectable antigen in PM supernatant fractions by 3- to 34-fold. Longer centrifugation of PM supernatant fractions yielded a pellet fraction that contained 92 +/- 1.2% of the ELISA-detectable antigen irrespective of the xenobiotic regimen. Relationships between xenobiotic induction of microsomal epoxide hydrolase activity and amount and hepatocarcinogenesis are discussed.

  20. A novel meta-cleavage product hydrolase from Flavobacterium sp. ATCC27551

    SciTech Connect

    Khajamohiddin, Syed; Babu, Pakala Suresh; Chakka, Deviprasanna; Merrick, Mike; Bhaduri, Anirban; Sowdhamini, Ramanathan; Siddavattam, Dayananda . E-mail: sdsl@uohyd.ernet.in

    2006-12-22

    The organophosphate degrading (opd) gene cluster of plasmid pPDL2 of Flavobacterium sp. ATCC27551 contains a novel open-reading frame, orf243. This was predicted to encode an {alpha}/{beta} hydrolase distantly related to the meta-fission product (MFP) hydrolases such as XylF, PhnD, and CumD. By homology modeling Orf243 has most of the structural features of MFP hydrolases including the characteristic active site catalytic triad. The purified protein (designated MfhA) is a homotetramer and shows similar affinity for 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD), 2-hydroxymuconic semialdehyde (HMSA), and 2-hydroxy-5-methylmuconic semialdehyde (HMMSA), the meta-fission products of 3-methyl catechol, catechol, and 4-methyl catechol. The unique catalytic properties of MfhA and the presence near its structural gene of cis-elements required for transposition suggest that mfhA has evolved towards encoding a common hydrolase that can act on meta-fission products containing either aldehyde or ketone groups.

  1. High-Throughput In Vitro Glycoside Hydrolase (HIGH) Screening for Enzyme Discovery

    SciTech Connect

    Kim, Tae-Wan; Chokhawala, Harshal A.; Hess, Matthias; Dana, Craig M.; Baer, Zachary; Sczyrba, Alexander; Rubin, Edward M.; Blanch, Harvey W.; Clark, Douglas S.

    2011-09-16

    A high-throughput protein-expression and screening method (HIGH method, see picture) provides a rapid approach to the discovery of active glycoside hydrolases in environmental samples. Finally, HIGH screening combines cloning, protein expression, and enzyme hydrolysis in one pot; thus, the entire process from gene expression to activity detection requires only three hours.

  2. Encoded Library Synthesis Using Chemical Ligation and the Discovery of sEH Inhibitors from a 334-Million Member Library.

    PubMed

    Litovchick, Alexander; Dumelin, Christoph E; Habeshian, Sevan; Gikunju, Diana; Guié, Marie-Aude; Centrella, Paolo; Zhang, Ying; Sigel, Eric A; Cuozzo, John W; Keefe, Anthony D; Clark, Matthew A

    2015-06-10

    A chemical ligation method for construction of DNA-encoded small-molecule libraries has been developed. Taking advantage of the ability of the Klenow fragment of DNA polymerase to accept templates with triazole linkages in place of phosphodiesters, we have designed a strategy for chemically ligating oligonucleotide tags using cycloaddition chemistry. We have utilized this strategy in the construction and selection of a small molecule library, and successfully identified inhibitors of the enzyme soluble epoxide hydrolase.

  3. Encoded Library Synthesis Using Chemical Ligation and the Discovery of sEH Inhibitors from a 334-Million Member Library

    NASA Astrophysics Data System (ADS)

    Litovchick, Alexander; Dumelin, Christoph E.; Habeshian, Sevan; Gikunju, Diana; Guié, Marie-Aude; Centrella, Paolo; Zhang, Ying; Sigel, Eric A.; Cuozzo, John W.; Keefe, Anthony D.; Clark, Matthew A.

    2015-06-01

    A chemical ligation method for construction of DNA-encoded small-molecule libraries has been developed. Taking advantage of the ability of the Klenow fragment of DNA polymerase to accept templates with triazole linkages in place of phosphodiesters, we have designed a strategy for chemically ligating oligonucleotide tags using cycloaddition chemistry. We have utilized this strategy in the construction and selection of a small molecule library, and successfully identified inhibitors of the enzyme soluble epoxide hydrolase.

  4. Characteristics, protein engineering and applications of microbial thermostable pullulanases and pullulan hydrolases.

    PubMed

    Nisha, M; Satyanarayana, T

    2016-07-01

    Pullulan hydrolyzing enzymes are endoacting, classified based on the substrate specificity and hydrolysis products as pullulanases (type I and II) and pullulan hydrolases (type I, II and III). Pullulanases and pullulan hydrolase type I are produced by bacteria and archaea. Among bacteria, many mesophilic, thermophilic and hyperthermophilic bacteria produce pullulanases and neopullulanases. While pullulan hydrolase type II and type III are produced by fungi and archaea, respectively. These are multi-domain proteins with three conserved catalytic acidic residues of the glycosyl hydrolases. The recent advances in molecular biology and protein engineering via mutagenesis and truncation led to improvement in thermostability, catalytic activity and substrate specificity. Pullulanases are debranching enzymes, which are widely employed in starch saccharification that minimizes the use of glucoamylase (approx. 50 %) and reduces the total reaction time of the industrial starch conversion process. The thermostable amylopullulanases are useful in one-step starch liquefaction and saccharification, which replaces amylolytic enzymes like α-amylase and glucoamylase, thus resulting in the reduction in the cost of sugar production. The enzymes also find application in making resistant starches and as an antistale in bread making. Panose and isopanose containing syrups are useful as prebiotics, while panose has also been reported to display anticarcinogenic activity. This review focuses on the distinguishing features of these enzymes based on the analysis of amino acid sequences and domain structure, besides highlighting recent advances in the molecular biology and protein engineering for enhancing their thermostability, catalytic activity and substrate specificity. This review also briefly summarizes the potential applications of pullulanases and pullulan hydrolases.

  5. γ-PGA Hydrolases of Phage Origin in Bacillus subtilis and Other Microbial Genomes

    PubMed Central

    Mamberti, Stefania; Prati, Paola; Cremaschi, Paolo; Seppi, Claudio; Morelli, Carlo F.; Galizzi, Alessandro; Fabbi, Massimo; Calvio, Cinzia

    2015-01-01

    Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the class Bacilli which forms a shield able to protect bacteria from phagocytosis and phages. Few enzymes are known to degrade γ-PGA; among them is a phage-encoded γ-PGA hydrolase, PghP. The supposed role of PghP in phages is to ensure access to the surface of bacterial cells by dismantling the γ-PGA barrier. We identified four unannotated B. subtilis genes through similarity of their encoded products to PghP; in fact these genes reside in prophage elements of B. subtilis genome. The recombinant products of two of them demonstrate efficient polymer degradation, confirming that sequence similarity reflects functional homology. Genes encoding similar γ-PGA hydrolases were identified in phages specific for the order Bacillales and in numerous microbial genomes, not only belonging to that order. The distribution of the γ-PGA biosynthesis operon was also investigated with a bioinformatics approach; it was found that the list of organisms endowed with γ-PGA biosynthetic functions is larger than expected and includes several pathogenic species. Moreover in non-Bacillales bacteria the predicted γ-PGA hydrolase genes are preferentially found in species that do not have the genetic asset for polymer production. Our findings suggest that γ-PGA hydrolase genes might have spread across microbial genomes via horizontal exchanges rather than via phage infection. We hypothesize that, in natural habitats rich in γ-PGA supplied by producer organisms, the availability of hydrolases that release glutamate oligomers from γ-PGA might be a beneficial trait under positive selection. PMID:26158264

  6. Different types of dienelactone hydrolase in 4-fluorobenzoate-utilizing bacteria.

    PubMed Central

    Schlömann, M; Schmidt, E; Knackmuss, H J

    1990-01-01

    Of various benzoate-utilizing bacteria tested, Alcaligenes eutrophus 335, A. eutrophus H16, A. eutrophus JMP222, A. eutrophus JMP134, Alcaligenes strain A7, and Pseudomonas cepacia were able to grow with 4-fluorobenzoate as the sole source of carbon and energy. P. cepacia also utilizes 3-fluorobenzoate. Except for A. eutrophus JMP134, which is known to grow with 2,4-dichlorophenoxyacetate and 3-chlorobenzoate (R. H. Don and J. M. Pemberton, J. Bacteriol. 145:681-686, 1981), the strains were unable to grow at the expense of these compounds or 4-chlorobenzoate. Assays of cell extracts revealed that all strains express dienelactone hydrolase and maleylacetate reductase activities in addition to enzymes of the catechol branch of the 3-oxoadipate pathway when growing with 4-fluorobenzoate. Induction of dienelactone hydrolase and maleylacetate reductase apparently is not necessarily connected to synthesis of catechol 1,2-dioxygenase type II and chloromuconate cycloisomerase activities, which are indispensable for the degradation of chlorocatechols. Substrate specificities of the dienelactone hydrolases provisionally differentiate among three types of this activity. (i) Extracts of A. eutrophus 335, A. eutrophus H16, A. eutrophus JMP222, and Alcaligenes strain A7 convert trans-4-carboxymethylenebut-2-en-4-olide (trans-dienelactone) much faster than the cis-isomer (type I). (ii) The enzyme present in P. cepacia shows the opposite preference for the isomeric substrates (type II). (iii) Cell extracts of A. eutrophus JMP134, as well as purified dienelactone hydrolase from Pseudomonas strain B13 (E. Schmidt and H.-J. Knackmuss, Biochem. J. 192:339-347, 1980), hydrolyze both dienelactones at rates that are of the same order of magnitude (type III). This classification implies that A. eutrophus JMP134 possesses at least two different dienelactone hydrolases, one of type III encoded by the plasmid pJP4 and one of type I, which is also present in the cured strain JMP222. PMID

  7. Post-synthetic modification of plant cell walls by expression of microbial hydrolases in the apoplast.

    PubMed

    Pogorelko, Gennady; Fursova, Oksana; Lin, Ming; Pyle, Eric; Jass, Johanna; Zabotina, Olga A

    2011-11-01

    The systematic creation of defined cell wall modifications in the model plant Arabidopsis thaliana by expression of microbial hydrolases with known specific activities is a promising approach to examine the impacts of cell wall composition and structure on both plant fitness and cell wall recalcitrance. Moreover, this approach allows the direct evaluation in living plants of hydrolase specificity, which can differ from in vitro specificity. To express genes encoding microbial hydrolases in A. thaliana, and target the hydrolases to the apoplast compartment, we constructed an expression cassette composed of the Cauliflower Mosaic Virus 35S RNA promoter, the A. thaliana β-expansin signal peptide, and the fluorescent marker protein YFP. Using this construct we successfully introduced into Colombia-0 plants three Aspergillus nidulans hydrolases, β-xylosidase/α-arabinosidase, feruloyl esterase, acetylxylan esterase, and a Xanthomonas oryzae putative a-L: -arabinofuranosidase. Fusion with YFP permitted quick and easy screening of transformants, detection of apoplastic localization, and protein size confirmation. Compared to wild-type Col-0, all transgenic lines showed a significant increase in the corresponding hydrolytic activity in the apoplast and changes in cell wall composition. Examination of hydrolytic activity in the transgenic plants also showed, for the first time, that the X. oryzae gene indeed encoded an enzyme with α-L: -arabinofuranosidase activity. None of the transgenic plants showed a visible phenotype; however, the induced compositional changes increased the degradability of biomass from plants expressing feruloyl esterase and β-xylosidase/α-arabinosidase. Our results demonstrate the viability of creating a set of transgenic A. thaliana plants with modified cell walls to use as a toolset for investigation of how cell wall composition contributes to recalcitrance and affects plant fitness.

  8. Supplementing with Non-Glycoside Hydrolase Proteins Enhances Enzymatic Deconstruction of Plant Biomass

    PubMed Central

    Su, Xiaoyun; Zhang, Jing; Mackie, Roderick I.; Cann, Isaac K. O.

    2012-01-01

    The glycoside hydrolases (GH) of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry. PMID:22952777

  9. Cell- and ligand-specific dephosphorylation of acid hydrolases: Evidence that the mannose 6-phosphatase is controlled by compartmentalization

    SciTech Connect

    Einstein, R.; Gabel, C.A. )

    1991-01-01

    Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum. To investigate the mechanism by which dephosphorylation competence is regulated, the dephosphorylation of individual acid hydrolases was studied in Man 6-P/IGF II receptor-positive and -deficient cell lines. 125I-labeled Man 6-P-containing acid hydrolases were proteolytically processed but remained phosphorylated when endocytosed by receptor-positive L cells maintained in the absence of serum; after the addition of serum, however, the cell-associated hydrolases were dephosphorylated. Individual hydrolases were dephosphorylated at distinct rates and to different extents. In contrast, the same hydrolases were dephosphorylated equally and completely after entry into Man 6-P/IGF II receptor-positive Chinese hamster ovary (CHO) cells. The dephosphorylation competence of Man 6-P/IGF II receptor-deficient mouse J774 cells was more limited. beta-Glucuronidase produced by these cells underwent a limited dephosphorylation in transit to lysosomes such that diphosphorylated oligosaccharides were converted to monophosphorylated species. The overall quantity of phosphorylated oligosaccharides associated with the enzyme, however, did not decrease within the lysosomal compartment. Likewise, beta-glucuronidase was not dephosphorylated when introduced into J774 cells via Fc receptor-mediated endocytosis. The CHO and J774 cell lysosomes, therefore, display opposite extremes with respect to their capacity to dephosphorylate acid hydrolases; within CHO cell lysosomes acid hydrolases are rapidly and efficiently dephosphorylated, but within J774 cell lysosomes the same acid hydrolases remain phosphorylated.

  10. Platelet aggregation Inhibitors from Hametophagous Animals

    PubMed Central

    Francischetti, Ivo M. B.

    2010-01-01

    Salivary glands from blood-sucking animals (e.g., mosquitoes, bugs, sandflies, fleas, ticks, leeches, hookworms, bats) are a rich source of bioactive molecules that counteract hemostasis in a redundant and synergistic manner. This review discusses recent progress in the identification of salivary inhibitors of platelet aggregation, their molecular characterization, and detailed mechanism of action. Diversity of inhibitors is remarkable, with distinct families of proteins characterized as apyrases that enzymatically degrade ADP or as collagen-binding proteins that prevent its interaction with vWF, or platelet integrin α2β1 or GPVI. Molecules that bind ADP, TXA2, epinephrine, or serotonin with high affinity have also been cloned, expressed, and their structure determined. In addition, a repertoire of antithrombins and an increasingly number of RGD and non-RGD disintegrins targeting platelet αIIbβ3 have been reported. Moreover, metalloproteases with fibrinogen(olytic) activity and PAF phosphorylcholine hydrolase are enzymes that have been recruited to the salivary gland to block platelet aggregation. Platelet inhibitory prostaglandins, lysophosphatydilcholine, adenosine, and nitric oxide (NO)-carrying proteins are other notable examples of molecules from hematophagous salivary secretions (herein named sialogenins) with antihemostatic properties. Sialogenins have been employed as tools in biochemistry and cell biology and also display potential therapeutic applications. PMID:20035779

  11. Positioning of aminopeptidase inhibitors in next generation cancer therapy.

    PubMed

    Hitzerd, Sarina M; Verbrugge, Sue Ellen; Ossenkoppele, Gert; Jansen, Gerrit; Peters, Godefridus J

    2014-04-01

    Aminopeptidases represent a class of (zinc) metalloenzymes that catalyze the cleavage of amino acids nearby the N-terminus of polypeptides, resulting in hydrolysis of peptide bonds. Aminopeptidases operate downstream of the ubiquitin-proteasome pathway and are implicated in the final step of intracellular protein degradation either by trimming proteasome-generated peptides for antigen presentation or full hydrolysis into free amino acids for recycling in renewed protein synthesis. This review focuses on the function and subcellular location of five key aminopeptidases (aminopeptidase N, leucine aminopeptidase, puromycin-sensitive aminopeptidase, leukotriene A4 hydrolase and endoplasmic reticulum aminopeptidase 1/2) and their association with different diseases, in particular cancer and their current position as target for therapeutic intervention by aminopeptidase inhibitors. Historically, bestatin was the first prototypical aminopeptidase inhibitor that entered the clinic 35 years ago and is still used for the treatment of lung cancer. More recently, new generation aminopeptidase inhibitors became available, including the aminopeptidase inhibitor prodrug tosedostat, which is currently tested in phase II clinical trials for acute myeloid leukemia. Beyond bestatin and tosedostat, medicinal chemistry has emerged with additional series of potential aminopeptidases inhibitors which are still in an early phase of (pre)clinical investigations. The expanded knowledge of the unique mechanism of action of aminopeptidases has revived interest in aminopeptidase inhibitors for drug combination regimens in anti-cancer treatment. In this context, this review will discuss relevant features and mechanisms of action of aminopeptidases and will also elaborate on factors contributing to aminopeptidase inhibitor efficacy and/or loss of efficacy due to drug resistance-related phenomena. Together, a growing body of data point to aminopeptidase inhibitors as attractive tools for

  12. Full Fatty Acid Amide Hydrolase Inhibition Combined with Partial Monoacylglycerol Lipase Inhibition: Augmented and Sustained Antinociceptive Effects with Reduced Cannabimimetic Side Effects in Mice

    PubMed Central

    Ghosh, Sudeshna; Kinsey, Steven G.; Liu, Qing-song; Hruba, Lenka; McMahon, Lance R.; Grim, Travis W.; Merritt, Christina R.; Wise, Laura E.; Abdullah, Rehab A.; Selley, Dana E.; Sim-Selley, Laura J.; Cravatt, Benjamin F.

    2015-01-01

    Inhibition of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the primary hydrolytic enzymes for the respective endocannabinoids N-arachidonoylethanolamine (AEA) and 2-arachidonylglycerol (2-AG), produces antinociception but with minimal cannabimimetic side effects. Although selective inhibitors of either enzyme often show partial efficacy in various nociceptive models, their combined blockade elicits augmented antinociceptive effects, but side effects emerge. Moreover, complete and prolonged MAGL blockade leads to cannabinoid receptor type 1 (CB1) receptor functional tolerance, which represents another challenge in this potential therapeutic strategy. Therefore, the present study tested whether full FAAH inhibition combined with partial MAGL inhibition would produce sustained antinociceptive effects with minimal cannabimimetic side effects. Accordingly, we tested a high dose of the FAAH inhibitor PF-3845 (N-​3-​pyridinyl-​4-​[[3-​[[5-​(trifluoromethyl)-​2-​pyridinyl]oxy]phenyl]methyl]-​1-​piperidinecarboxamide; 10 mg/kg) given in combination with a low dose of the MAGL inhibitor JZL184 [4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate] (4 mg/kg) in mouse models of inflammatory and neuropathic pain. This combination of inhibitors elicited profound increases in brain AEA levels (>10-fold) but only 2- to 3-fold increases in brain 2-AG levels. This combination produced significantly greater antinociceptive effects than single enzyme inhibition and did not elicit common cannabimimetic effects (e.g., catalepsy, hypomotility, hypothermia, and substitution for Δ9-tetrahydrocannabinol in the drug-discrimination assay), although these side effects emerged with high-dose JZL184 (i.e., 100 mg/kg). Finally, repeated administration of this combination did not lead to tolerance to its antiallodynic actions in the carrageenan assay or CB1 receptor functional tolerance. Thus, full FAAH inhibition combined

  13. Cathepsin D-mediated yolk protein degradation is blocked by acid phosphatase inhibitors.

    PubMed

    Fialho, Eliane; Nakamura, Angelica; Juliano, Luiz; Masuda, Hatisaburo; Silva-Neto, Mário A C

    2005-04-15

    Vitellin (VT) is a lipoglycophosphoprotein stored inside the eggs of every oviparous organism during oogenesis. In the blood-sucking bug Rhodnius prolixus, VT is deposited inside growing oocytes together with two acid hydrolases: acid phosphatase (AP) and cathepsin D (CD). Egg fertilization triggers AP activity and VT proteolysis in vivo [Insect Biochem. Mol. Biol. 2002 (32) 847]. Here, we show that CD is the main protease targeting VT proteolysis during egg development. CD activity in total egg homogenates is blocked by the classical aspartyl protease inhibitor, pepstatin A. Surprisingly, AP inhibitors such as NaF, Na+/K+ tartrate, and inorganic phosphate also block VT proteolysis, whereas this effect is not observed when tyrosine phosphatase inhibitors such as vanadate and phenylarsine oxide or an inhibitor of alkaline phosphatases such as levamisole are used in a VT proteolysis assay. NaF concentrations that block isolated AP activity do not affect the activity of partially purified CD. Therefore, a specific repressor of VT proteolysis must be dephosphorylated by AP in vivo. In conclusion, these results demonstrate for the first time that acid hydrolases act cooperatively to promote yolk degradation during egg development in arthropods.

  14. Detoxification of Acetylcholinesterase Inhibitors.

    DTIC Science & Technology

    1987-02-19

    from Pseudomronas diminuta and Flavobacterium species. It has been posible to -isolate the enzyme in associaTowith a sm1Th-mbrEFane fraction and initiate...Toxicol. 34:809-814. 2. Identification of a Plasmid-borne Parathion Hydrolase Gene from Flavobacterium by Southern Hybridization with opd from...Pseudcmonas diwinuta, 9; and Flavobacterium , 10). The genes have been designated q d (organophosphate degrading) and have were transferred to plasnid

  15. Identification of selective covalent inhibitors of platelet activating factor acetylhydrolase 1B2 from the screening of an oxadiazolone-capped peptoid-azapeptoid hybrid library.

    PubMed

    Sarma, Bani Kanta; Liu, Xiaodan; Kodadek, Thomas

    2016-09-01

    A potent and selective inhibitor of platelet-activating factor acetylhydrolase 1B2 (PAFAH1B2) is described. The compound was derived by improvement of a modest affinity primary hit isolated from the screening of a bead-displayed peptoid-azapeptoid hybrid library tethered to an oxadiazolone 'warhead'. The oxadiazolone moiety of the inhibitors was found to react covalently with the active site serine residue of PAFAH1B2. This screening strategy may be useful for the identification of many selective, covalent inhibitors of serine hydrolases.

  16. Blocking of fatty acid amide hydrolase activity with PF-04457845 in human brain: a positron emission tomography study with the novel radioligand [(11)C]CURB.

    PubMed

    Boileau, Isabelle; Rusjan, Pablo M; Williams, Belinda; Mansouri, Esmaeil; Mizrahi, Romina; De Luca, Vincenzo; Johnson, Douglas S; Wilson, Alan A; Houle, Sylvain; Kish, Stephen J; Tong, Junchao

    2015-11-01

    Positron emission tomography with [(11)C]CURB was recently developed to quantify fatty acid amide hydrolase (FAAH), the enzyme responsible for hydrolyzing the endocannabinoid anandamide. This study investigated the test-retest reliability of [(11)C]CURB as well as its in vivo specificity and the validity of the kinetic model by using the highly specific FAAH inhibitor, PF-04457845. Five healthy volunteers completed test-retest [(11)C]CURB scans 1 to 2 months apart and six subjects completed baseline and blocking scans on the same day after PF-04457845 (p.o.) administration (1, 4, or 20 mg; n=2 each). The composite parameter λk3 (an index of FAAH activity, λ=K1/k2) was estimated using an irreversible two-tissue compartment model with plasma input function. There were no clinically observable responses to oral PF-04457845 or [(11)C]CURB injection. Oral administration of PF-04457845 reduced [(11)C]CURB binding to a homogeneous level at all three doses, with λk3 values decreased by ⩾91%. Excellent reproducibility and good reliability (test-retest variability=9%; intraclass correlation coefficient=0.79) were observed across all regions of interest investigated. Our findings suggest that λk3/[(11)C]CURB is a reliable, highly sensitive, and selective tool to measure FAAH activity in human brain in vivo. Moreover, PF-04457845 is a highly potent FAAH inhibitor (>95% inhibition at 1 mg) in living human brain.

  17. High-performance liquid chromatography-tandem mass spectrometry assay of fatty acid amide hydrolase (FAAH) in blood: FAAH inhibition as clinical biomarker.

    PubMed

    Yapa, Udeni; Prusakiewicz, Jeffery J; Wrightstone, Ann D; Christine, Lori J; Palandra, Joe; Groeber, Elizabeth; Wittwer, Arthur J

    2012-02-15

    Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for the degradation of the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA). FAAH inhibitors may be useful in treating many disorders involving inflammation and pain. Although brain FAAH may be the relevant target for inhibition, rat studies show a correlation between blood and brain FAAH inhibition, allowing blood FAAH activity to be used as a target biomarker. Building on experience with a rat leukocyte FAAH activity assay using [³H]AEA, we have developed a human leukocyte assay using stably labeled [²H₄]AEA as substrate. The deuterium-labeled ethanolamine reaction product ([²H₄]EA) was analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in the positive electrospray ionization (ESI) mode. The response for [²H₄]EA was linear from 10 nM to 10 μM, and the analysis time was less than 6 min/sample. Results using the [²H₄]AEA and HPLC-MS/MS method agreed well with those obtained using the [³H]AEA radiometric assay. In addition to using a nonradioactive substrate, the HPLC-MS/MS method had increased sensitivity with lower background. Importantly, the assay preserved partial FAAH inhibition resulting from ex vivo treatment with a time-dependent irreversible inhibitor, suggesting its utility with clinical samples. The assay has been used to profile the successful inhibition of FAAH in recent clinical trials.

  18. De novo synthesis of ubiquitin carboxyl-terminal hydrolase isozyme l1 in rostral ventrolateral medulla is crucial to survival during mevinphos intoxication.

    PubMed

    Chang, Chi; Chang, Alice Y W; Chan, Samuel H H

    2004-12-01

    Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1) is a deubiquitinating enzyme that is responsible for making ubiquitin, which is required to target proteins for degradation by the ubiquitin-proteasome pathway in neurons, available. We investigated whether UCH-L1 plays a neuroprotective role at the rostral ventrolateral medulla (RVLM), the origin of sympathetic neurogenic vasomotor tone in the medulla oblongata where the organophosphate insecticide mevinphos (Mev) acts to elicit cardiovascular toxicity. In Sprague-Dawley rats maintained under propofol anesthesia, Mev (960 microg/kg, i.v.) induced a parallel and progressive augmentation in UCH-L1 or ubiquitin expression at the ventrolateral medulla during the course of Mev intoxication. The increase in UCH-L1 level was significantly blunted on pretreatment with bilateral microinjection into the RVLM of a transcription inhibitor, actinomycin D (5 nmol), or a translation inhibitor, cycloheximide (20 nmol). Compared with aCSF or sense oligonucleotide (100 pmol) pretreatment, microinjection of an antisense uch-L1 oligonucleotide (100 pmol) bilaterally into the RVLM significantly increased mortality, reduced the duration of the "pro-life" phase, blunted the increase in ubiquitin expression in ventrolateral medulla, and augmented the induced hypotension in rats that received Mev. These findings suggest that de novo synthesis of UCH-L1, leading to an enhanced disassembly of ubiquitin-protein conjugates in the RVLM, is essential to maintenance of the "pro-life" phase of Mev intoxication via prevention of cardiovascular depression, leading to neuroprotection.

  19. Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

    SciTech Connect

    Germane, Katherine L.; Servinsky, Matthew D.; Gerlach, Elliot S.; Sund, Christian J.; Hurley, Margaret M.

    2015-07-29

    The crystal structure of the protein product of the C. acetobutylicum ATCC 824 gene CA-C0359 is structurally similar to YteR, an unsaturated rhamnogalacturonyl hydrolase from B. subtilis strain 168. Substrate modeling and electrostatic studies of the active site of the structure of CA-C0359 suggests that the protein can now be considered to be part of CAZy glycoside hydrolase family 105. Clostridium acetobutylicum ATCC 824 gene CA-C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA-C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry (http://scripts.iucr.org/cgi-bin/cr.cgi?rm)) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA-C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate

  20. The putative α/β-hydrolases of Dietzia cinnamea P4 strain as potential enzymes for biocatalytic applications.

    PubMed

    Procópio, Luciano; Macrae, Andrew; van Elsas, Jan Dirk; Seldin, Lucy

    2013-03-01

    The draft genome of the soil actinomycete Dietzia cinnamea P4 reveals a versatile group of α/β-hydrolase fold enzymes. Phylogenetic and comparative sequence analyses were used to classify the α/β-hydrolases of strain P4 into six different groups: (i) lipases, (ii) esterases, (iii) epoxide hydrolases, (iv) haloacid dehalogenases, (v) C-C breaking enzymes and (vi) serine peptidases. The high number of lipases/esterases (41) and epoxide hydrolase enzymes (14) present in the relatively small (3.6 Mb) P4 genome is unusual; it is likely to be linked to the survival of strain P4 in its natural environment. Strain P4 is thus equipped with a large number of genes which would appear to confer survivability in harsh hot tropical soil. As such, this highly resilient soil bacterial strain provides an interesting genome for enzyme mining for applications in the field of biotransformations of polymeric compounds.

  1. Structure Determination and Characterization of the Vitamin B[superscript 6] Degradative Enzyme (E)-2-(Acetamidomethylene)succinate Hydrolase

    SciTech Connect

    McCulloch, Kathryn M.; Mukherjee, Tathagata; Begley, Tadhg P.; Ealick, Steven E.

    2010-06-22

    The gene identification and kinetic characterization of (E)-2-(acetamidomethylene)succinate (E-2AMS) hydrolase has recently been described. This enzyme catalyzes the final reaction in the degradation of vitamin B{sub 6} and produces succinic semialdehyde, acetate, ammonia, and carbon dioxide from E-2AMS. The structure of E-2AMS hydrolase was determined to 2.3 {angstrom} using SAD phasing. E-2AMS hydrolase is a member of the {alpha}/{beta} hydrolase superfamily and utilizes a serine/histidine/aspartic acid catalytic triad. Mutation of either the nucleophilic serine or the aspartate resulted in inactive enzyme. Mutation of an additional serine residue in the active site causes the enzyme to be unstable and is likely structurally important. The structure also provides insight into the mechanism of hydrolysis of E-2AMS and identifies several potential catalytically important residues.

  2. Effect of Soluble Epoxide Hydrolase on the Modulation of Coronary Reactive Hyperemia: Role of Oxylipins and PPARγ

    PubMed Central

    Hanif, Ahmad; Edin, Matthew L.; Zeldin, Darryl C.; Morisseau, Christophe; Nayeem, Mohammed A.

    2016-01-01

    Coronary reactive hyperemia (CRH) is a physiological response to ischemic insult that prevents the potential harm associated with an interruption of blood supply. The relationship between the pharmacologic inhibition of soluble epoxide hydrolase (sEH) and CRH response to a brief ischemia is not known. sEH is involved in the main catabolic pathway of epoxyeicosatrienoic acids (EETs), which are converted into dihydroxyeicosatrienoic acids (DHETs). EETs protect against ischemia/reperfusion injury and have numerous beneficial physiological effects. We hypothesized that inhibition of sEH by t-AUCB enhances CRH in isolated mouse hearts through changing the oxylipin profiles, including an increase in EETs/DHETs ratio. Compared to controls, t-AUCB–treated mice had increased CRH, including repayment volume (RV), repayment duration, and repayment/debt ratio (p < 0.05). Treatment with t-AUCB significantly changed oxylipin profiles, including an increase in EET/DHET ratio, increase in EpOME/DiHOME ratio, increase in the levels of HODEs, decrease in the levels of mid-chain HETEs, and decrease in prostanoids (p < 0.05). Treatment with MS-PPOH (CYP epoxygenase inhibitor) reduced CRH, including RV (p < 0.05). Involvement of PPARγ in the modulation of CRH was demonstrated using a PPARγ-antagonist (T0070907) and a PPARγ-agonist (rosiglitazone). T0070907 reduced CRH (p < 0.05), whereas rosiglitazone enhanced CRH (p < 0.05) in isolated mouse hearts compared to the non-treated. These data demonstrate that sEH inhibition enhances, whereas CYP epoxygenases-inhibition attenuates CRH, PPARγ mediate CRH downstream of the CYP epoxygenases-EET pathway, and the changes in oxylipin profiles associated with sEH-inhibition collectively contributed to the enhanced CRH. PMID:27583776

  3. Impaired neurogenesis by HIV-1-Gp120 is rescued by genetic deletion of fatty acid amide hydrolase enzyme

    PubMed Central

    Avraham, H K; Jiang, S; Fu, Y; Rockenstein, E; Makriyannis, A; Wood, J; Wang, L; Masliah, E; Avraham, S

    2015-01-01

    Background and Purpose The HIV-envelope glycoprotein Gp120 is involved in neuronal injury and is associated with neuro-AIDS pathogenesis in the brain. Endocannabinoids are important lipid ligands in the CNS regulating neural functions, and their degeneration is controlled by hydrolysing enzymes such as the fatty acid amide hydrolase (FAAH). Here, we examined whether in vivo genetic deletion of Faah gene prevents HIV-1 Gp120-mediated effects on neurogenesis. Experimental Approach We generated new GFAP/Gp120 transgenic (Tg) mice that have genetic deletion of Faah gene by mating glial fribillary acidic protein (GFAP)/Gp120 Tg mice with Faah−/− mice. Neurogenesis and cell death were assessed by immunocytochemical analysis. Key Results Endocannabinoid levels in the brain of the double GFAP/Gp120//Faah−/− mice were similar to those observed in Faah−/− mice. However, unlike the impaired neurogenesis observed in GFAP/Gp120 Tg mice and Faah−/− mice, these GFAP/Gp120//Faah-/ mice showed significantly improved neurogenesis in the hippocampus, indicated by a significant increase in neuroblasts and neuronal cells, an increase in BrdU+ cells and doublecortin positive cells (DCX+), and an increase in the number of PCNA. Furthermore, a significant decrease in astrogliosis and gliogenesis was observed in GFAP/Gp120//Faah−/−mice and neurogenesis was stimulated by neural progenitor cells (NPCs) and/or the newly formed NPC niches characterized by increased COX-2 expression and elevated levels of PGE2. Conclusions and Implications In vivo genetic ablation of Faah, resulted in enhanced neurogenesis through modulation of the newly generated NPC niches in GFAP/Gp120//Faah−/− mice. This suggests a novel approach of using FAAH inhibitors to enhance neurogenesis in HIV-1 infected brain. PMID:24571443

  4. Effect of Tris, MOPS, and phosphate buffers on the hydrolysis of polyethylene terephthalate films by polyester hydrolases.

    PubMed

    Schmidt, Juliane; Wei, Ren; Oeser, Thorsten; Belisário-Ferrari, Matheus Regis; Barth, Markus; Then, Johannes; Zimmermann, Wolfgang

    2016-09-01

    The enzymatic degradation of polyethylene terephthalate (PET) occurs at mild reaction conditions and may find applications in environmentally friendly plastic waste recycling processes. The hydrolytic activity of the homologous polyester hydrolases LC cutinase (LCC) from a compost metagenome and TfCut2 from Thermobifida fusca KW3 against PET films was strongly influenced by the reaction medium buffers tris(hydroxymethyl)aminomethane (Tris), 3-(N-morpholino)propanesulfonic acid (MOPS), and sodium phosphate. LCC showed the highest initial hydrolysis rate of PET films in 0.2 m Tris, while the rate of TfCut2 was 2.1-fold lower at this buffer concentration. At a Tris concentration of 1 m, the hydrolysis rate of LCC decreased by more than 90% and of TfCut2 by about 80%. In 0.2 m MOPS or sodium phosphate buffer, no significant differences in the maximum initial hydrolysis rates of PET films by both enzymes were detected. When the concentration of MOPS was increased to 1 m, the hydrolysis rate of LCC decreased by about 90%. The activity of TfCut2 remained low compared to the increasing hydrolysis rates observed at higher concentrations of sodium phosphate buffer. In contrast, the activity of LCC did not change at different concentrations of this buffer. An inhibition study suggested a competitive inhibition of TfCut2 and LCC by Tris and MOPS. Molecular docking showed that Tris and MOPS interfered with the binding of the polymeric substrate in a groove located at the protein surface. A comparison of the K i values and the average binding energies indicated MOPS as the stronger inhibitor of the both enzymes.

  5. Ubiquitin C-terminal hydrolase-L1 protects cystic fibrosis transmembrane conductance regulator from early stages of proteasomal degradation.

    PubMed

    Henderson, Mark J; Vij, Neeraj; Zeitlin, Pamela L

    2010-04-09

    DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) degradation involves ubiquitin modification and efficient proteasomal targeting of the nascent misfolded protein. We show that a deubiquitinating enzyme, ubiquitin C-terminal hydrolase-L1 (UCH-L1), is highly expressed in cystic fibrosis (CF) airway epithelial cells in vitro and in vivo. We hypothesized that the elevation in UCH-L1 in CF cells represents a cellular adaptation to counterbalance excessive proteasomal degradation. The bronchial epithelial cell lines IB3-1 (CF, high UCH-L1 expression) and S9 (non-CF, low UCH-L1 expression) were transiently transfected with wild type (WT) or DeltaF508 CFTR, WT UCH-L1 or small interfering RNA-UCH-L1, and a variety of ubiquitin mutants. We observed a positive correlation between UCH-L1 expression and steady state levels of WT- or DeltaF508-CFTR, and this stabilizing effect was confined to the early stages of CFTR synthesis. Immunolocalization of UCH-L1 by confocal microscopy revealed a partial co-localization with a ribosomal subunit and the endoplasmic reticulum. The UCH-L1-associated increase in CFTR levels was correlated with an increase in ubiquitinated CFTR (CFTR-Ub). Co-transfection with mutant ubiquitins and treatment with proteasome inhibitors suggested that UCH-L1 was reducing the proteasomal targeting of CFTR during synthesis by shortening conjugated polyubiquitin chains. Although not sufficient by itself to rescue mutant CFTR therapeutically, the elevation of UCH-L1 and its effect on CFTR processing provides insight into its potential roles in CF and other diseases.

  6. Proteins with an alpha/beta hydrolase fold: Relationships between subfamilies in an ever-growing superfamily.

    PubMed

    Lenfant, Nicolas; Hotelier, Thierry; Bourne, Yves; Marchot, Pascale; Chatonnet, Arnaud

    2013-03-25

    Alpha/beta hydrolases function as hydrolases, lyases, transferases, hormone precursors or transporters, chaperones or routers of other proteins. The amount of structural and functional available data related to this protein superfamily expands exponentially, as does the number of proteins classified as alpha/beta hydrolases despite poor sequence similarity and lack of experimental data. However the superfamily can be rationally divided according to sequence or structural homologies, leading to subfamilies of proteins with potentially similar functions. Since the discovery of proteins homologous to cholinesterases but devoid of enzymatic activity (e.g., the neuroligins), divergent functions have been ascribed to members of other subfamilies (e.g., lipases, dipeptidylaminopeptidase IV, etc.). To study the potentially moonlighting properties of alpha/beta hydrolases, the ESTHER database (for ESTerase and alpha/beta Hydrolase Enzymes and Relatives; http://bioweb.ensam.inra.fr/esther), which collects, organizes and disseminates structural and functional information related to alpha/beta hydrolases, has been updated with new tools and the web server interface has been upgraded. A new Overall Table along with a new Tree based on HMM models has been included to tentatively group subfamilies. These tools provide starting points for phylogenetic studies aimed at pinpointing the origin of duplications leading to paralogous genes (e.g., acetylcholinesterase versus butyrylcholinesterase, or neuroligin versus carboxylesterase). Another of our goals is to implement new tools to distinguish catalytically active enzymes from non-catalytic proteins in poorly studied or annotated subfamilies.

  7. A New Insight into the Physiological Role of Bile Salt Hydrolase among Intestinal Bacteria from the Genus Bifidobacterium

    PubMed Central

    Jarocki, Piotr; Podleśny, Marcin; Glibowski, Paweł; Targoński, Zdzisław

    2014-01-01

    This study analyzes the occurrence of bile salt hydrolase in fourteen strains belonging to the genus Bifidobacterium. Deconjugation activity was detected using a plate test, two-step enzymatic reaction and activity staining on a native polyacrylamide gel. Subsequently, bile salt hydrolases from B. pseudocatenulatum and B. longum subsp. suis were purified using a two-step chromatographic procedure. Biochemical characterization of the bile salt hydrolases showed that the purified enzymes hydrolyzed all of the six major human bile salts under the pH and temperature conditions commonly found in the human gastrointestinal tract. Next, the dynamic rheometry was applied to monitor the gelation process of deoxycholic acid under different conditions. The results showed that bile acids displayed aqueous media gelating properties. Finally, gel-forming abilities of bifidobacteria exhibiting bile salt hydrolase activity were analyzed. Our investigations have demonstrated that the release of deconjugated bile acids led to the gelation phenomenon of the enzymatic reaction solution containing purified BSH. The presented results suggest that bile salt hydrolase activity commonly found among intestinal microbiota increases hydrogel-forming abilities of certain bile salts. To our knowledge, this is the first report showing that bile salt hydrolase activity among Bifidobacterium is directly connected with the gelation process of bile salts. In our opinion, if such a phenomenon occurs in physiological conditions of human gut, it may improve bacterial ability to colonize the gastrointestinal tract and their survival in this specific ecological niche. PMID:25470405

  8. Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids.

    PubMed Central

    Stahl, U.; Banas, A.; Stymne, S.

    1995-01-01

    Developing endosperms and embryos accumulating triacylglycerols rich in caproyl (decanoyl) groups (i.e. developing embryos of Cuphea procumbens and Ulmus glabra) had microsomal acyl hydrolases with high selectivities toward phosphatidylcholine with this acyl group. Similarly, membranes from Euphorbia lagascae and Ricinus communis endosperms, which accumulate triacylglycerols with vernoleate (12-epoxy-octadeca-9-enoate) and ricinoleate (12-hydroxy-octadeca-9-enoate), respectively, had acyl hydrolases that selectively removed their respective oxygenated acyl group from the phospholipids. The activities toward phospholipid substrates with epoxy, hydroxy, and medium-chain acyl groups varied greatly between microsomal preparations from different plant species. Epoxidated and hydroxylated acyl groups in sn-1 and sn-2 positions of phosphatidylcholine and in sn-1-lysophosphatidylcholine were hydrolyzed to a similar extent, whereas the hydrolysis of caproyl groups was highly dependent on the positional localization. PMID:12228415

  9. Production of monospecific antiserum to a cytosolic epoxide hydrolase from human liver.

    PubMed

    Qato, M K; Reinmund, S G; Guenthner, T M

    1990-01-15

    A method for the purification to apparent homogeneity of cytosolic trans-stilbene oxide hydrolase from human liver is presented. The method employed ion exchange and gel filtration chromatography. From 50 g of human liver, 4.9 mg of homogenous enzyme protein was obtained. Although the enzyme had lost much of its catalytic activity during purification, it was nevertheless suitable for the preparation of antibodies to the enzyme. Only one immunogenic species was present in the antigen preparation, but some antibodies that were cross-reactive to sites on catalase were present in the antiserum. These catalase-specific antibodies were removed by immunoaffinity chromatography, and an IgG fraction that is monospecific to the cytosolic epoxide hydrolase was obtained. The usefulness of antibodies to this enzyme in immunoblotting experiments, following either sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focussing, as well as in enzyme-linked immunosorbent assays, is demonstrated.

  10. Tolerance of β-diketone hydrolases as representatives of the crotonase superfamily towards organic solvents.

    PubMed

    Siirola, Elina; Grischek, Barbara; Clay, Dorina; Frank, Annika; Grogan, Gideon; Kroutil, Wolfgang

    2011-12-01

    Crotonase superfamily enzymes catalyze a wide variety of reactions, including hydrolytic C-C bond cleavage in symmetrical β-diketones by 6-oxo camphor hydrolase (OCH) from Rhodococcus sp. The organic solvent tolerance and temperature stability of OCH and its structurally related ortholog Anabaena β-diketone hydrolase have been investigated. Both enzymes showed excellent tolerance toward organic solvents; for instance, even in the presence of 80% (v/v) THF or dioxane, OCH was still active. In most solvent mixtures, except methanol, the stereospecificity was conserved (>99% e.e. of product), hence neither the type of solvent nor its concentration appeared to have an effect on the stereoselectivity of the enzyme. Attempts to correlate the observed activities with log P, functional solvent group or denaturing capacity (DC) of the solvent were only successful in the case of DC for water miscible solvents. This study represents the first investigation of organic solvent stability for members of the crotonase superfamily.

  11. Crystallization and preliminary X-ray analysis of neoagarobiose hydrolase from Saccharophagus degradans 2-40

    PubMed Central

    Lee, Saeyoung; Lee, Jonas Yun; Ha, Sung Chul; Jung, Jina; Shin, Dong Hae; Kim, Kyoung Heon; Choi, In-Geol

    2009-01-01

    Many agarolytic bacteria degrade agar polysaccharide into the disaccharide unit neoagarobiose [O-3,6-anhydro-α-l-galactopyranosyl-(1→3)-d-galactose] using various β-agarases. Neoagarobiose hydrolase is an enzyme that acts on the α-­1,3 linkage in neoagarobiose to yield d-galactose and 3,6-anhydro-l-galactose. This activity is essential in both the metabolism of agar by agarolytic bacteria and the production of fermentable sugars from agar biomass for bioenergy production. Neoagarobiose hydrolase from the marine bacterium Saccharophagus degradans 2-40 was overexpressed in Escherichia coli and crystallized in the monoclinic space group C2, with unit-cell parameters a = 129.83, b = 76.81, c = 90.11 Å, β = 101.86°. The crystals diffracted to 1.98 Å resolution and possibly contains two molecules in the asymmetric unit. PMID:20054134

  12. A chemical proteomic atlas of brain serine hydrolases identifies cell type-specific pathways regulating neuroinflammation.

    PubMed

    Viader, Andreu; Ogasawara, Daisuke; Joslyn, Christopher M; Sanchez-Alavez, Manuel; Mori, Simone; Nguyen, William; Conti, Bruno; Cravatt, Benjamin F

    2016-01-18

    Metabolic specialization among major brain cell types is central to nervous system function and determined in large part by the cellular distribution of enzymes. Serine hydrolases are a diverse enzyme class that plays fundamental roles in CNS metabolism and signaling. Here, we perform an activity-based proteomic analysis of primary mouse neurons, astrocytes, and microglia to furnish a global portrait of the cellular anatomy of serine hydrolases in the brain. We uncover compelling evidence for the cellular compartmentalization of key chemical transmission pathways, including the functional segregation of endocannabinoid (eCB) biosynthetic enzymes diacylglycerol lipase-alpha (DAGLα) and -beta (DAGLβ) to neurons and microglia, respectively. Disruption of DAGLβ perturbed eCB-eicosanoid crosstalk specifically in microglia and suppressed neuroinflammatory events in vivo independently of broader effects on eCB content. Mapping the cellular distribution of metabolic enzymes thus identifies pathways for regulating specialized inflammatory responses in the brain while avoiding global alterations in CNS function.

  13. Effects of proteins and polynucleotides on the activity of various hydrolases

    PubMed Central

    Palmieri, M. J.; Koldovský, O.

    1972-01-01

    The effect of various macromolecules on the activity of several hydrolases was studied. Dilution of partially purified acid β-galactosidase from ileal mucosa of suckling rats resulted in a decrease of specific activity. The relationship between specific activity and dilution of the enzyme suggests a dissociation of the enzyme. This could be prevented by addition of several proteins tested. However, addition of DNA to the assay mixture for acid β-galactosidase caused an inhibition. This inhibition could be prevented by addition of proteins. Other polynucleotides and tRNA also exert an inhibitory effect that is prevented by albumin, but nucleotides have no effect. This inhibition occurs maximally at a low pH (3.0–4.0); no inhibition is observed at pH5.5. A similar pH-dependent inhibition by DNA was also found with various other acid hydrolases. PMID:5076227

  14. Physical comparison of parathion hydrolase plasmids from Pseudomonas diminuta and Flavobacterium sp.

    PubMed

    Mulbry, W W; Kearney, P C; Nelson, J O; Karns, J S

    1987-09-01

    Restriction maps of two plasmids encoding parathion hydrolase have been determined. pPDL2 is a 39-kb plasmid harbored by Flavobacterium sp. (ATCC 27551), while pCMS1 is a 70-kb plasmid found in Pseudomonas diminuta (strain MG). Both plasmids previously have been shown to share homologous parathion hydrolase genes (termed opd for organophosphate degradation) as judged by DNA-DNA hybridization and restriction mapping. In the present study, we conducted DNA hybridization experiments using each of nine PstI restriction fragments from pCMS1 as probes against Flavobacterium plasmid DNA. The opd genes of both plasmids are located within a highly conserved region of approximately 5.1 kb. This region of homology extends approximately 2.6 kb upstream and 1.7 kb downstream from the opd genes. No homology between the two plasmids is evident outside of this region.

  15. Dual roles of brain serine hydrolase KIAA1363 in ether lipid metabolism and organophosphate detoxification

    SciTech Connect

    Nomura, Daniel K.; Fujioka, Kazutoshi; Issa, Roger S.; Ward, Anna M.; Cravatt, Benjamin F.; Casida, John E.

    2008-04-01

    Serine hydrolase KIAA1363 is an acetyl monoalkylglycerol ether (AcMAGE) hydrolase involved in tumor cell invasiveness. It is also an organophosphate (OP) insecticide-detoxifying enzyme. The key to understanding these dual properties was the use of KIAA1363 +/+ (wildtype) and -/- (gene deficient) mice to define the role of this enzyme in brain and other tissues and its effectiveness in vivo in reducing OP toxicity. KIAA1363 was the primary AcMAGE hydrolase in brain, lung, heart and kidney and was highly sensitive to inactivation by chlorpyrifos oxon (CPO) (IC{sub 50} 2 nM) [the bioactivated metabolite of the major insecticide chlorpyrifos (CPF)]. Although there was no difference in hydrolysis product monoalkylglycerol ether (MAGE) levels in +/+ and -/- mouse brains in vivo, isopropyl dodecylfluorophosphonate (30 mg/kg) and CPF (100 mg/kg) resulted in 23-51% decrease in brain MAGE levels consistent with inhibition of AcMAGE hydrolase activity. On incubating +/+ and -/- brain membranes with AcMAGE and cytidine-5'-diphosphocholine, the absence of KIAA1363 activity dramatically increased de novo formation of platelet-activating factor (PAF) and lyso-PAF, signifying that metabolically-stabilized AcMAGE can be converted to this bioactive lipid in brain. On considering detoxification, KIAA1363 -/- mice were significantly more sensitive than +/+ mice to ip-administered CPF (100 mg/kg) and parathion (10 mg/kg) with increased tremoring and mortality that correlated for CPF with greater brain acetylcholinesterase inhibition. Docking AcMAGE and CPO in a KIAA1363 active site model showed similar positioning of their acetyl and trichloropyridinyl moieties, respectively. This study establishes the relevance of KIAA1363 in ether lipid metabolism and OP detoxification.

  16. Trigger factor assisted folding of the recombinant epoxide hydrolases identified from C. pelagibacter and S. nassauensis.

    PubMed

    Saini, Priya; Wani, Shadil Ibrahim; Kumar, Ranjai; Chhabra, Ravneet; Chimni, Swapandeep Singh; Sareen, Dipti

    2014-12-01

    Epoxide hydrolases (EHs), are enantioselective enzymes as they catalyze the kinetic resolution of racemic epoxides into the corresponding enantiopure vicinal diols, which are useful precursors in the synthesis of chiral pharmaceutical compounds. Here, we have identified and cloned two putative epoxide hydrolase genes (cpeh and sneh) from marine bacteria, Candidatus pelagibacter ubique and terrestrial bacteria, Stackebrandtia nassauensis, respectively and overexpressed them in pET28a vector in Escherichia coli BL21(DE3). The CPEH protein (42kDa) was found to be overexpressed as inactive inclusion bodies while SNEH protein (40kDa) was found to form soluble aggregates. In this study, the recombinant CPEH was successfully transformed from insoluble aggregates to the soluble and functionally active form, using pCold TF vector, though with low EH activity. To prevent the soluble aggregate formation of SNEH, it was co-expressed with GroEL/ES chaperone and was also fused with trigger factor (TF) chaperone at its N-terminus. The TF chaperone-assisted correct folding of SNEH led to a purified active EH with a specific activity of 3.85μmol/min/mg. The pure enzyme was further used to biocatalyze the hydrolysis of 10mM benzyl glycidyl ether (BGE) and α-methyl styrene oxide (MSO) with an enantiomeric excess of the product (eep) of 86% and 73% in 30 and 15min, respectively. In conclusion, this is the first report about the heterologous expression of epoxide hydrolases using TF as a molecular chaperone in pCold TF expression vector, resulting in remarkable increase in the solubility and activity of the otherwise improperly folded recombinant epoxide hydrolases.

  17. Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

    PubMed Central

    Germane, Katherine L.; Servinsky, Matthew D.; Gerlach, Elliot S.; Sund, Christian J.; Hurley, Margaret M.

    2015-01-01

    Clostridium acetobutylicum ATCC 824 gene CA_C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA_C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry 1nc5) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA_C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate specificity from that of YteR. PMID:26249707

  18. A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad

    PubMed Central

    Sarian, Fean D.; Janeček, Štefan; Pijning, Tjaard; Ihsanawati; Nurachman, Zeily; Radjasa, Ocky K.; Dijkhuizen, Lubbert; Natalia, Dessy; van der Maarel, Marc J. E. C.

    2017-01-01

    α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)8-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical α-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillus megaterium strain NL3, a bacterial isolate from a sea anemone of Kakaban landlocked marine lake, Derawan Island, Indonesia. In BmaN1 the third residue, the aspartic acid that acts as the transition state stabilizer, was replaced by a histidine. Three-dimensional structure modeling of the BmaN1 amino acid sequence confirmed the aberrant catalytic triad. Glucose and maltose were found as products of the action of the novel α-amylase on soluble starch, demonstrating that it is active in spite of the peculiar catalytic triad. This novel BmaN1 α-amylase is part of a group of α-amylases that all have this atypical catalytic triad, consisting of aspartic acid, glutamic acid and histidine. Phylogenetic analysis showed that this group of α-amylases comprises a new subfamily of the glycoside hydrolase family 13. PMID:28287181

  19. Sph3 Is a Glycoside Hydrolase Required for the Biosynthesis of Galactosaminogalactan in Aspergillus fumigatus.

    PubMed

    Bamford, Natalie C; Snarr, Brendan D; Gravelat, Fabrice N; Little, Dustin J; Lee, Mark J; Zacharias, Caitlin A; Chabot, Josée C; Geller, Alexander M; Baptista, Stefanie D; Baker, Perrin; Robinson, Howard; Howell, P Lynne; Sheppard, Donald C

    2015-11-13

    Aspergillus fumigatus is the most virulent species within the Aspergillus genus and causes invasive infections with high mortality rates. The exopolysaccharide galactosaminogalactan (GAG) contributes to the virulence of A. fumigatus. A co-regulated five-gene cluster has been identified and proposed to encode the proteins required for GAG biosynthesis. One of these genes, sph3, is predicted to encode a protein belonging to the spherulin 4 family, a protein family with no known function. Construction of an sph3-deficient mutant demonstrated that the gene is necessary for GAG production. To determine the role of Sph3 in GAG biosynthesis, we determined the structure of Aspergillus clavatus Sph3 to 1.25 Å. The structure revealed a (β/α)8 fold, with similarities to glycoside hydrolase families 18, 27, and 84. Recombinant Sph3 displayed hydrolytic activity against both purified and cell wall-associated GAG. Structural and sequence alignments identified three conserved acidic residues, Asp-166, Glu-167, and Glu-222, that are located within the putative active site groove. In vitro and in vivo mutagenesis analysis demonstrated that all three residues are important for activity. Variants of Asp-166 yielded the greatest decrease in activity suggesting a role in catalysis. This work shows that Sph3 is a glycoside hydrolase essential for GAG production and defines a new glycoside hydrolase family, GH135.

  20. Exported Epoxide Hydrolases Modulate Erythrocyte Vasoactive Lipids during Plasmodium falciparum Infection

    PubMed Central

    Dalmia, Varun K.

    2016-01-01

    ABSTRACT Erythrocytes are reservoirs of important epoxide-containing lipid signaling molecules, including epoxyeicosatrienoic acids (EETs). EETs function as vasodilators and anti-inflammatory modulators in the bloodstream. Bioactive EETs are hydrolyzed to less active diols (dihydroxyeicosatrienoic acids) by epoxide hydrolases (EHs). The malaria parasite Plasmodium falciparum infects host red blood cells (RBCs) and exports hundreds of proteins into the RBC compartment. In this study, we show that two parasite epoxide hydrolases, P. falciparum epoxide hydrolases 1 (PfEH1) and 2 (PfEH2), both with noncanonical serine nucleophiles, are exported to the periphery of infected RBCs. PfEH1 and PfEH2 were successfully expressed in Escherichia coli, and they hydrolyzed physiologically relevant erythrocyte EETs. Mutations in active site residues of PfEH1 ablated the ability of the enzyme to hydrolyze an epoxide substrate. Overexpression of PfEH1 or PfEH2 in parasite-infected RBCs resulted in a significant alteration in the epoxide fatty acids stored in RBC phospholipids. We hypothesize that the parasite disruption of epoxide-containing signaling lipids leads to perturbed vascular function, creating favorable conditions for binding and sequestration of infected RBCs to the microvascular endothelium. PMID:27795395

  1. LytF, a Novel Competence-Regulated Murein Hydrolase in the Genus Streptococcus

    PubMed Central

    Berg, Kari Helene; Ohnstad, Hilde Solheim

    2012-01-01

    Streptococcus pneumoniae and probably most other members of the genus Streptococcus are competent for natural genetic transformation. During the competent state, S. pneumoniae produces a murein hydrolase, CbpD, that kills and lyses noncompetent pneumococci and closely related species. Previous studies have shown that CbpD is essential for efficient transfer of genomic DNA from noncompetent to competent cells in vitro. Consequently, it has been proposed that CbpD together with the cognate immunity protein ComM constitutes a DNA acquisition mechanism that enables competent pneumococci to capture homologous DNA from closely related streptococci sharing the same habitat. Although genes encoding CbpD homologs or CbpD-related proteins are present in many different streptococcal species, the genomes of a number of streptococci do not encode CbpD-type proteins. In the present study we show that the genomes of nearly all species lacking CbpD encode an unrelated competence-regulated murein hydrolase termed LytF. Using Streptococcus gordonii as a model system, we obtained evidence indicating that LytF is a functional analogue of CbpD. In sum, our results show that a murein hydrolase gene is part of the competence regulon of most or all streptococcal species, demonstrating that these muralytic enzymes constitute an essential part of the streptococcal natural transformation system. PMID:22123253

  2. A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad.

    PubMed

    Sarian, Fean D; Janeček, Štefan; Pijning, Tjaard; Ihsanawati; Nurachman, Zeily; Radjasa, Ocky K; Dijkhuizen, Lubbert; Natalia, Dessy; van der Maarel, Marc J E C

    2017-03-13

    α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)8-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical α-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillus megaterium strain NL3, a bacterial isolate from a sea anemone of Kakaban landlocked marine lake, Derawan Island, Indonesia. In BmaN1 the third residue, the aspartic acid that acts as the transition state stabilizer, was replaced by a histidine. Three-dimensional structure modeling of the BmaN1 amino acid sequence confirmed the aberrant catalytic triad. Glucose and maltose were found as products of the action of the novel α-amylase on soluble starch, demonstrating that it is active in spite of the peculiar catalytic triad. This novel BmaN1 α-amylase is part of a group of α-amylases that all have this atypical catalytic triad, consisting of aspartic acid, glutamic acid and histidine. Phylogenetic analysis showed that this group of α-amylases comprises a new subfamily of the glycoside hydrolase family 13.

  3. Brucella abortus Choloylglycine Hydrolase Affects Cell Envelope Composition and Host Cell Internalization

    PubMed Central

    Marchesini, María Inés; Connolly, Joseph; Delpino, María Victoria; Baldi, Pablo C.; Mujer, Cesar V.; DelVecchio, Vito G.; Comerci, Diego J.

    2011-01-01

    Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization. PMID:22174816

  4. Brucella abortus choloylglycine hydrolase affects cell envelope composition and host cell internalization.

    PubMed

    Marchesini, María Inés; Connolly, Joseph; Delpino, María Victoria; Baldi, Pablo C; Mujer, Cesar V; DelVecchio, Vito G; Comerci, Diego J

    2011-01-01

    Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization.

  5. Structure of the Cyanuric Acid Hydrolase TrzD Reveals Product Exit Channel

    PubMed Central

    Bera, Asim K; Aukema, Kelly G.; Elias, Mikael; Wackett, Lawrence P.

    2017-01-01

    Cyanuric acid hydrolases are of industrial importance because of their use in aquatic recreational facilities to remove cyanuric acid, a stabilizer for the chlorine. Degradation of excess cyanuric acid is necessary to maintain chlorine disinfection in the waters. Cyanuric acid hydrolase opens the cyanuric acid ring hydrolytically and subsequent decarboxylation produces carbon dioxide and biuret. In the present study, we report the X-ray structure of TrzD, a cyanuric acid hydrolase from Acidovorax citrulli. The crystal structure at 2.19 Å resolution shows a large displacement of the catalytic lysine (Lys163) in domain 2 away from the active site core, whereas the two other active site lysines from the two other domains are not able to move. The lysine displacement is proposed here to open up a channel for product release. Consistent with that, the structure also showed two molecules of the co-product, carbon dioxide, one in the active site and another trapped in the proposed exit channel. Previous data indicated that the domain 2 lysine residue plays a role in activating an adjacent serine residue carrying out nucleophilic attack, opening the cyanuric acid ring, and the mobile lysine guides products through the exit channel. PMID:28345631

  6. Purification, crystallization and preliminary crystallographic studies of plant S-adenosyl-l-homocysteine hydrolase (Lupinus luteus)

    SciTech Connect

    Brzezinski, Krzysztof; Bujacz, Grzegorz; Jaskolski, Mariusz

    2008-07-01

    Single crystals of recombinant S-adenosyl-l-homocysteine hydrolase from L. luteus in complex with adenosine diffract X-rays to 1.17 Å resolution at 100 K. The crystals are tetragonal, space group P4{sub 3}2{sub 1}2, and contain one copy of the dimeric enzyme in the asymmetric unit. By degrading S-adenosyl-l-homocysteine, which is a byproduct of S-adenosyl-l-methionine-dependent methylation reactions, S-adenosyl-l-homocysteine hydrolase (SAHase) acts as a regulator of cellular methylation processes. S-Adenosyl-l-homocysteine hydrolase from the leguminose plant yellow lupin (Lupinus luteus), LlSAHase, which is composed of 485 amino acids and has a molecular weight of 55 kDa, has been cloned, expressed in Escherichia coli and purified. Crystals of LlSAHase in complex with adenosine were obtained by the hanging-drop vapour-diffusion method using 20%(w/v) PEG 4000 and 10%(v/v) 2-propanol as precipitants in 0.1 M Tris–HCl buffer pH 8.0. The crystals were tetragonal, space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = 122.4, c = 126.5 Å and contained two protein molecules in the asymmetric unit, corresponding to the functional dimeric form of the enzyme. Atomic resolution (1.17 Å) X-ray diffraction data have been collected using synchrotron radiation.

  7. Thermal unfolding of nucleoside hydrolases from the hyperthermophilic archaeon Sulfolobus solfataricus: role of disulfide bonds.

    PubMed

    Porcelli, Marina; De Leo, Ester; Del Vecchio, Pompea; Fuccio, Francesca; Cacciapuoti, Giovanna

    2012-03-01

    Nucleoside hydrolases are metalloproteins that hydrolyze the N-glycosidic bond of β-ribonucleosides, forming the free purine/pyrimidine base and ribose. We report the stability of the two hyperthermophilic enzymes Sulfolobus solfataricus pyrimidine-specific nucleoside hydrolase (SsCU-NH) and Sulfolobus solfataricus purine-specific inosineadenosine- guanosine nucleoside hydrolase (SsIAG-NH) against the denaturing action of temperature and guanidine hydrochloride by means of circular dichroism and fluorescence spectroscopy. The guanidine hydrochloride-induced unfolding is reversible for both enzymes as demonstrated by the analysis of the refolding process by activity assays and fluorescence measurements. The evidence that the denaturation of SsIAG-NH carried out in the presence of reducing agents proved to be reversible indicates that the presence of disulfide bonds interferes with the refolding process of this enzyme. Both enzymes are highly thermostable and no thermal unfolding transition can be obtained up to 108°C. SsIAG-NH is thermally denatured under reducing conditions (T(m)=93°C) demonstrating the contribution of disulfide bridges to enzyme thermostability.

  8. Bacterial Cell Enlargement Requires Control of Cell Wall Stiffness Mediated by Peptidoglycan Hydrolases

    PubMed Central

    Wheeler, Richard; Turner, Robert D.; Bailey, Richard G.; Salamaga, Bartłomiej; Mesnage, Stéphane; Mohamad, Sharifah A. S.; Hayhurst, Emma J.; Horsburgh, Malcolm; Hobbs, Jamie K.

    2015-01-01

    ABSTRACT Most bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology of Staphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan. PMID:26220963

  9. Ubiquitin dimers control the hydrolase activity of UCH-L3.

    PubMed

    Setsuie, Rieko; Sakurai, Mikako; Sakaguchi, Yuriko; Wada, Keiji

    2009-01-01

    Ubiquitin (Ub) carboxy terminal hydrolase (UCH)-L1 and UCH-L3 are two of the deubiquitinating enzymes expressed in the brain. Both gad mice, which lack UCH-L1 expression and Uchl3 knockout mice exhibit neurodegeneration, although at distinct areas. These phenotypes indicate the importance of UCH-L1 and UCH-L3 in the regulation of the central nervous system. However, molecular substrates and the molecular regulators of UCH-L1 and UCH-L3 remain poorly identified. Here we show that Ub dimers interact non-covalently with UCH-L3 in vitro and in cells. These interactions were not observed with UCH-L1 in cells. In vitro, K48-linked Ub dimers pronouncedly inhibited the hydrolase activity of UCH-L3, while mono-Ub, a previously identified interacting protein, inhibited the hydrolase activity of UCH-L1. These results indicate that mono-Ub and Ub dimers may regulate the enzymatic functions of UCH-L1 and UCH-L3, respectively, in vivo.

  10. Production of polyclonal anti-dUCH (Drosophila ubiquitin carboxyl-terminal hydrolase) antibodies.

    PubMed

    Tram, Nguyen Thi Quynh; Trang, Nguyen Thi Thu; Thao, Dang Thi Phuong; Thuoc, Tran Linh

    2013-04-01

    Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), which is a member of the ubiquitin carboxyl-terminal hydrolase (UCH) family, is highly expressed in neurons. In vitro, UCH- L1 exhibits both ubiquitin hydrolase and ligase activity. Many studies have suggested that UCH-L1 is involved in the pathogenesis of Parkinson's disease and some different human cancer diseases, but its role in a living system is still unclear. Recently, Drosophila melanogaster has been shown to be a compatible model for studying human diseases. To investigate the role of UCH-L1 in a living system, the UCH-L1 homologous protein in Drosophila melanogaster (dUCH) is used for analyzing the role of the protein's function in transgenic Drosophila. Here, we used DNA molecular techniques to clone, express, and purify dUCH protein from Escherichia coli. The purified dUCH protein was injected into a rabbit to produce an anti-dUCH antibody, which was shown to have high specificity and sensitivity to the dUCH protein. The affinity of the antibody is 1:320,000 at 7.81 ng/μL antigen concentration. The 1:40,000 dilution-produced antibodies can detect antigen at a low concentration of 0.98 ng/μL. Success in producing this antibody provides good material for further experiments in the study of the role of UCH-L1 by a Drosophila model.

  11. Strategies to reduce end-product inhibition in family 48 glycoside hydrolases

    DOE PAGES

    Chen, Mo; Bu, Lintao; Alahuhta, Markus; ...

    2016-02-01

    Family 48 cellobiohydrolases are some of the most abundant glycoside hydrolases in nature. They are able to degrade cellulosic biomass and therefore serve as good enzyme candidates for biofuel production. Family 48 cellulases hydrolyze cellulose chains via a processive mechanism, and produce end products composed primarily of cellobiose as well as other cellooligomers (dp ≤ 4). The challenge of utilizing cellulases in biofuel production lies in their extremely slow turnover rate. A factor contributing to the low enzyme activity is suggested to be product binding to enzyme and the resulting performance inhibition. In this study, we quantitatively evaluated the productmore » inhibitory effect of four family 48 glycoside hydrolases using molecular dynamics simulations and product expulsion free-energy calculations. We also suggested a series of single mutants of the four family 48 glycoside hydrolases with theoretically reduced level of product inhibition. As a result, the theoretical calculations provide a guide for future experimental studies designed to produce mutant cellulases with enhanced activity.« less

  12. Juggling jobs: roles and mechanisms of multifunctional protease inhibitors in plants.

    PubMed

    Grosse-Holz, Friederike M; van der Hoorn, Renier A L

    2016-05-01

    Multifunctional protease inhibitors juggle jobs by targeting different enzymes and thereby often controlling more than one biological process. Here, we discuss the biological functions, mechanisms and evolution of three types of multifunctional protease inhibitors in plants. The first type is double-headed inhibitors, which feature two inhibitory sites targeting proteases with different specificities (e.g. Bowman-Birk inhibitors) or even different hydrolases (e.g. α-amylase/protease inhibitors preventing both early germination and seed predation). The second type consists of multidomain inhibitors which evolved by intragenic duplication and are released by processing (e.g. multicystatins and potato inhibitor II, implicated in tuber dormancy and defence, respectively). The third type consists of promiscuous inhibitory folds which resemble mouse traps that can inhibit different proteases cleaving the bait they offer (e.g. serpins, regulating cell death, and α-macroglobulins). Understanding how multifunctional inhibitors juggle biological jobs increases our knowledge of the connections between the networks they regulate. These examples show that multifunctionality evolved independently from a remarkable diversity of molecular mechanisms that can be exploited for crop improvement and provide concepts for protein design.

  13. Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors.

    PubMed

    Iglesias, Jose; Lamontagne, Julien; Erb, Heidi; Gezzar, Sari; Zhao, Shangang; Joly, Erik; Truong, Vouy Linh; Skorey, Kathryn; Crane, Sheldon; Madiraju, S R Murthy; Prentki, Marc

    2016-01-01

    Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors.

  14. Soluble epoxide hydrolase inhibition improves coronary endothelial function and prevents the development of cardiac alterations in obese insulin-resistant mice

    PubMed Central

    Roche, Clothilde; Besnier, Marie; Cassel, Roméo; Harouki, Najah; Coquerel, David; Guerrot, Dominique; Nicol, Lionel; Loizon, Emmanuelle; Remy-Jouet, Isabelle; Morisseau, Christophe; Mulder, Paul; Ouvrard-Pascaud, Antoine; Madec, Anne-Marie; Richard, Vincent

    2015-01-01

    This study addressed the hypothesis that inhibiting the soluble epoxide hydrolase (sEH)-mediated degradation of epoxy-fatty acids, notably epoxyeicosatrienoic acids, has an additional impact against cardiovascular damage in insulin resistance, beyond its previously demonstrated beneficial effect on glucose homeostasis. The cardiovascular and metabolic effects of the sEH inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB; 10 mg/l in drinking water) were compared with those of the sulfonylurea glibenclamide (80 mg/l), both administered for 8 wk in FVB mice subjected to a high-fat diet (HFD; 60% fat) for 16 wk. Mice on control chow diet (10% fat) and nontreated HFD mice served as controls. Glibenclamide and t-AUCB similarly prevented the increased fasting glycemia in HFD mice, but only t-AUCB improved glucose tolerance and decreased gluconeogenesis, without modifying weight gain. Moreover, t-AUCB reduced adipose tissue inflammation, plasma free fatty acids, and LDL cholesterol and prevented hepatic steatosis. Furthermore, only the sEH inhibitor improved endothelium-dependent relaxations to acetylcholine, assessed by myography in isolated coronary arteries. This improvement was related to a restoration of epoxyeicosatrienoic acid and nitric oxide pathways, as shown by the increased inhibitory effects of the nitric oxide synthase and cytochrome P-450 epoxygenase inhibitors l-NA and MSPPOH on these relaxations. Moreover, t-AUCB decreased cardiac hypertrophy, fibrosis, and inflammation and improved diastolic function, as demonstrated by the increased E/A ratio (echocardiography) and decreased slope of the end-diastolic pressure-volume relation (invasive hemodynamics). These results demonstrate that sEH inhibition improves coronary endothelial function and prevents cardiac remodeling and diastolic dysfunction in obese insulin-resistant mice. PMID:25724490

  15. Discovery of PF-04457845: A Highly Potent, Orally Bioavailable, and Selective Urea FAAH Inhibitor.

    PubMed

    Johnson, Douglas S; Stiff, Cory; Lazerwith, Scott E; Kesten, Suzanne R; Fay, Lorraine K; Morris, Mark; Beidler, David; Liimatta, Marya B; Smith, Sarah E; Dudley, David T; Sadagopan, Nalini; Bhattachar, Shobha N; Kesten, Stephen J; Nomanbhoy, Tyzoon K; Cravatt, Benjamin F; Ahn, Kay

    2011-02-10

    Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic and anti-inflammatory phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for the treatment of inflammatory pain and other nervous system disorders. Herein, we report the discovery and characterization of a highly efficacious and selective FAAH inhibitor PF-04457845 (23). Compound 23 inhibits FAAH by a covalent, irreversible mechanism involving carbamylation of the active-site serine nucleophile of FAAH with high in vitro potency (k(inact)/K(i) and IC(50) values of 40300 M(-1) s(-1) and 7.2 nM, respectively, for human FAAH). Compound 23 has exquisite selectivity for FAAH relative to other members of the serine hydrolase superfamily as demonstrated by competitive activity-based protein profiling. Oral administration of 23 at 0.1 mg/kg results in efficacy comparable to that of naproxen at 10 mg/kg in a rat model of inflammatory pain. Compound 23 is being evaluated in human clinical trials.

  16. Post-exposure administration of diazepam combined with soluble epoxide hydrolase inhibition stops seizures and modulates neuroinflammation in a murine model of acute TETS intoxication

    SciTech Connect

    Vito, Stephen T.; Austin, Adam T.; Banks, Christopher N.; Inceoglu, Bora; Bruun, Donald A.; Zolkowska, Dorota; Tancredi, Daniel J.; Rogawski, Michael A.; Hammock, Bruce D.; Lein, Pamela J.

    2014-12-01

    Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison for which there is currently no approved antidote. The convulsant action of TETS is thought to be mediated by inhibition of type A gamma-aminobutyric acid receptor (GABA{sub A}R) function. We, therefore, investigated the effects of post-exposure administration of diazepam, a GABA{sub A}R positive allosteric modulator, on seizure activity, death and neuroinflammation in adult male Swiss mice injected with a lethal dose of TETS (0.15 mg/kg, ip). Administration of a high dose of diazepam (5 mg/kg, ip) immediately following the second clonic seizure (approximately 20 min post-TETS injection) effectively prevented progression to tonic seizures and death. However, this treatment did not prevent persistent reactive astrogliosis and microglial activation, as determined by GFAP and Iba-1 immunoreactivity and microglial cell morphology. Inhibition of soluble epoxide hydrolase (sEH) has been shown to exert potent anti-inflammatory effects and to increase survival in mice intoxicated with other GABA{sub A}R antagonists. The sEH inhibitor TUPS (1 mg/kg, ip) administered immediately after the second clonic seizure did not protect TETS-intoxicated animals from tonic seizures or death. Combined administration of diazepam (5 mg/kg, ip) and TUPS (1 mg/kg, ip, starting 1 h after diazepam and repeated every 24 h) prevented TETS-induced lethality and influenced signs of neuroinflammation in some brain regions. Significantly decreased microglial activation and enhanced reactive astrogliosis were observed in the hippocampus, with no changes in the cortex. Combining an agent that targets specific anti-inflammatory mechanisms with a traditional antiseizure drug may enhance treatment outcome in TETS intoxication. - Highlights: • Acute TETS intoxication causes delayed and persistent neuroinflammation. • Diazepam given post-TETS prevents lethal tonic seizures but not neuroinflammation. • A soluble epoxide hydrolase

  17. The Potential of Inhibitors of Endocannabinoid Metabolism for Drug Development: A Critical Review.

    PubMed

    Fowler, Christopher J

    2015-01-01

    The endocannabinoids anandamide and 2-arachidonoylglycerol are metabolised by both hydrolytic enzymes (primarily fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL)) and oxygenating enzymes (e.g. cyclooxygenase-2, COX-2). In the present article, the in vivo data for compounds inhibiting endocannabinoid metabolism have been reviewed, focussing on inflammation and pain. Potential reasons for the failure of an FAAH inhibitor in a clinical trial in patients with osteoarthritic pain are discussed. It is concluded that there is a continued potential for compounds inhibiting endocannabinoid metabolism in terms of drug development, but that it is wise not to be unrealistic in terms of expectations of success.

  18. Preclinical Characterization of the FAAH Inhibitor JNJ-42165279

    PubMed Central

    2015-01-01

    The pre-clinical characterization of the aryl piperazinyl urea inhibitor of fatty acid amide hydrolase (FAAH) JNJ-42165279 is described. JNJ-42165279 covalently inactivates the FAAH enzyme, but is highly selective with regard to other enzymes, ion channels, transporters, and receptors. JNJ-42165279 exhibited excellent ADME and pharmacodynamic properties as evidenced by its ability to block FAAH in the brain and periphery of rats and thereby cause an elevation of the concentrations of anandamide (AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The compound was also efficacious in the spinal nerve ligation (SNL) model of neuropathic pain. The combination of good physical, ADME, and PD properties of JNJ-42165279 supported it entering the clinical portfolio. PMID:26713105

  19. Preclinical Characterization of the FAAH Inhibitor JNJ-42165279.

    PubMed

    Keith, John M; Jones, William M; Tichenor, Mark; Liu, Jing; Seierstad, Mark; Palmer, James A; Webb, Michael; Karbarz, Mark; Scott, Brian P; Wilson, Sandy J; Luo, Lin; Wennerholm, Michelle L; Chang, Leon; Rizzolio, Michele; Rynberg, Raymond; Chaplan, Sandra R; Breitenbucher, J Guy

    2015-12-10

    The pre-clinical characterization of the aryl piperazinyl urea inhibitor of fatty acid amide hydrolase (FAAH) JNJ-42165279 is described. JNJ-42165279 covalently inactivates the FAAH enzyme, but is highly selective with regard to other enzymes, ion channels, transporters, and receptors. JNJ-42165279 exhibited excellent ADME and pharmacodynamic properties as evidenced by its ability to block FAAH in the brain and periphery of rats and thereby cause an elevation of the concentrations of anandamide (AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The compound was also efficacious in the spinal nerve ligation (SNL) model of neuropathic pain. The combination of good physical, ADME, and PD properties of JNJ-42165279 supported it entering the clinical portfolio.

  20. The exploration of thienothiazines as selective butyrylcholinesterase inhibitors.

    PubMed

    Karlsson, Daniela; Fallarero, Adyary; Brunhofer, Gerda; Mayer, Claudia; Prakash, Ohm; Mohan, C Gopi; Vuorela, Pia; Erker, Thomas

    2012-08-30

    The role of butyrylcholinesterase (BChE) in the progression of Alzheimer's disease (AD) has recently become more crucial. In the AD brain, selective BChE inhibitors have been demonstrated to have a beneficial effect in vivo, probably by recovering cholinergic activity and/or by restoring AChE:BChE activity ratios to the levels observed in the healthy brain. Thienothiazines are compounds sharing some structural features with phenothiazines, which are known to be potent BChE inhibitors. Thus, in this contribution 45 thienothiazines were investigated for their BChE inhibitory activity. Six of them were proven to be potent and selective inhibitors of equine BChE's hydrolase activity. Structure-activity relationships were laid out, and a tentative pharmacophore model for BChE inhibitors of the thienothiazine type was proposed. The most active compound, 3f, displayed a mixed type of inhibition and was also active against the human BChE (huBChE) with an IC(50) huBChE of 0.51 ± 0.07 μM. Computational studies suggested that 3f likely binds to the catalytic site and nearby to the peripheral site of the huBChE in an extended form. In addition, the chemical space occupied by the active thienothiazines, as opposed to phenothiazines and other representative chemical classes of BChE inhibitors, was explored with the aid of ChemGPS-NP, and the relevant chemical space regions were identified. This study shows for the first time that thienothiazines represent a new group of BChE inhibitors that can be used as molecular probes for studying the role of BChE in the brain or for developing newer drug leads for AD therapy.

  1. Proton pump inhibitors

    MedlinePlus

    Proton pump inhibitors (PPIs) are medicines that work by reducing the amount of stomach acid made by ... Proton pump inhibitors are used to: Relieve symptoms of acid reflux, or gastroesophageal reflux disease (GERD). This ...

  2. Design and screening strategies for alpha-glucosidase inhibitors based on enzymological information.

    PubMed

    Hakamata, Wataru; Kurihara, Masaaki; Okuda, Haruhiro; Nishio, Toshiyuki; Oku, Tadatake

    2009-01-01

    Alpha-glucosidase inhibitors are marketed as therapeutic drugs for diabetes that act through the inhibition of carbohydrate metabolism. Inhibitors of the alpha-glucosidases that are involved in the biosynthesis of N-linked oligosaccharide chains have been reported to have antitumor, antiviral, and apoptosis-inducing activities, and some have been used clinically. alpha-Glucosidase inhibitors have interesting biological activities, and their design, synthesis, and screening are being actively performed. In quite a few reports, however, alpha-glucosidases with different origins than the target alpha-glucosidases, have been used to evaluate inhibitory activities. There might be confusion regarding the naming of alpha-glucosidases. For example, the term alpha-glucosidase is sometimes used as a generic name for alpha-glucoside hydrolases. Moreover, IUBMB recommends the use of "alpha-glucosidase" (EC 3.2.1.20) for exo-alpha-1,4-glucosidases, which are further classified into four families based on amino acid sequence similarities. Accordingly, substrate specificity and susceptibility to inhibitors varies markedly among enzymes in the IUBMB alpha-glucosidases. The design and screening of inhibitors without consideration of these differences is not efficient. For the development of a practical inhibitor that is operational in cells, HTS using the target alpha-glucosidase and the computer-aided design of inhibitors based on enzymatic information concerning the same alpha-glucosidase are essential.

  3. Enzymic hydrolysis of barley and other β-glucans by a β-(1→4)-glucan hydrolase

    PubMed Central

    Clarke, A. E.; Stone, B. A.

    1966-01-01

    1. A barley glucan with 68% of β-(1→4)-linkages and 32% of β-(1→3)-linkages was exhaustively hydrolysed with an Aspergillus niger β-(1→4)-glucan 4-glucanohydrolase (EC 3.2.1.4) (Clarke & Stone, 1965b). The hydrolysis products were separated and estimated. 2. The lower-molecular-weight products were identified as: glucose, 1·4%; cellobiose, 11·9%; 32-O-β-glucosylcellobiose, 45·0%; a tetrasaccharide(s), which was a substituted cellobiose, 16·4%. A series of unidentified higher-molecular-weight products (26·5%) were also found. 3. The identity of the products suggests that the A. niger β-(1→4)-glucan hydrolase hydrolyses β-glucosidic linkages joining 4-O-substituted glucose residues. 4. When an enzyme fraction containing the β-(1→4)-glucan hydrolase and an exo-β-(1→3)-glucan hydrolase was used, the same products were found, but the higher-molecular-weight products were observed to have only a transient existence in the hydrolysate and were virtually absent after prolonged incubation. It is suggested that these oligosaccharides are resistant to attack by β-(1→4)-glucan hydrolase but are partially hydrolysed by the exo-β-(1→3)-glucan hydrolase and therefore possess one or more (1→3)-linked glucose residues at their non-reducing end. PMID:4290404

  4. Defining sequence space and reaction products within the cyanuric acid hydrolase (AtzD)/barbiturase protein family.

    PubMed

    Seffernick, Jennifer L; Erickson, Jasmine S; Cameron, Stephan M; Cho, Seunghee; Dodge, Anthony G; Richman, Jack E; Sadowsky, Michael J; Wackett, Lawrence P

    2012-09-01

    Cyanuric acid hydrolases (AtzD) and barbiturases are homologous, found almost exclusively in bacteria, and comprise a rare protein family with no discernible linkage to other protein families or an X-ray structural class. There has been confusion in the literature and in genome projects regarding the reaction products, the assignment of individual sequences as either cyanuric acid hydrolases or barbiturases, and spurious connection of this family to another protein family. The present study has addressed those issues. First, the published enzyme reaction products of cyanuric acid hydrolase are incorrectly identified as biuret and carbon dioxide. The current study employed (13)C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry to show that cyanuric acid hydrolase releases carboxybiuret, which spontaneously decarboxylates to biuret. This is significant because it revealed that homologous cyanuric acid hydrolases and barbiturases catalyze completely analogous reactions. Second, enzymes that had been annotated incorrectly in genome projects have been reassigned here by bioinformatics, gene cloning, and protein characterization studies. Third, the AtzD/barbiturase family has previously been suggested to consist of members of the amidohydrolase superfamily, a large class of metallohydrolases. Bioinformatics and the lack of bound metals both argue against a connection to the amidohydrolase superfamily. Lastly, steady-state kinetic measurements and observations of protein stability suggested that the AtzD/barbiturase family might be an undistinguished protein family that has undergone some resurgence with the recent introduction of industrial s-triazine compounds such as atrazine and melamine into the environment.

  5. Systemic and spinal administration of FAAH, MAGL inhibitors and dual FAAH/MAGL inhibitors produce antipruritic effect in mice.

    PubMed

    Yesilyurt, Ozgur; Cayirli, Mutlu; Sakin, Yusuf Serdar; Seyrek, Melik; Akar, Ahmet; Dogrul, Ahmet

    2016-07-01

    The increase of endocannabinoid tonus by inhibiting fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL) represents a promising therapeutic approach in a variety of disease to overcome serious central side effects of exocannabinoids. Recent studies reported that systemic administration of FAAH and MAGL inhibitors produce antipruritic action. Dual FAAH/MAGL inhibitors have also been described to get enhanced endocannabinoid therapeutic effect. In this study, we examined and compared dose-related antipruritic effects of systemic (intraperitoneal; ip) or intrathecal (it) administration of selective FAAH inhibitor PF-3845 (5, 10, and 20 mg/kg, i.p.; 1, 5, and 10 µg, i.t.), MAGL inhibitor JZL184 (4, 20, and 40 mg/kg, i.p.; 1, 5, and 10 µg, i.t.) and dual FAAH/MAGL inhibitor JZL195 (2, 5, and 20 mg/kg, i.p.; 1, 5, and 10 µg, i.t.) on serotonin (5-HT)-induced scratching model. Serotonin (25 μg) was injected intradermally in a volume of 50 μl into the rostral part of skin on the back of male Balb-C mice. Both systemic or intrathecal administration of PF-3845, JZL184 or JZL195 produced similar dose-dependent antipruritic effects. Our results suggest that endocannabinoid-degrading enzymes FAAH and MAGL are involved in pruritic process at spinal level. FAAH, MAGL or dual FAAH/MAGL inhibitors have promising antipruritic effects, at least, in part through spinal site of action.

  6. Substrate ambiguity among the nudix hydrolases: biologically significant, evolutionary remnant, or both?

    PubMed

    McLennan, Alexander G

    2013-02-01

    Many members of the nudix hydrolase family exhibit considerable substrate multispecificity and ambiguity, which raises significant issues when assessing their functions in vivo and gives rise to errors in database annotation. Several display low antimutator activity when expressed in bacterial tester strains as well as some degree of activity in vitro towards mutagenic, oxidized nucleotides such as 8-oxo-dGTP. However, many of these show greater activity towards other nucleotides such as ADP-ribose or diadenosine tetraphosphate (Ap(4)A). The antimutator activities have tended to gain prominence in the literature, whereas they may in fact represent the residual activity of an ancestral antimutator enzyme that has become secondary to the more recently evolved major activity after gene duplication. Whether any meaningful antimutagenic function has also been retained in vivo requires very careful assessment. Then again, other examples of substrate ambiguity may indicate as yet unexplored regulatory systems. For example, bacterial Ap(4)A hydrolases also efficiently remove pyrophosphate from the 5' termini of mRNAs, suggesting a potential role for Ap(4)A in the control of bacterial mRNA turnover, while the ability of some eukaryotic mRNA decapping enzymes to degrade IDP and dIDP or diphosphoinositol polyphosphates (DIPs) may also be indicative of new regulatory networks in RNA metabolism. DIP phosphohydrolases also degrade diadenosine polyphosphates and inorganic polyphosphates, suggesting further avenues for investigation. This article uses these and other examples to highlight the need for a greater awareness of the possible significance of substrate ambiguity among the nudix hydrolases as well as the need to exert caution when interpreting incomplete analyses.

  7. A real-time fluorogenic assay for the visualization of glycoside hydrolase activity in planta.

    PubMed

    Ibatullin, Farid M; Banasiak, Alicja; Baumann, Martin J; Greffe, Lionel; Takahashi, Junko; Mellerowicz, Ewa J; Brumer, Harry

    2009-12-01

    There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e.g. protein-glucuronidase fusions). In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin beta-glycoside of a xylogluco-oligosaccharide (XXXG-beta-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pK(a) (5.8) and large extinction coefficient (epsilon 62,000 M(-1) cm(-1)), long-wavelength fluorescence (excitation 571 nm/emission 585 nm), and high quantum yield (0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-beta-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. The observation that XXXG-beta-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.

  8. A novel and enantioselective epoxide hydrolase from Aspergillus brasiliensis CCT 1435: purification and characterization.

    PubMed

    Beloti, Lilian L; Costa, Bruna Z; Toledo, Marcelo A S; Santos, Clelton A; Crucello, Aline; Fávaro, Marianna T P; Santiago, André S; Mendes, Juliano S; Marsaioli, Anita J; Souza, Anete P

    2013-10-01

    A novel epoxide hydrolase from Aspergillus brasiliensis CCT1435 (AbEH) was cloned and overexpressed in Escherichia coli cells with a 6xHis-tag and purified by nickel affinity chromatography. Gel filtration analysis and circular dichroism measurements indicated that this novel AbEH is a homodimer in aqueous solution and contains the typical secondary structure of an α/β hydrolase fold. The activity of AbEH was initially assessed using the fluorogenic probe O-(3,4-epoxybutyl) umbelliferone and was active in a broad range of pH (6-9) and temperature (25-45°C); showing optimum performance at pH 6.0 and 30°C. The Michaelis constant (KM) and maximum rate (Vmax) values were 495μM and 0.24μM/s, respectively. Racemic styrene oxide (SO) was used as a substrate to assess the AbEH activity and enantioselectivity, and 66% of the SO was hydrolyzed after only 5min of reaction, with the remaining (S)-SO ee exceeding 99% in a typical kinetic resolution behavior. The AbEH-catalyzed hydrolysis of SO was also evaluated in a biphasic system of water:isooctane; (R)-diol in 84% ee and unreacted (S)-SO in 36% ee were produced, with 43% conversion in 24h, indicating a discrete enantioconvergent behavior for AbEH. This novel epoxide hydrolase has biotechnological potential for the preparation of enantiopure epoxides or vicinal diols.

  9. Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms

    PubMed Central

    Baker, Perrin; Hill, Preston J.; Snarr, Brendan D.; Alnabelseya, Noor; Pestrak, Matthew J.; Lee, Mark J.; Jennings, Laura K.; Tam, John; Melnyk, Roman A.; Parsek, Matthew R.; Sheppard, Donald C.; Wozniak, Daniel J.; Howell, P. Lynne

    2016-01-01

    Bacterial biofilms present a significant medical challenge because they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases PelAh and PslGh encoded in the pel and psl biosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to target P. aeruginosa biofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix. PelAh and PslGh inhibit biofilm formation over a 24-hour period with a half maximal effective concentration (EC50) of 69.3 ± 1.2 and 4.1 ± 1.1 nM, respectively, and are capable of disrupting preexisting biofilms in 1 hour with EC50 of 35.7 ± 1.1 and 12.9 ± 1.1 nM, respectively. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58 to 94%. These noncytotoxic enzymes potentiated antibiotics because the addition of either enzyme to a sublethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude when used either prophylactically or on established 24-hour biofilms. In addition, PelAh was able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases to develop novel antibiofilm therapeutics. PMID:27386527

  10. Development of organophosphate hydrolase activity in a bacterial homolog of human cholinesterase

    NASA Astrophysics Data System (ADS)

    Legler, Patricia; Boisvert, Susanne; Compton, Jaimee; Millard, Charles

    2014-07-01

    We applied a combination of rational design and directed evolution (DE) to Bacillus subtilis p-nitrobenzyl esterase (pNBE) with the goal of enhancing organophosphorus acid anhydride hydrolase (OPAAH) activity. DE started with a designed variant, pNBE A107H, carrying a histidine homologous with human butyrylcholinesterase G117H to find complementary mutations that further enhance its OPAAH activity. Five sites were selected (G105, G106, A107, A190, and A400) within a 6.7 Å radius of the nucleophilic serine O?. All 95 variants were screened for esterase activity with a set of five substrates: pNP-acetate, pNP-butyrate, acetylthiocholine, butyrylthiocholine, or benzoylthiocholine. A microscale assay for OPAAH activity was developed for screening DE libraries. Reductions in esterase activity were generally concomitant with enhancements in OPAAH activity. One variant, A107K, showed an unexpected 7-fold increase in its kcat/Km for benzoylthiocholine, demonstrating that it is also possible to enhance the cholinesterase activity of pNBE. Moreover, DE resulted in at least three variants with modestly enhanced OPAAH activity compared to wild type pNBE. A107H/A190C showed a 50-fold increase in paraoxonase activity and underwent a slow time- and temperature-dependent change affecting the hydrolysis of OPAA and ester substrates. Structural analysis suggests that pNBE may represent a precursor leading to human cholinesterase and carboxylesterase 1 through extension of two vestigial specificity loops; a preliminary attempt to transfer the Ω-loop of BChE into pNBE is described. pNBE was tested as a surrogate scaffold for mammalian esterases. Unlike butyrylcholinesterase and pNBE, introducing a G143H mutation (equivalent to G117H) did not confer detectable OP hydrolase activity on human carboxylesterase 1. We discuss the importance of the oxyanion-hole residues for enhancing the OPAAH activity of selected serine hydrolases.

  11. Polyglycine hydrolases secreted by Pleosporineae fungi that target the linker region of plant class IV chitinases.

    PubMed

    Naumann, Todd A; Wicklow, Donald T; Price, Neil P J

    2014-06-01

    Cmps (chitinase-modifying proteins) are fungal proteases that truncate plant class IV chitinases by cleaving near their N-termini. We previously described Fv-cmp, a fungalysin protease that cleaves a conserved glycine-cysteine bond within the hevein domain. In the present paper we describe a new type of cmp, polyglycine hydrolases, as proteases that selectively cleave glycine-glycine peptide bonds within the polyglycine linker of plant class IV chitinases. Polyglycine hydrolases were purified from Cochliobolus carbonum (syn. Bipolaris zeicola; Bz-cmp) and Epicoccum sorghi (syn. Phoma sorghina; Es-cmp) and were shown to cleave three different maize class IV chitinase substrates. The proteolytic cleavage sites were assessed by SDS/PAGE and MALDI-TOF-MS and indicated the cleavage of multiple peptide bonds within the polyglycine linker regions. Site-directed mutagenesis was used to produce mutants of maize ChitB chitinase in which two serine residues in its linker were systematically modified to glycine. Serine to glycine changes in the ChitB linker resulted in higher susceptibility to truncation by Bz-cmp and altered substrate specificity for Bz-cmp and Es-cmp, such that different glycine-glycine peptide bonds were cleaved. Removal of the hevein domain led to loss of Es-cmp activity, indicating that interactions outside of the active site are important for recognition. Our findings demonstrate that plant class IV chitinases with polyglycine linkers are targeted for truncation by selective polyglycine hydrolases that are secreted by plant pathogenic fungi. This novel proteolysis of polyglycine motifs is previously unreported, but the specificity is similar to that of bacterial lysostaphin proteases, which cleave pentaglycine cross-links from peptidoglycan.

  12. Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms.

    PubMed

    Baker, Perrin; Hill, Preston J; Snarr, Brendan D; Alnabelseya, Noor; Pestrak, Matthew J; Lee, Mark J; Jennings, Laura K; Tam, John; Melnyk, Roman A; Parsek, Matthew R; Sheppard, Donald C; Wozniak, Daniel J; Howell, P Lynne

    2016-05-01

    Bacterial biofilms present a significant medical challenge because they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases PelAh and PslGh encoded in the pel and psl biosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to target P. aeruginosa biofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix. PelAh and PslGh inhibit biofilm formation over a 24-hour period with a half maximal effective concentration (EC50) of 69.3 ± 1.2 and 4.1 ± 1.1 nM, respectively, and are capable of disrupting preexisting biofilms in 1 hour with EC50 of 35.7 ± 1.1 and 12.9 ± 1.1 nM, respectively. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58 to 94%. These noncytotoxic enzymes potentiated antibiotics because the addition of either enzyme to a sublethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude when used either prophylactically or on established 24-hour biofilms. In addition, PelAh was able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases to develop novel antibiofilm therapeutics.

  13. Gene expression of 5-lipoxygenase and LTA4 hydrolase in renal tissue of nephrotic syndrome patients

    PubMed Central

    Menegatti, E; Roccatello, D; Fadden, K; Piccoli, G; De Rosa, G; Sena, L M; Rifai, A

    1999-01-01

    Leukotrienes (LT) of the 5-lipoxygenase pathway constitute a class of potent biological lipid mediators of inflammation implicated in the pathogenesis of different models of experimental glomerulonephritis. The key enzyme, 5-lipoxygenase (5-LO), catalyses oxygenation of arachidonic acid to generate the primary leukotriene LTA4. This LT, in turn, serves as a substrate for either LTA4 hydrolase, to form the potent chemoattractant LTB4, or LTC4 synthase, to produce the powerful vasoconstrictor LTC4. To investigate the potential role of LT in the pathogenesis of human glomerulonephritis with nephrotic syndrome, we examined the gene expression of 5-LO and LTA4 hydrolase in renal tissue of 21 adult patients with nephrotic syndrome and 11 controls. The patients consisted of 11 cases of membranous nephropathy (MN), seven focal and segmental glomerulosclerosis (FSGS), two non-IgA mesangial glomerulonephritis and one minimal change disease. Total RNA purified from renal tissue was reverse transcribed into cDNA and amplified with specific primers in a polymerase chain reaction (RT-PCR). Eight patients' renal tissue, four MN and four FSGS, co-expressed 5-LO and LTA4 hydrolase. In situ hybridization analysis revealed 5-LO expression and distribution limited to the interstitial cells surrounding the peritubular capillaries. Comparative clinical and immunohistological data showed that these eight patients had impaired renal function and interstitial changes that significantly correlated with 5-LO expression. These findings suggest that leukotrienes may play an important role in the pathogenesis of MN and FSGS. These results are also relevant to elucidating the pathophysiologic mechanisms which underlie progression to renal failure in these diseases. PMID:10337029

  14. Expression and purification of an engineered, yeast-expressed Leishmania donovani nucleoside hydrolase with immunogenic properties

    PubMed Central

    Hudspeth, Elissa M.; Wang, Qian; Seid, Christopher A.; Hammond, Molly; Wei, Junfei; Liu, Zhuyun; Zhan, Bin; Pollet, Jeroen; Heffernan, Michael J.; McAtee, C. Patrick; Engler, David A.; Matsunami, Risë K.; Strych, Ulrich; Asojo, Oluwatoyin A.; Hotez, Peter J.; Bottazzi, Maria Elena

    2016-01-01

    ABSTRACT Leishmania donovani is the major cause of visceral leishmaniasis (kala-azar), now recognized as the parasitic disease with the highest level of mortality second only to malaria. No human vaccine is currently available. A 36 kDa L. donovani nucleoside hydrolase (LdNH36) surface protein has been previously identified as a potential vaccine candidate antigen. Here we present data on the expression of LdNH36 in Pichia pastoris and its purification at the 20 L scale to establish suitability for future pilot scale manufacturing. To improve efficiency of process development and ensure reproducibility, 4 N-linked glycosylation sites shown to contribute to heterogeneous high-mannose glycosylation were mutated to glutamine residues. The mutant LdNH36 (LdNH36-dg2) was expressed and purified to homogeneity. Size exclusion chromatography and light scattering demonstrated that LdNH36-dg2 existed as a tetramer in solution, similar to the wild-type recombinant L. major nucleoside hydrolase. The amino acid mutations do not affect the tetrameric interface as confirmed by theoretical modeling, and the mutated amino acids are located outside the major immunogenic domain. Immunogenic properties of the LdNH36-dg2 recombinant protein were evaluated in BALB/c mice using formulations that included a synthetic CpG oligodeoxynucleotide, together with a microparticle delivery platform (poly(lactic-co-glycolic acid)). Mice exhibited high levels of IgG1, IgG2a, and IgG2b antibodies that were reactive to both LdNH36-dg2 and LdNH36 wild-type. While the point mutations did affect the hydrolase activity of the enzyme, the IgG antibodies elicited by LdNH36-dg2 were shown to inhibit the hydrolase activity of the wild-type LdNH36. The results indicate that LdNH36-dg2 as expressed in and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing in support of future first-in-humans phase 1 clinical trials. PMID:26839079

  15. Biosensing Paraoxon in Simulated Environmental Samples by Immobilized Organophosphorus Hydrolase in Functionalized Mesoporous Silica

    SciTech Connect

    Lei, Chenghong; Valenta, Michelle M.; Saripalli, Prasad; Ackerman, Eric J.

    2007-01-01

    There is a critical need for highly sensitive, cost-effective sensors to conduct ecological analyses for environmental and homeland security related applications. We report here on a method which significantly overcomes this difficulty, and demonstrate its application in a biosensor for aquatic environmental applications. A fast-responding and stable biosensor was developed via immobilization of organophosphorus hydrolase (OPH) in functionalized mesoporous silica (FMS) with pore sizes in tens of nanometers. The sensor was tested for detection of paraoxon in simulated environmental samples, under wide ranging physico-chemical conditions.

  16. Neutron diffraction analysis of Pseudomonas aeruginosa peptidyl-tRNA hydrolase 1.

    PubMed

    McFeeters, Hana; Vandavasi, Venu Gopal; Weiss, Kevin L; Coates, Leighton; McFeeters, Robert L

    2016-03-01

    Perdeuterated peptidyl-tRNA hydrolase 1 from Pseudomonas aeruginosa was crystallized for structural analysis using neutron diffraction. Crystals of perdeuterated protein were grown to 0.15 mm(3) in size using batch crystallization in 22.5% polyethylene glycol 4000, 100 mM Tris pH 7.5, 10%(v/v) isopropyl alcohol with a 20-molar excess of trilysine as an additive. Neutron diffraction data were collected from a crystal at room temperature using the MaNDi single-crystal diffractometer at Oak Ridge National Laboratory.

  17. Enantioselective hydrolysis of racemic epichlorohydrin using an epoxide hydrolase from Novosphingobium aromaticivorans.

    PubMed

    Woo, Jung-Hee; Hwang, Young-Ok; Kang, Ji-Hyun; Lee, Hyun Sook; Kim, Sang-Jin; Kang, Sung Gyun

    2010-09-01

    Previously we reported that an epoxide hydrolase (EHase) from Novosphingobium aromaticivorans could preferentially hydrolyze (R)-styrene oxide. In this study, we demonstrate that the purified NEH could be also effective in chiral resolution of racemic epichlorohydrin (ECH). Particularly, the purified NEH showed excellent hydrolyzing activity toward ECH to complete the reaction at a short period of incubation time. Enantiopure (S)-ECH could be obtained with a high enantiopurity of more than 99.99% enantiomeric excess (ee) and yield of 20.7% (theoretical, 50%). The chiral resolution of the purified NEH toward ECH was not susceptible to substrate inhibition by 500 mM racemic ECH.

  18. Some hydrolase activities from the tick Hyalomma lusitanicum koch, 1844 (Ixodoidea: Ixodida).

    PubMed

    Giménez-Pardo, C; Martínez-Grueiro, M M

    2008-12-01

    In this work has been made a detection and preliminary characterization of some hydrolases in whole extracts from unfed adult males and females of Hyalomma lusitanicum, one of the vectors for Theileria annulata that causes Mediterranean theileriosis in cattle. We have elected as targets, proteases as enzymes implicated in the nutritional processes of ticks, esterases that are usually implicated in resistance to organophosphates and phosphatises often implicated in protein phosphorilation and control of ticks salivary gland. The biological role and physiological significance are discussed in terms of the possibility of use these enzymes as possible in future anti-tick vaccination or acaricide resistance.

  19. Characterization of organophosphorus hydrolases and the genetic manipulation of the phosphotriesterase from pseudomonas diminuta

    SciTech Connect

    Dave, K.I.; Miller, C.E.; Wild, J.R.

    1993-12-31

    There are a variety of enzymes which are specifically capable of hydrolyzing organophosphorus esters with different phosphoryl bonds from the typical phosphotriester bonds of common insecticidal neurotoxins (e.g. paraoxon or coumaphos) to the phosphonate-fluoride bonds of chemical warfare agents (e.g. soman or sarin). These enzymes comprise a diverse set of enzymes whose basic architecture and substrate specificities vary dramatically, yet they appear to be ubiquitous throughout nature. The most thoroughly studied of these enzymes is the organophosphate hydrolase (opd gene product) of Pseudomonas diminuta and Ftavobacterium sp. ATCC 27551, and the heterologous expression, post-translational modification, and genetic engineering studies undertaken with this enzyme are described.

  20. Characterization of organophosphorus hydrolases and the genetic manipulation of the phosphotriesterase from Pseudomonas diminuta.

    PubMed

    Dave, K I; Miller, C E; Wild, J R

    1993-06-01

    There are a variety of enzymes which are specifically capable of hydrolyzing organophosphorus esters with different phosphoryl bonds from the typical phosphotriester bonds of common insecticidal neurotoxins (e.g. paraoxon or coumaphos) to the phosphonate-fluoride bonds of chemical warfare agents (e.g. soman or sarin). These enzymes comprise a diverse set of enzymes whose basic architecture and substrate specificities vary dramatically, yet they appear to be ubiquitous throughout nature. The most thoroughly studied of these enzymes is the organophosphate hydrolase (opd gene product) of Pseudomonas diminuta and Flavobacterium sp. ATCC 27551, and the heterologous expression, post-translational modification, and genetic engineering studies undertaken with this enzyme are described.

  1. Acyl hydrolases from trans-AT polyketide synthases target acetyl units on acyl carrier proteins.

    PubMed

    Jenner, Matthew; Afonso, Jose P; Kohlhaas, Christoph; Karbaum, Petra; Frank, Sarah; Piel, Jörn; Oldham, Neil J

    2016-04-18

    Acyl hydrolase (AH) domains are a common feature of trans-AT PKSs. They have been hypothesised to perform a proofreading function by removing acyl chains from stalled sites. This study determines the substrate tolerance of the AH PedC for a range of acyl-ACPs. Clear preference towards short, linear acyl-ACPs is shown, with acetyl-ACP the best substrate. These results imply a more targeted housekeeping role for PedC: namely the removal of unwanted acetyl groups from ACP domains caused by erroneous transfer of acetyl-CoA, or possibly by decarboxylation of malonyl-ACP.

  2. [Research advances on ubiquitin C-terminal hydrolase in oncogenesis and progression].

    PubMed

    Yu, Juan; Chen, Wei-lin

    2015-03-01

    By regulating the ubiquitination and deubiquitination of key proteins, ubiquitin-proteasome system mediates a variety of cellular activities. Ubiquitin C-terminal hydrolase (UCH) is a deubiquitinating enzyme which can remove ubiquitin chains at the end of ubiquited proteins. The abnormal expression of UCH has been found in a variety of tumor tissues, indicating that it participates in the process of tumor development. Here we review the characteristics of UCH members and current understanding about the role of UCH in tumor development, and the potential target for cancer treatment.

  3. Remodeling Natural Products: Chemistry and Serine Hydrolase Activity of a Rocaglate-Derived β-Lactone

    PubMed Central

    2015-01-01

    Flavaglines are a class of natural products with potent insecticidal and anticancer activities. β-Lactones are a privileged structural motif found in both therapeutic agents and chemical probes. Herein, we report the synthesis, unexpected light-driven di-epimerization, and activity-based protein profiling of a novel rocaglate-derived β-lactone. In addition to in vitro inhibition of the serine hydrolases ABHD10 and ACOT1/2, the most potent β-lactone enantiomer was also found to inhibit these enzymes, as well as the serine peptidases CTSA and SCPEP1, in PC3 cells. PMID:24447064

  4. New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

    DOE PAGES

    Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth; ...

    2015-12-18

    In this study, non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall.

  5. Effect of aloe vera leaf gel extract on membrane bound phosphatases and lysosomal hydrolases in rats with streptozotocin diabetes.

    PubMed

    Rajasekaran, S; Sriram, N; Arulselvan, P; Subramanian, S

    2007-03-01

    Diabetes mellitus is known to promote deterioration of membrane function and impair intra cellular metabolism in the organism. The aim of the present study was to examine the effect of the ethanolic extract from Aloe vera leaf gel on membrane bound phosphatases and lysosomal hydrolases in the liver and kidney of streptozotocin (STZ)-induced diabetic rats. The rats treated with STZ showed significant alterations in the activities of membrane bound phosphatases and lysosomal hydrolases in the liver and kidney. Oral administration of Aloe vera gel extract at a dose of 300 mg/kg body weight/day to STZ-induced diabetic rats for a period of 21 days significantly restored the alterations in enzymes activity to near normalcy. These results were compared with glibenclamide, a reference drug. Thus, the present study confirms that Aloe vera gel extract possesses a significant beneficial effect on membrane bound phosphatases and lysosomal hydrolases.

  6. A chemical genetic screen uncovers a small molecule enhancer of the N-acylethanolamine degrading enzyme, fatty acid amide hydrolase, in Arabidopsis

    PubMed Central

    Khan, Bibi Rafeiza; Faure, Lionel; Chapman, Kent D.; Blancaflor, Elison B.

    2017-01-01

    N-Acylethanolamines (NAEs) are a group of fatty acid amides that play signaling roles in diverse physiological processes in eukaryotes. Fatty acid amide hydrolase (FAAH) degrades NAE into ethanolamine and free fatty acid to terminate its signaling function. In animals, chemical inhibitors of FAAH have been used for therapeutic treatment of pain and as tools to probe deeper into biochemical properties of FAAH. In a chemical genetic screen for small molecules that dampened the inhibitory effect of N-lauroylethanolamine (NAE 12:0) on Arabidopsis thaliana seedling growth, we identified 6-(2-methoxyphenyl)-1,3-dimethyl-5-phenyl-1H-pyrrolo[3,4-d]pyrimidine-2,4(3 H,6 H)-dione (or MDPD). MDPD alleviated the growth inhibitory effects of NAE 12:0, in part by enhancing the enzymatic activity of Arabidopsis FAAH (AtFAAH). In vitro, biochemical assays showed that MDPD enhanced the apparent Vmax of AtFAAH but did not alter the affinity of AtFAAH for its NAE substrates. Structural analogs of MDPD did not affect AtFAAH activity or dampen the inhibitory effect of NAE 12:0 on seedling growth indicating that MDPD is a specific synthetic chemical activator of AtFAAH. Collectively, our study demonstrates the feasibility of using an unbiased chemical genetic approach to identify new pharmacological tools for manipulating FAAH- and NAE-mediated physiological processes in plants. PMID:28112243

  7. β-Amyloid (Aβ) Oligomers Impair Brain-derived Neurotrophic Factor Retrograde Trafficking by Down-regulating Ubiquitin C-terminal Hydrolase, UCH-L1*

    PubMed Central

    Poon, Wayne W.; Carlos, Anthony J.; Aguilar, Brittany L.; Berchtold, Nicole C.; Kawano, Crystal K.; Zograbyan, Vahe; Yaopruke, Tim; Shelanski, Michael; Cotman, Carl W.

    2013-01-01

    We previously found that BDNF-dependent retrograde trafficking is impaired in AD transgenic mouse neurons. Utilizing a novel microfluidic culture chamber, we demonstrate that Aβ oligomers compromise BDNF-mediated retrograde transport by impairing endosomal vesicle velocities, resulting in impaired downstream signaling driven by BDNF/TrkB, including ERK5 activation, and CREB-dependent gene regulation. Our data suggest that a key mechanism mediating the deficit involves ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that functions to regulate cellular ubiquitin. Aβ-induced deficits in BDNF trafficking and signaling are mimicked by LDN (an inhibitor of UCH-L1) and can be reversed by increasing cellular UCH-L1 levels, demonstrated here using a transducible TAT-UCH-L1 strategy. Finally, our data reveal that UCH-L1 mRNA levels are decreased in the hippocampi of AD brains. Taken together, our data implicate that UCH-L1 is important for regulating neurotrophin receptor sorting to signaling endosomes and supporting retrograde transport. Further, our results support the idea that in AD, Aβ may down-regulate UCH-L1 in the AD brain, which in turn impairs BDNF/TrkB-mediated retrograde signaling, compromising synaptic plasticity and neuronal survival. PMID:23599427

  8. Inhibition of soluble epoxide hydrolase after cardiac arrest/cardiopulmonary resuscitation induces a neuroprotective phenotype in activated microglia and improves neuronal survival.

    PubMed

    Wang, Jianming; Fujiyoshi, Tetsuhiro; Kosaka, Yasuharu; Raybuck, Jonathan D; Lattal, K Matthew; Ikeda, Mizuko; Herson, Paco S; Koerner, Ines P

    2013-10-01

    Cardiac arrest (CA) causes hippocampal neuronal death that frequently leads to severe loss of memory function in survivors. No specific treatment is available to reduce neuronal death and improve functional outcome. The brain's inflammatory response to ischemia can exacerbate injury and provides a potential treatment target. We hypothesized that microglia are activated by CA and contribute to neuronal loss. We used a mouse model to determine whether pharmacologic inhibition of the proinflammatory microglial enzyme soluble epoxide hydrolase (sEH) after CA alters microglial activation and neuronal death. The sEH inhibitor 4-phenylchalcone oxide (4-PCO) was administered after successful cardiopulmonary resuscitation (CPR). The 4-PCO treatment significantly reduced neuronal death and improved memory function after CA/CPR. We found early activation of microglia and increased expression of inflammatory tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the hippocampus after CA/CPR, which was unchanged after 4-PCO treatment, while expression of antiinflammatory IL-10 increased significantly. We conclude that sEH inhibition after CA/CPR can alter the transcription profile in activated microglia to selectively induce antiinflammatory and neuroprotective IL-10 and reduce subsequent neuronal death. Switching microglial gene expression toward a neuroprotective phenotype is a promising new therapeutic approach for ischemic brain injury.

  9. A chemical genetic screen uncovers a small molecule enhancer of the N-acylethanolamine degrading enzyme, fatty acid amide hydrolase, in Arabidopsis

    DOE PAGES

    Khan, Bibi Rafeiza; Faure, Lionel; Chapman, Kent D.; ...

    2017-01-23

    N-Acylethanolamines (NAEs) are a group of fatty acid amides that play signaling roles in diverse physiological processes in eukaryotes. We used fatty acid amide hydrolase (FAAH) degrades NAE into ethanolamine and free fatty acid to terminate its signaling function. In animals, chemical inhibitors of FAAH for therapeutic treatment of pain and as tools to probe deeper into biochemical properties of FAAH. In a chemical genetic screen for small molecules that dampened the inhibitory effect of N-lauroylethanolamine (NAE 12:0) on Arabidopsis thaliana seedling growth, we identified 6-(2-methoxyphenyl)-1,3-dimethyl-5-phenyl-1H-pyrrolo[3,4-d]pyrimidine-2,4(3 H,6 H)-dione (or MDPD). MDPD alleviated the growth inhibitory effects of NAE 12:0, inmore » part by enhancing the enzymatic activity of Arabidopsis FAAH (AtFAAH). In vitro, biochemical assays showed that MDPD enhanced the apparent Vmax of AtFAAH but did not alter the affinity of AtFAAH for its NAE substrates. Furthermore, structural analogs of MDPD did not affect AtFAAH activity or dampen the inhibitory effect of NAE 12:0 on seedling growth indicating that MDPD is a specific synthetic chemical activator of AtFAAH. Our study demonstrates the feasibility of using an unbiased chemical genetic approach to identify new pharmacological tools for manipulating FAAH- and NAE-mediated physiological processes in plants.« less

  10. β-Amyloid (Aβ) oligomers impair brain-derived neurotrophic factor retrograde trafficking by down-regulating ubiquitin C-terminal hydrolase, UCH-L1.

    PubMed

    Poon, Wayne W; Carlos, Anthony J; Aguilar, Brittany L; Berchtold, Nicole C; Kawano, Crystal K; Zograbyan, Vahe; Yaopruke, Tim; Shelanski, Michael; Cotman, Carl W

    2013-06-07

    We previously found that BDNF-dependent retrograde trafficking is impaired in AD transgenic mouse neurons. Utilizing a novel microfluidic culture chamber, we demonstrate that Aβ oligomers compromise BDNF-mediated retrograde transport by impairing endosomal vesicle velocities, resulting in impaired downstream signaling driven by BDNF/TrkB, including ERK5 activation, and CREB-dependent gene regulation. Our data suggest that a key mechanism mediating the deficit involves ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that functions to regulate cellular ubiquitin. Aβ-induced deficits in BDNF trafficking and signaling are mimicked by LDN (an inhibitor of UCH-L1) and can be reversed by increasing cellular UCH-L1 levels, demonstrated here using a transducible TAT-UCH-L1 strategy. Finally, our data reveal that UCH-L1 mRNA levels are decreased in the hippocampi of AD brains. Taken together, our data implicate that UCH-L1 is important for regulating neurotrophin receptor sorting to signaling endosomes and supporting retrograde transport. Further, our results support the idea that in AD, Aβ may down-regulate UCH-L1 in the AD brain, which in turn impairs BDNF/TrkB-mediated retrograde signaling, compromising synaptic plasticity and neuronal survival.

  11. The case for the development of novel analgesic agents targeting both fatty acid amide hydrolase and either cyclooxygenase or TRPV1

    PubMed Central

    Fowler, CJ; Naidu, PS; Lichtman, A; Onnis, V

    2009-01-01

    Although the dominant approach to drug development is the design of compounds selective for a given target, compounds targeting more than one biological process may have superior efficacy, or alternatively a better safety profile than standard selective compounds. Here, this possibility has been explored with respect to the endocannabinoid system and pain. Compounds inhibiting the enzyme fatty acid amide hydrolase (FAAH), by increasing local endocannabinoid tone, produce potentially useful effects in models of inflammatory and possibly neuropathic pain. Local increases in levels of the endocannabinoid anandamide potentiate the actions of cyclooxygenase inhibitors, raising the possibility that compounds inhibiting both FAAH and cyclooxygenase can be as effective as non-steroidal anti-inflammatory drugs but with a reduced cyclooxygenase inhibitory ‘load’. An ibuprofen analogue active in models of visceral pain and with FAAH and cyclooxygenase inhibitory properties has been identified. Another approach, built in to the experimental analgesic compound N-arachidonoylserotonin, is the combination of FAAH inhibitory and transient receptor potential vanilloid type 1 antagonist properties. Although finding the right balance of actions upon the two targets is a key to success, it is hoped that dual-action compounds of the types illustrated in this review will prove to be useful analgesic drugs. PMID:19226258

  12. Co-purification of microsomal epoxide hydrolase with the warfarin-sensitive vitamin K1 oxide reductase of the vitamin K cycle.

    PubMed

    Guenthner, T M; Cai, D; Wallin, R

    1998-01-15

    Vitamin K1 oxide reductase activity has been partially purified from rat liver microsomes. A three-step procedure produced a preparation in which warfarin-sensitive vitamin K1 oxide reductase activity was 118-fold enriched over the activity in intact rat liver microsomes. A major component of the multi-protein mixture was identified as a 50 kDa protein that strongly cross-reacts with antiserum prepared against homogeneous rat liver microsomal epoxide hydrolase. The reductase preparation also had a high level or epoxide hydrolase activity against two xenobiotic epoxide substrates. The K(m) values for hydrolysis by the reductase preparation were similar to those for homogeneous microsomal epoxide hydrolase itself, and the specific hydrolase activities of the reductase preparation were 25-35% of the specific activities measured for the homogeneous hydrolase preparation. Antibodies prepared against homogeneous microsomal epoxide hydrolase inhibited up to 80% of reductase activity of the reductase preparation. Homogeneous microsomal epoxide hydrolase had no vitamin K1 oxide reductase activity. This evidence suggests that microsomal epoxide hydrolase, or a protein that is very similar to it, is a major functional component of a multi-protein complex that is responsible for vitamin K1 oxide reduction in rat liver microsomes.

  13. Cloning and expression of a phloretin hydrolase gene from Eubacterium ramulus and characterization of the recombinant enzyme.

    PubMed

    Schoefer, Lilian; Braune, Annett; Blaut, Michael

    2004-10-01

    Phloretin hydrolase catalyzes the hydrolytic C-C cleavage of phloretin to phloroglucinol and 3-(4-hydroxyphenyl)propionic acid during flavonoid degradation in Eubacterium ramulus. The gene encoding the enzyme was cloned by screening a gene library for hydrolase activity. The insert of a clone conferring phloretin hydrolase activity was sequenced. Sequence analysis revealed an open reading frame of 822 bp (phy), a putative promoter region, and a terminating stem-loop structure. The deduced amino acid sequence of phy showed similarities to a putative protein of the 2,4-diacetylphloroglucinol biosynthetic operon from Pseudomonas fluorescens. The phloretin hydrolase was heterologously expressed in Escherichia coli and purified. The molecular mass of the native enzyme was approximately 55 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of phy indicated molecular masses of 30 and 30.8 kDa, respectively, suggesting that the enzyme is a homodimer. The recombinant phloretin hydrolase catalyzed the hydrolysis of phloretin to equimolar amounts of phloroglucinol and 3-(4-hydroxyphenyl)propionic acid. The optimal temperature and pH of the catalyzed reaction mixture were 37 degrees C and 7.0, respectively. The K(m) for phloretin was 13 +/- 3 microM and the k(cat) was 10 +/- 2 s(-1). The enzyme did not transform phloretin-2'-glucoside (phloridzin), neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, or trans-1,3-diphenyl-2,3-epoxy-propan-1-one. The catalytic activity of the phloretin hydrolase was reduced by N-bromosuccinimide, o-phenanthroline, N-ethylmaleimide, and CuCl(2) to 3, 20, 35, and 85%, respectively. Phloroglucinol and 3-(4-hydroxyphenyl)propionic acid reduced the activity to 54 and 70%, respectively.

  14. Genetic surface-display of methyl parathion hydrolase on Yarrowia lipolytica for removal of methyl parathion in water.

    PubMed

    Wang, Xing-Xing; Chi, Zhe; Ru, Shao-Guo; Chi, Zhen-Ming

    2012-09-01

    In this study, the mph gene encoding methyl parathion hydrolase from Pseudomonas sp. WBC-3 was expressed in Yarrowia lipolytica and the expressed methyl parathion hydrolase was displayed on cell surface of Y. lipolytica. The activity of methyl parathion hydrolase displayed on the yeast cells of the transformant Z51 was 59.5 U mg⁻¹ of cell dry cells (450.6 U per mL of the culture) in the presence of 5.0 mM of Co²⁺. The displayed methyl parathion hydrolase had the optimal pH of 9.5 and the optimal temperature of 40 °C, respectively and was stable in the pH range of 4.5-11 and up to 40 °C. The displayed methyl parathion hydrolase was also stimulated by Co²⁺, Cu²⁺, Ni²⁺ and Mn²⁺, and was not affected by Fe²⁺, Fe³⁺, Na⁺, K⁺, Ca²⁺ and Zn²⁺, but was inhibited by other cations tested. Under the optimal conditions (OD(600 nm) = 2.6, the substrate concentration = 100 mg L⁻¹ and 40 °C), 90.8 % of methyl parathion was hydrolyzed within 30 min. Under the similar conditions, 98.7, 97.0, 96.5 and 94.4 % of methyl parathion in tap water (pH 9.5), tap water (pH 6.8), seawater (pH 9.5) and natural seawater (pH 8.2) were hydrolyzed, respectively, suggesting that the methyl parathion hydrolase displayed on the yeast cells can effectively remove methyl parathion in water.

  15. Inhibition of soluble epoxide hydrolase modulates inflammation and autophagy in obese adipose tissue and liver: role for omega-3 epoxides.

    PubMed

    López-Vicario, Cristina; Alcaraz-Quiles, José; García-Alonso, Verónica; Rius, Bibiana; Hwang, Sung H; Titos, Esther; Lopategi, Aritz; Hammock, Bruce D; Arroyo, Vicente; Clària, Joan

    2015-01-13

    Soluble epoxide hydrolase (sEH) is an emerging therapeutic target in a number of diseases that have inflammation as a common underlying cause. sEH limits tissue levels of cytochrome P450 (CYP) epoxides derived from omega-6 and omega-3 polyunsaturated fatty acids (PUFA) by converting these antiinflammatory mediators into their less active diols. Here, we explored the metabolic effects of a sEH inhibitor (t-TUCB) in fat-1 mice with transgenic expression of an omega-3 desaturase capable of enriching tissues with endogenous omega-3 PUFA. These mice exhibited increased CYP1A1, CYP2E1, and CYP2U1 expression and abundant levels of the omega-3-derived epoxides 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic (19,20-EDP) in insulin-sensitive tissues, especially liver, as determined by LC-ESI-MS/MS. In obese fat-1 mice, t-TUCB raised hepatic 17,18-EEQ and 19,20-EDP levels and reinforced the omega-3-dependent reduction observed in tissue inflammation and lipid peroxidation. t-TUCB also produced a more intense antisteatotic action in obese fat-1 mice, as revealed by magnetic resonance spectroscopy. Notably, t-TUCB skewed macrophage polarization toward an antiinflammatory M2 phenotype and expanded the interscapular brown adipose tissue volume. Moreover, t-TUCB restored hepatic levels of Atg12-Atg5 and LC3-II conjugates and reduced p62 expression, indicating up-regulation of hepatic autophagy. t-TUCB consistently reduced endoplasmic reticulum stress demonstrated by the attenuation of IRE-1α and eIF2α phosphorylation. These actions were recapitulated in vitro in palmitate-primed hepatocytes and adipocytes incubated with 19,20-EDP or 17,18-EEQ. Relatively similar but less pronounced actions were observed with the omega-6 epoxide, 14,15-EET, and nonoxidized DHA. Together, these findings identify omega-3 epoxides as important regulators of inflammation and autophagy in insulin-sensitive tissues and postulate sEH as a druggable target in metabolic

  16. Citrate-capped gold nanoparticles for the label-free detection of ubiquitin C-terminal hydrolase-1.

    PubMed

    Agarwal, Srishti; Mishra, Priyanka; Shivange, Gururaj; Kodipelli, Naveena; Moros, María; de la Fuente, Jesús M; Anindya, Roy

    2015-02-21

    Ubiquitin C-terminal hydrolase-1 (UCH-L1) is a specific neuronal endoprotease that cleaves the specific peptide bond between ubiquitin molecules. UCH-L1 is released in serum and cerebrospinal fluid after severe brain injury and is considered to be an important biomarker of brain injury. A common polymorphism of UCH-L1 (S18Y) is also linked to a reduced risk of Parkinson's disease. In addition to its function in neuronal tissues, UCH-L1 may also play a part in the progression of certain non-neuronal cancers. UCH-L1 is highly expressed in primary lung tumors and colo-rectal cancers, suggesting a role in tumorigenesis. We report here the development of a sensitive and accurate UCH-L1 assay based on the surface plasmon resonance (SPR) absorbance of gold nanoparticles. We created a unique UCH-L1 substrate containing a ubiquitin molecule with two terminal thiol groups. This UCH-L1 substrate interacted with gold nanoparticles via the terminal thiol groups and induced clustering of the nanoparticles, which was detected by SPR absorbance at 650 nm. UCH-L1 proteolytically cleaved the substrate and the clustered gold nanoparticles were dispersed and could be detected by a shift in the SPR absorbance to 530 nm. This change in absorbance was proportional to the concentration of UCH-L1 and can be used for the quantification of functional UCH-L1. The currently available fluorescence-based UCH-L1 assay is affected by a high background signal and a poor detection limit, especially in the presence of serum. The assay reported here can detect concentrations of UCH-L1 as low as 20 ng ml(-1) (0.8 nM) and the presence of serum had no effect on the detection limit. This assay could be adapted for the rapid determination of the severity of brain injury and could also be applied to high-throughput screening of inhibitors of UCH-L1 enzymatic activity in Parkinson's disease and cancer.

  17. Blocking of fatty acid amide hydrolase activity with PF-04457845 in human brain: a positron emission tomography study with the novel radioligand [11C]CURB

    PubMed Central

    Boileau, Isabelle; Rusjan, Pablo M; Williams, Belinda; Mansouri, Esmaeil; Mizrahi, Romina; De Luca, Vincenzo; Johnson, Douglas S; Wilson, Alan A; Houle, Sylvain; Kish, Stephen J; Tong, Junchao

    2015-01-01

    Positron emission tomography with [11C]CURB was recently developed to quantify fatty acid amide hydrolase (FAAH), the enzyme responsible for hydrolyzing the endocannabinoid anandamide. This study investigated the test–retest reliability of [11C]CURB as well as its in vivo specificity and the validity of the kinetic model by using the highly specific FAAH inhibitor, PF-04457845. Five healthy volunteers completed test–retest [11C]CURB scans 1 to 2 months apart and six subjects completed baseline and blocking scans on the same day after PF-04457845 (p.o.) administration (1, 4, or 20 mg; n=2 each). The composite parameter λk3 (an index of FAAH activity, λ=K1/k2) was estimated using an irreversible two-tissue compartment model with plasma input function. There were no clinically observable responses to oral PF-04457845 or [11C]CURB injection. Oral administration of PF-04457845 reduced [11C]CURB binding to a homogeneous level at all three doses, with λk3 values decreased by ⩾91%. Excellent reproducibility and good reliability (test–retest variability=9% intraclass correlation coefficient=0.79) were observed across all regions of interest investigated. Our findings suggest that λk3/[11C]CURB is a reliable, highly sensitive, and selective tool to measure FAAH activity in human brain in vivo. Moreover, PF-04457845 is a highly potent FAAH inhibitor (>95% inhibition at 1 mg) in living human brain. PMID:26082009

  18. Pharmacological blockade of the fatty acid amide hydrolase (FAAH) alters neural proliferation, apoptosis and gliosis in the rat hippocampus, hypothalamus and striatum in a negative energy context

    PubMed Central

    Rivera, Patricia; Bindila, Laura; Pastor, Antoni; Pérez-Martín, Margarita; Pavón, Francisco J.; Serrano, Antonia; de la Torre, Rafael; Lutz, Beat; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2015-01-01

    Endocannabinoids participate in the control of neurogenesis, neural cell death and gliosis. The pharmacological effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which limits the endocannabinoid degradation, was investigated in the present study. Cell proliferation (phospho-H3+ or BrdU+ cells) of the main adult neurogenic zones as well as apoptosis (cleaved caspase-3+), astroglia (GFAP+), and microglia (Iba1+ cells) were analyzed in the hippocampus, hypothalamus and striatum of rats intraperitoneally treated with URB597 (0.3 mg/kg/day) at one dose/4-days resting or 5 doses (1 dose/day). Repeated URB597 treatment increased the plasma levels of the N-acylethanolamines oleoylethanolamide, palmitoylethanolamide and arachidonoylethanolamine, reduced the plasma levels of glucose, triglycerides and cholesterol, and induced a transitory body weight decrease. The hippocampi of repeated URB597-treated rats showed a reduced number of phospho-H3+ and BrdU+ subgranular cells as well as GFAP+, Iba1+ and cleaved caspase-3+ cells, which was accompanied with decreased hippocampal expression of the cannabinoid CB1 receptor gene Cnr1 and Faah. In the hypothalami of these rats, the number of phospho-H3+, GFAP+ and 3-weeks-old BrdU+ cells was specifically decreased. The reduced striatal expression of CB1 receptor in repeated URB597-treated rats was only associated with a reduced apoptosis. In contrast, the striatum of acute URB597-treated rats showed an increased number of subventricular proliferative, astroglial and apoptotic cells, which was accompanied with increased Faah expression. Main results indicated that FAAH inhibitor URB597 decreased neural proliferation, glia and apoptosis in a brain region-dependent manner, which were coupled to local changes in Faah and/or Cnr1 expression and a negative energy context. PMID:25870539

  19. A multi-target approach for pain treatment: dual inhibition of fatty acid amide hydrolase and TRPV1 in a rat model of osteoarthritis.

    PubMed

    Malek, Natalia; Mrugala, Monika; Makuch, Wioletta; Kolosowska, Natalia; Przewlocka, Barbara; Binkowski, Marcin; Czaja, Martyna; Morera, Enrico; Di Marzo, Vincenzo; Starowicz, Katarzyna

    2015-05-01

    The pharmacological inhibition of anandamide (AEA) hydrolysis by fatty acid amide hydrolase (FAAH) attenuates pain in animal models of osteoarthritis (OA) but has failed in clinical trials. This may have occurred because AEA also activates transient receptor potential vanilloid type 1 (TRPV1), which contributes to pain development. Therefore, we investigated the effectiveness of the dual FAAH-TRPV1 blocker OMDM-198 in an MIA-model of osteoarthritic pain. We first investigated the MIA-induced model of OA by (1) characterizing the pain phenotype and degenerative changes within the joint using X-ray microtomography and (2) evaluating nerve injury and inflammation marker (ATF-3 and IL-6) expression in the lumbar dorsal root ganglia of osteoarthritic rats and differences in gene and protein expression of the cannabinoid CB1 receptors FAAH and TRPV1. Furthermore, we compared OMDM-198 with compounds acting exclusively on FAAH or TRPV1. Osteoarthritis was accompanied by the fragmentation of bone microstructure and destroyed cartilage. An increase of the mRNA levels of ATF3 and IL-6 and an upregulation of AEA receptors and FAAH in the dorsal root ganglia were observed. OMDM-198 showed antihyperalgesic effects in the OA model, which were comparable with those of a selective TRPV1 antagonist, SB-366,791, and a selective FAAH inhibitor, URB-597. The effect of OMDM-198 was attenuated by the CB1 receptor antagonist, AM-251, and by the nonpungent TRPV1 agonist, olvanil, suggesting its action as an "indirect" CB1 agonist and TRPV1 antagonist. These results suggest an innovative strategy for the treatment of OA, which may yield more satisfactory results than those obtained so far with selective FAAH inhibitors in human OA.

  20. Pharmacological blockade of the fatty acid amide hydrolase (FAAH) alters neural proliferation, apoptosis and gliosis in the rat hippocampus, hypothalamus and striatum in a negative energy context.

    PubMed

    Rivera, Patricia; Bindila, Laura; Pastor, Antoni; Pérez-Martín, Margarita; Pavón, Francisco J; Serrano, Antonia; de la Torre, Rafael; Lutz, Beat; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2015-01-01

    Endocannabinoids participate in the control of neurogenesis, neural cell death and gliosis. The pharmacological effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which limits the endocannabinoid degradation, was investigated in the present study. Cell proliferation (phospho-H3(+) or BrdU(+) cells) of the main adult neurogenic zones as well as apoptosis (cleaved caspase-3(+)), astroglia (GFAP(+)), and microglia (Iba1(+) cells) were analyzed in the hippocampus, hypothalamus and striatum of rats intraperitoneally treated with URB597 (0.3 mg/kg/day) at one dose/4-days resting or 5 doses (1 dose/day). Repeated URB597 treatment increased the plasma levels of the N-acylethanolamines oleoylethanolamide, palmitoylethanolamide and arachidonoylethanolamine, reduced the plasma levels of glucose, triglycerides and cholesterol, and induced a transitory body weight decrease. The hippocampi of repeated URB597-treated rats showed a reduced number of phospho-H3(+) and BrdU(+) subgranular cells as well as GFAP(+), Iba1(+) and cleaved caspase-3(+) cells, which was accompanied with decreased hippocampal expression of the cannabinoid CB1 receptor gene Cnr1 and Faah. In the hypothalami of these rats, the number of phospho-H3(+), GFAP(+) and 3-weeks-old BrdU(+) cells was specifically decreased. The reduced striatal expression of CB1 receptor in repeated URB597-treated rats was only associated with a reduced apoptosis. In contrast, the striatum of acute URB597-treated rats showed an increased number of subventricular proliferative, astroglial and apoptotic cells, which was accompanied with increased Faah expression. Main results indicated that FAAH inhibitor URB597 decreased neural proliferation, glia and apoptosis in a brain region-dependent manner, which were coupled to local changes in Faah and/or Cnr1 expression and a negative energy context.

  1. Ubiquitin hydrolase Dub3 promotes oncogenic transformation by stabilizing Cdc25A.

    PubMed

    Pereg, Yaron; Liu, Bob Y; O'Rourke, Karen M; Sagolla, Meredith; Dey, Anwesha; Komuves, Laszlo; French, Dorothy M; Dixit, Vishva M

    2010-04-01

    The dual specificity (Tyr/Thr) phosphatase Cdc25A activates cyclin-dependent kinases (Cdks) to promote cell-cycle progression and has significant oncogenic potential. Cdc25A protein levels are regulated tightly in normal tissues, but many human cancers overexpress Cdc25A. The underlying mechanism for overexpression has been enigmatic. Here we show that Cdc25A is stabilized by the ubiquitin hydrolase Dub3. Upon binding Cdc25A, Dub3 removes the polyubiquitin modifications that mark Cdc25A for proteasomal degradation. Dub3 knockdown in cells increased Cdc25A ubiquitylation and degradation, resulting in reduced Cdk/Cyclin activity and arrest at G1/S and G2/M phases of the cell cycle. In contrast, acute Dub3 overexpression produced a signature response to oncogene induction: cells accumulated in S and G2 because of replication stress, and activated a DNA damage response. Dub3 also transformed NIH-3T3 cells and cooperated with activated H-Ras to promote growth in soft agar. Importantly, we show that Dub3 overexpression is responsible for an abnormally high level of Cdc25A in a subset of human breast cancers. Moreover, Dub3 knockdown significantly retarded the growth of breast tumour xenografts in nude mice. As a major regulator of Cdc25A, Dub3 is an example of a transforming ubiquitin hydrolase that subverts a key component of the cell cycle machinery.

  2. Structural and kinetic insights into the mechanism of 5-hydroxyisourate hydrolase from Klebsiella pneumoniae

    SciTech Connect

    French, Jarrod B.; Ealick, Steven E.

    2011-08-01

    The crystal structure of 5-hydroxyisourate hydrolase from K. pneumoniae and the steady-state kinetic parameters of the native enzyme as well as several mutants provide insights into the catalytic mechanism of this enzyme and the possible roles of the active-site residues. The stereospecific oxidative degradation of uric acid to (S)-allantoin has recently been demonstrated to proceed via two unstable intermediates and requires three separate enzymatic reactions. The second step of this reaction, the conversion of 5-hydroxyisourate (HIU) to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, is catalyzed by HIU hydrolase (HIUH). The high-resolution crystal structure of HIUH from the opportunistic pathogen Klebsiella pneumoniae (KpHIUH) has been determined. KpHIUH is a homotetrameric protein that, based on sequence and structural similarity, belongs to the transthyretin-related protein family. In addition, the steady-state kinetic parameters for this enzyme and four active-site mutants have been measured. These data provide valuable insight into the functional roles of the active-site residues. Based upon the structural and kinetic data, a mechanism is proposed for the KpHIUH-catalyzed reaction.

  3. Molecular cloning, expression and characterization of acylpeptide hydrolase in the silkworm, Bombyx mori.

    PubMed

    Fu, Ping; Sun, Wei; Zhang, Ze

    2016-04-10

    Acylpeptide hydrolase (APH) can catalyze the release of the N-terminal amino acid from acetylated peptides. There were many documented examples of this enzyme in various prokaryotic and eukaryotic organisms. However, knowledge about APH in insects still remains unknown. In this study, we cloned and sequenced a putative silkworm Bombyx mori APH (BmAPH) gene. The BmAPH gene encodes a protein of 710 amino acids with a predicted molecular mass of 78.5kDa. The putative BmAPH and mammal APHs share about 36% amino acid sequence identity, yet key catalytic residues are conserved (Ser566, Asp654, and His686). Expression and purification of the recombinant BmAPH in Escherichia coli showed that it has acylpeptide hydrolase activity toward the traditional substrate, Ac-Ala-pNA. Furthermore, organophosphorus (OP) insecticides, chlorpyrifos, phoxim, and malathion, significantly inhibited the activity of the APH both in vitro and in vivo. In addition, BmAPH was expressed in all tested tissues and developmental stages of the silkworm. Finally, immunohistochemistry analysis showed that BmAPH protein was localized in the basement membranes. These results suggested that BmAPH may be involved in enhancing silkworm tolerance to the OP insecticides. In a word, our results provide evidence for understanding of the biological function of APH in insects.

  4. Purification and characterisation of a novel enantioselective epoxide hydrolase from Aspergillus niger M200.

    PubMed

    Kotik, Michael; Kyslík, Pavel

    2006-02-01

    Purification of a novel enantioselective epoxide hydrolase from Aspergillus niger M200 has been achieved using ammonium sulphate precipitation, ionic exchange, hydrophobic interaction, and size-exclusion chromatography, in conjunction with two additional chromatographic steps employing hydroxylapatite, and Mimetic Green. The enzyme was purified 186-fold with a yield of 15%. The apparent molecular mass of the enzyme was determined to be 77 kDa under native conditions and 40 kDa under denaturing conditions, implying a dimeric structure of the native enzyme. The isoelectric point of the enzyme was estimated to be 4.0 by isoelectric focusing electrophoresis. The enzyme has a broad substrate specificity with highest specificities towards tert-butyl glycidyl ether, para-nitrostyrene oxide, benzyl glycidyl ether, and styrene oxide. Enantiomeric ratios of 30 to more than 100 were determined for the hydrolysis reactions of 4 epoxidic substrates using the purified enzyme at a reaction temperature of 10 degrees C. Product inhibition studies suggest that the enzyme is able to differentiate to a high degree between the (R)-diol and (S)-diol product of the hydrolysis reaction with tert-butyl glycidyl ether as the substrate. The highest activity of the enzyme was at 42 degrees C and a pH of 6.8. Six peptide sequences, which were obtained by cleavage of the purified enzyme with trypsin and mass spectrometry analysis of the tryptic peptides, show high similarity with corresponding sequences originated from the epoxide hydrolase from Aspergillus niger LCP 521.

  5. Bacillus licheniformis trehalose-6-phosphate hydrolase structures suggest keys to substrate specificity.

    PubMed

    Lin, Min Guan; Chi, Meng Chun; Naveen, Vankadari; Li, Yi Ching; Lin, Long Liu; Hsiao, Chwan Deng

    2016-01-01

    Trehalose-6-phosphate hydrolase (TreA) belongs to glycoside hydrolase family 13 (GH13) and catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to yield glucose and glucose 6-phosphate. The products of this reaction can be further metabolized by the energy-generating glycolytic pathway. Here, crystal structures of Bacillus licheniformis TreA (BlTreA) and its R201Q mutant complexed with p-nitrophenyl-α-D-glucopyranoside (R201Q-pPNG) are presented at 2.0 and 2.05 Å resolution, respectively. The overall structure of BlTreA is similar to those of other GH13 family enzymes. However, detailed structural comparisons revealed that the catalytic site of BlTreA contains a long loop that adopts a different conformation from those of other GH13 family members. Unlike the homologous regions of Bacillus cereus oligo-1,6-glucosidase (BcOgl) and Erwinia rhapontici isomaltulose synthase (NX-5), the surface potential of the BlTreA active site exhibits a largely positive charge contributed by the four basic residues His281, His282, Lys284 and Lys292. Mutation of these residues resulted in significant decreases in the enzymatic activity of BlTreA. Strikingly, the (281)HHLK(284) motif and Lys292 play critical roles in substrate discrimination by BlTreA.

  6. Nostoc commune UTEX 584 gene expressing indole phosphate hydrolase activity in Escherichia coli.

    PubMed Central

    Xie, W Q; Whitton, B A; Simon, J W; Jäger, K; Reed, D; Potts, M

    1989-01-01

    A gene encoding an enzyme capable of hydrolyzing indole phosphate was isolated from a recombinant gene library of Nostoc commune UTEX 584 DNA in lambda gt10. The gene (designated iph) is located on a 2.9-kilobase EcoRI restriction fragment and is present in a single copy in the genome of N. commune UTEX 584. The iph gene was expressed when the purified 2.9-kilobase DNA fragment, free of any vector sequences, was added to a cell-free coupled transcription-translation system. A polypeptide with an Mr of 74,000 was synthesized when the iph gene or different iph-vector DNA templates were expressed in vitro. When carried by different multicopy plasmids and phagemids (pMP005, pBH6, pB8) the cyanobacterial iph gene conferred an Iph+ phenotype upon various strains of Escherichia coli, including a phoA mutant. Hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate was detected in recombinant E. coli strains grown in phosphate-rich medium, and the activity persisted in assay buffers that contained phosphate. In contrast, indole phosphate hydrolase activity only developed in cells of N. commune UTEX 584, when they were partially depleted of phosphorus, and the activity associated with these cells was suppressed partially by the addition of phosphate to assay buffers. Indole phosphate hydrolase activity was detected in periplasmic extracts from E. coli (Iph+) transformants. Images PMID:2536677

  7. Mycobacterium tuberculosis FtsX extracellular domain activates the peptidoglycan hydrolase, RipC

    PubMed Central

    Mavrici, Daniela; Marakalala, Mohlopheni J.; Holton, James M.; Prigozhin, Daniil M.; Gee, Christine L.; Zhang, Yanjia J.; Rubin, Eric J.; Alber, Tom

    2014-01-01

    Bacterial growth and cell division are coordinated with hydrolysis of the peptidoglycan (PG) layer of the cell wall, but the mechanisms of regulation of extracellular PG hydrolases are not well understood. Here we report the biochemical, structural, and genetic analysis of the Mycobacterium tuberculosis homolog of the transmembrane PG-hydrolase regulator, FtsX. The purified FtsX extracellular domain binds the PG peptidase Rv2190c/RipC N-terminal segment, causing a conformational change that activates the enzyme. Deletion of ftsEX and ripC caused similar phenotypes in Mycobacterium smegmatis, as expected for genes in a single pathway. The crystal structure of the FtsX extracellular domain reveals an unprecedented fold containing two lobes connected by a flexible hinge. Mutations in the hydrophobic cleft between the lobes reduce RipC binding in vitro and inhibit FtsX function in M. smegmatis. These studies suggest how FtsX recognizes RipC and support a model in which a conformational change in FtsX links the cell division apparatus with PG hydrolysis. PMID:24843173

  8. The role of human glutathione transferases and epoxide hydrolases in the metabolism of xenobiotics.

    PubMed Central

    Seidegård, J; Ekström, G

    1997-01-01

    Human glutathione transferases (GSTs) are a multigene family of enzymes that are involved in the metabolism of a wide range of electrophilic compounds of both exogenous and endogenous origin. GSTs are generally recognized as detoxifying enzymes by catalyzing the conjugation of these compounds with glutathione, but they may also be involved in activation of some carcinogens. The memmalian GSTs can be differentiated into four classes of cytosolic enzymes and two membrane bound enzymes. Human epoxide hydrolases (EHs) catalyze the addition of water to epoxides to form the corresponding dihydrodiol. The enzymatic hydration is essentially irreversible and produces mainly metabolites of lower reactivity that can be conjugated and excreted. The reaction of EHs is therefore generally regarded as detoxifying. The mammalian EHs can be distinguished by their physical and enzymatic properties. Microsomal EH (mEH) exhibits a broad substrate specificity, while the soluble EH (sEH) is an enzyme with a "complementary" substrate specificity to mEH. Cholesterol EH and leukotriene A4 hydrolase are two EHs with very limited substrate specificity. The activities of either GSTs or EHs expressed in vivo exhibit a relatively large interindividual variation, which might be explained by induction, inhibition, or genetic factors. These variations in levels or activities of individual isoenzymes are of importance with respect to an individual's susceptibility to genotoxic effects. This article gives a general overview of GSTs and EHs, discussing the modulation of activities, determination of these enzymes ex vivo, and the polymorphic expression of some isoenzymes. PMID:9255563

  9. Molecular and functional characterization of a unique sucrose hydrolase from Xanthomonas axonopodis pv. glycines.

    PubMed

    Kim, Hong-Suk; Park, Hyoung-Joon; Heu, Sunggi; Jung, Jin

    2004-01-01

    A novel sucrose hydrolase (SUH) from Xanthomonas axonopodis pv. glycines, a causative agent of bacterial pustule disease on soybeans, was studied at the functional and molecular levels. SUH was shown to act rather specifically on sucrose (K(m) = 2.5 mM) but not on sucrose-6-phosphate. Protein analysis of purified SUH revealed that, in this monomeric enzyme with an estimated molecular mass of 70,223 +/- 12 Da, amino acid sequences determined for several segments have corresponding nucleotide sequences in XAC3490, a protein-coding gene found in the genome of X. axonopodis pv. citri. Based on this information, the SUH gene, consisting of an open reading frame of 1,935 bp, was cloned by screening a genomic library of X. axonopodis pv. glycines 8ra. Database searches and sequence comparison revealed that SUH has significant homology to some family 13 enzymes, with all of the crucial invariant residues involved in the catalytic mechanism conserved, but it shows no similarity to known invertases belonging to family 32. suh expression in X. axonopodis pv. glycines requires sucrose induction, and insertional mutagenesis resulted in an absence of sucrose-inducible sucrose hydrolase activity in crude protein extracts and a sucrose-negative phenotype. Recombinant SUH, overproduced in Escherichia coli and purified, was shown to have the same enzymatic characteristics in terms of kinetic parameters.

  10. The role of a purine-specific nucleoside hydrolase in spore germination of Bacillus thuringiensis.

    PubMed

    Liang, Liang; He, Xihong; Liu, Gang; Tan, Huarong

    2008-05-01

    A homologous gene (iunH) of a putative nucleoside hydrolase (NH), which had been identified from the exosporia of Bacillus cereus and Bacillus anthracis spores, was cloned from Bacillus thuringiensis subsp. kurstaki. Disruption of iunH did not affect the vegetative growth and sporulation of Bacillus thuringiensis, but promoted both inosine- and adenosine-induced spore germination. The inosine- or adenosine-induced germination rate decreased when the wild-type iunH gene was overexpressed in Bacillus thuringiensis. The iunH gene product was characterized as a purine-specific NH. The kinetic parameters of IunH with inosine as substrate were K(m)=399+/-115 microM, k(cat)=48.9+/-8.5 s(-1) and k(cat)/K(m)=1.23 x 10(5) M(-1) s(-1). The optimal pH and temperature for IunH were found to be pH 6 and 80 degrees C. Meanwhile, the specific activity of inosine hydrolase in intact spores of the wild-type strain with inosine as substrate was 2.89+/-0.23x10(-2) micromol min(-1) (mg dry wt)(-1). These results indicate that IunH is important in moderating inosine- or adenosine-induced germination of Bacillus thuringiensis spores.

  11. Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

    SciTech Connect

    Ortega, Corrie; Anderson, Lindsey N.; Frando, Andrew; Sadler, Natalie C.; Brown, Robert W.; Smith, Richard D.; Wright, Aaron T.; Grundner, Christoph

    2016-02-01

    The transition between replication and non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenicity, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating, persistent populations is a priority for tuberculosis treatment, but only few drug targets in non-replicating Mtb are currently known. Here, we directly measure the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication by activity-based proteomics. We predict serine hydrolase activity for 78 proteins, including 27 proteins with previously unknown function, and identify 37 SHs that remain active even in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with large shifts in the activity of the majority of SHs. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.

  12. Targeted Discovery of Glycoside Hydrolases from a Switchgrass-Adapted Compost Community

    SciTech Connect

    Reddy, Amitha; Allgaier, Martin; Park, Joshua I.; Ivanoval, Natalia; Dhaeseleer, Patrik; Lowry, Steve; Sapra, Rajat; Hazen, Terry C.; Simmons, Blake A.; VanderGheynst, Jean S.; Hugenholtz, Philip

    2011-05-11

    Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Smallsubunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, ,10percent were putative cellulasesmostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50uC and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

  13. Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community

    SciTech Connect

    Allgaier, M.; Reddy, A.; Park, J. I.; Ivanova, N.; D'haeseleer, P.; Lowry, S.; Sapra, R.; Hazen, T.C.; Simmons, B.A.; VanderGheynst, J. S.; Hugenholtz, P.

    2009-11-15

    Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, {approx}10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50 C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

  14. Hepatic Retinyl Ester Hydrolases and the Mobilization of Retinyl Ester Stores

    PubMed Central

    Grumet, Lukas; Taschler, Ulrike; Lass, Achim

    2016-01-01

    For mammals, vitamin A (retinol and metabolites) is an essential micronutrient that is required for the maintenance of life. Mammals cannot synthesize vitamin A but have to obtain it from their diet. Resorbed dietary vitamin A is stored in large quantities in the form of retinyl esters (REs) in cytosolic lipid droplets of cells to ensure a constant supply of the body. The largest quantities of REs are stored in the liver, comprising around 80% of the body’s total vitamin A content. These hepatic vitamin A stores are known to be mobilized under times of insufficient dietary vitamin A intake but also under pathological conditions such as chronic alcohol consumption and different forms of liver diseases. The mobilization of REs requires the activity of RE hydrolases. It is astounding that despite their physiological significance little is known about their identities as well as about factors or stimuli which lead to their activation and consequently to the mobilization of hepatic RE stores. In this review, we focus on the recent advances for the understanding of hepatic RE hydrolases and discuss pathological conditions which lead to the mobilization of hepatic RE stores. PMID:28035980

  15. Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates

    PubMed Central

    Kong, Xu-Dong; Yuan, Shuguang; Li, Lin; Chen, She; Xu, Jian-He; Zhou, Jiahai

    2014-01-01

    Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S)-alprenolol, and other β-adrenergic receptor blocking drugs. Although the enzymatic production of these bulky epoxides has proven difficult, here we report a method to effectively improve the activity of BmEH, an epoxide hydrolase from Bacillus megaterium ECU1001 toward α-naphthyl glycidyl ether, the precursor of (S)-propranolol, by eliminating the steric hindrance near the potential product-release site. Using X-ray crystallography, mass spectrum, and molecular dynamics calculations, we have identified an active tunnel for substrate access and product release of this enzyme. The crystal structures revealed that there is an independent product-release site in BmEH that was not included in other reported epoxide hydrolase structures. By alanine scanning, two mutants, F128A and M145A, targeted to expand the potential product-release site displayed 42 and 25 times higher activities toward α-naphthyl glycidyl ether than the wild-type enzyme, respectively. These results show great promise for structure-based rational design in improving the catalytic efficiency of industrial enzymes for bulky substrates. PMID:25331869

  16. Identification of a novel peptidoglycan hydrolase CwlM in Mycobacterium tuberculosis.

    PubMed

    Deng, Lingyi Lynn; Humphries, Donald E; Arbeit, Robert D; Carlton, Laura E; Smole, Sandra C; Carroll, J David

    2005-02-14

    Mycobacterium tuberculosis is a major global pathogen whose threat has increased with the emergence of multidrug-resistant strains. The cell wall of M. tuberculosis is thick, rigid, and hydrophobic, which serves to protect the organism from the environment and makes it highly impermeable to conventional antimicrobial agents. There is little known about cell wall autolysins (also referred to as peptidoglycan hydrolases) of mycobacteria. We identified an open reading frame (Rv3915) in the M. tuberculosis genome designated cwlM that appeared consistent with a peptidoglycan hydrolase. The 1218-bp gene was amplified by PCR, cloned and expressed in E. coli strain HMS174(DE-3), and its gene product, a 47-kDa recombinant protein, was purified and partially characterized. Purified CwlM was able to lyse whole mycobacteria, release peptidoglycan from the cell wall of Micrococcus luteus and Mycobacterium smegmatis, and cleave N-acetylmuramoyl-L-alanyl-D-isoglutamine, releasing free N-acetylmuramic acid. These results indicate that CwlM is a novel autolysin and identify cwlM as the first, to our knowledge, autolysin gene identified and cloned from M. tuberculosis. CwlM offers a new target for a unique class of drugs that could alter the permeability of the mycobacterial cell wall and enhance the effectiveness of treatments for tuberculosis.

  17. A chemical proteomic atlas of brain serine hydrolases identifies cell type-specific pathways regulating neuroinflammation

    PubMed Central

    Viader, Andreu; Ogasawara, Daisuke; Joslyn, Christopher M; Sanchez-Alavez, Manuel; Mori, Simone; Nguyen, William; Conti, Bruno; Cravatt, Benjamin F

    2016-01-01

    Metabolic specialization among major brain cell types is central to nervous system function and determined in large part by the cellular distribution of enzymes. Serine hydrolases are a diverse enzyme class that plays fundamental roles in CNS metabolism and signaling. Here, we perform an activity-based proteomic analysis of primary mouse neurons, astrocytes, and microglia to furnish a global portrait of the cellular anatomy of serine hydrolases in the brain. We uncover compelling evidence for the cellular compartmentalization of key chemical transmission pathways, including the functional segregation of endocannabinoid (eCB) biosynthetic enzymes diacylglycerol lipase-alpha (DAGLα) and –beta (DAGLβ) to neurons and microglia, respectively. Disruption of DAGLβ perturbed eCB-eicosanoid crosstalk specifically in microglia and suppressed neuroinflammatory events in vivo independently of broader effects on eCB content. Mapping the cellular distribution of metabolic enzymes thus identifies pathways for regulating specialized inflammatory responses in the brain while avoiding global alterations in CNS function. DOI: http://dx.doi.org/10.7554/eLife.12345.001 PMID:26779719

  18. Signature motifs identify an Acinetobacter Cif virulence factor with epoxide hydrolase activity.

    PubMed

    Bahl, Christopher D; Hvorecny, Kelli L; Bridges, Andrew A; Ballok, Alicia E; Bomberger, Jennifer M; Cady, Kyle C; O'Toole, George A; Madden, Dean R

    2014-03-14

    Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii ("aCif"). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog ("aCifR") and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections.

  19. Organophosphate and Pyrethroid Hydrolase Activities of Mutant Esterases from the Cotton Bollworm Helicoverpa armigera

    PubMed Central

    Li, Yongqiang; Farnsworth, Claire A.; Coppin, Chris W.; Teese, Mark G.; Liu, Jian-Wei; Scott, Colin; Zhang, Xing; Russell, Robyn J.; Oakeshott, John G.

    2013-01-01

    Two mutations have been found in five closely related insect esterases (from four higher Diptera and a hymenopteran) which each confer organophosphate (OP) hydrolase activity on the enzyme and OP resistance on the insect. One mutation converts a Glycine to an Aspartate, and the other converts a Tryptophan to a Leucine in the enzymes’ active site. One of the dipteran enzymes with the Leucine mutation also shows enhanced activity against pyrethroids. Introduction of the two mutations in vitro into eight esterases from six other widely separated insect groups has also been reported to increase substantially the OP hydrolase activity of most of them. These data suggest that the two mutations could contribute to OP, and possibly pyrethroid, resistance in a variety of insects. We therefore introduced them in vitro into eight Helicoverpa armigera esterases from a clade that has already been implicated in OP and pyrethroid resistance. We found that they do not generally enhance either OP or pyrethroid hydrolysis in these esterases but the Aspartate mutation did increase OP hydrolysis in one enzyme by about 14 fold and the Leucine mutation caused a 4–6 fold increase in activity (more in one case) of another three against some of the most insecticidal isomers of fenvalerate and cypermethrin. The Aspartate enzyme and one of the Leucine enzymes occur in regions of the H. armigera esterase isozyme profile that have been previously implicated in OP and pyrethroid resistance, respectively. PMID:24204917

  20. Novel Strategies for Upstream and Downstream Processing of Tannin Acyl Hydrolase

    PubMed Central

    Rodríguez-Durán, Luis V.; Valdivia-Urdiales, Blanca; Contreras-Esquivel, Juan C.; Rodríguez-Herrera, Raúl; Aguilar, Cristóbal N.

    2011-01-01

    Tannin acyl hydrolase also referred as tannase is an enzyme with important applications in several science and technology fields. Due to its hydrolytic and synthetic properties, tannase could be used to reduce the negative effects of tannins in beverages, food, feed, and tannery effluents, for the production of gallic acid from tannin-rich materials, the elucidation of tannin structure, and the synthesis of gallic acid esters in nonaqueous media. However, industrial applications of tannase are still very limited due to its high production cost. Thus, there is a growing interest in the production, recovery, and purification of this enzyme. Recently, there have been published a number of papers on the improvement of upstream and downstream processing of the enzyme. These papers dealt with the search for new tannase producing microorganisms, the application of novel fermentation systems, optimization of culture conditions, the production of the enzyme by recombinant microorganism, and the design of efficient protocols for tannase recovery and purification. The present work reviews the state of the art of basic and biotechnological aspects of tannin acyl hydrolase, focusing on the recent advances in the upstream and downstream processing of the enzyme. PMID:21941633

  1. Mutational and nucleotide sequence analysis of S-adenosyl-L-homocysteine hydrolase from Rhodobacter capsulatus.

    PubMed Central

    Sganga, M W; Aksamit, R R; Cantoni, G L; Bauer, C E

    1992-01-01

    The genetic locus ahcY, encoding the enzyme S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) from the bacterium Rhodobacter capsulatus, has been mapped by mutational analysis to within a cluster of genes involved in regulating the induction and maintenance of the bacterial photosynthetic apparatus. Sequence analysis demonstrates that ahcY encodes a 51-kDa polypeptide that displays 64% sequence identity to its human homolog. Insertion mutants in ahcY lack detectable S-adenosyl-L-homocysteine hydrolase activity and, as a consequence, S-adenosyl-L-homocysteine accumulates in the cells, resulting in a 16-fold decrease in the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine as compared to wild-type cells. The ahcY disrupted strain fails to grow in minimal medium; however, growth is restored in minimal medium supplemented with methionine or homocysteine or in a complex medium, thereby indicating that the hydrolysis of S-adenosyl-L-homocysteine plays a key role in the metabolism of sulfur-containing amino acids. The ahcY mutant, when grown in supplemented medium, synthesizes significantly reduced levels of bacteriochlorophyll, indicating that modulation of the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine may be an important factor in regulating bacteriochlorophyll biosynthesis. PMID:1631127

  2. Probing the mechanisms for the selectivity and promiscuity of methyl parathion hydrolase

    PubMed Central

    Purg, Miha; Pabis, Anna; Baier, Florian; Tokuriki, Nobuhiko; Jackson, Colin

    2016-01-01

    Diverse organophosphate hydrolases have convergently evolved the ability to hydrolyse man-made organophosphates. Thus, these enzymes are attractive model systems for studying the factors shaping enzyme functional evolution. Methyl parathion hydrolase (MPH) is an enzyme from the metallo-β-lactamase superfamily, which hydrolyses a wide range of organophosphate, aryl ester and lactone substrates. In addition, MPH demonstrates metal-ion-dependent selectivity patterns. The origins of this remain unclear, but are linked to open questions about the more general role of metal ions in functional evolution and divergence within enzyme superfamilies. Here, we present detailed mechanistic studies of the paraoxonase and arylesterase activities of MPH complexed with five different transition metal ions, and demonstrate that the hydrolysis reactions proceed via similar pathways and transition states. However, while it is possible to discern a clear structural origin for the selectivity between different substrates, the selectivity between different metal ions appears to lie instead in the distinct electrostatic properties of the metal ions themselves, which causes subtle changes in transition state geometries and metal–metal distances at the transition state rather than significant structural changes in the active site. While subtle, these differences can be significant for shaping the metal-ion-dependent activity patterns observed for this enzyme. This article is part of the themed issue ‘Multiscale modelling at the physics–chemistry–biology interface’. PMID:27698033

  3. Cold-active hydrolases producing bacteria from two different sub-glacial Himalayan lakes.

    PubMed

    Sahay, Harmesh; Babu, Bandamaravuri Kishore; Singh, Surendra; Kaushik, Rajeev; Saxena, Anil K; Arora, Dilip K

    2013-08-01

    Microorganisms, native to the cold environments have successfully acclimatized their physiological, metabolic, and biological features, exhibiting uniqueness in their enzymes, proteins, and membrane structures. These cold-active enzymes have immense biotechnological potential. The diversity of culturable bacteria in two different water lakes (the sub-glacial freshwater and the brackish) of Himalayas was analyzed using SYBR green staining and cultural methods. A total of 140 bacteria were isolated and were grouped as psychrophiles, psychrotrophs, and psychrotolerant organisms, based on their optimal temperature for growth. The amplified ribosomal DNA restriction analysis using three restriction enzymes facilitated the grouping of these isolates into 96 genotypes at ≥85% polymorphism. Phylogenetic analysis using 16S rRNA gene sequences revealed that the bacterial strains from both lakes belonged to Firmicutes, Proteobacteria (α, β, and γ) or Actinobacteria. Screening of the germplasm for the activity of different cold-active hydrolases such as protease, amylase, xylanase, and cellulase, revealed that about 16 isolates were positive, and exhibiting a wide range of stability at various temperature and pH. Our results suggest that the distinctly different ecosystems of sub-glacial freshwater and brackish water lakes have diverse groups of bacteria, which can be an excellent source of extracellular hydrolases with a wide range of thermal stability.

  4. Heavy Chain Single Domain Antibodies to Detect Native Human Soluble Epoxide Hydrolase

    PubMed Central

    Cui, Yongliang; Li, Dongyang; Morisseau, Christophe; Yang, Jun; Wan, Debin; Rossotti, Martín A.; Gee, Shirley J.; González-Sapienza, Gualberto G.; Hammock, Bruce D.

    2015-01-01

    The soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, pain, cancer and other diseases. However, there is not a simple, inexpensive and reliable method to estimate levels of active sEH in tissues. Toward developing such an assay, a polyclonal-variable domain of heavy chain antibody (VHH) sandwich immunoassay was developed. Ten VHHs, which are highly selective for native human sEH, were isolated from a phage displayed library. The ten VHHs have no significant cross-reactivity with human microsomal epoxide hydrolase, rat and mouse sEH, and denatured human sEH. There is a high correlation between protein levels of the sEH determined by the ELISA and the catalytic activity of the enzyme in S9 fractions of human tissues (liver, kidney and lung). The VHH based ELISA appears to be a new reliable method for monitoring the sEH, and may be useful as a diagnostic tool for diseases influenced by sEH. This study also demonstrates the broad utility of VHH in biochemical and pharmacological research. PMID:26229025

  5. Improving the thermostability of a methyl parathion hydrolase by adding the ionic bond on protein surface.

    PubMed

    Su, Yidan; Tian, Jian; Wang, Ping; Chu, Xiaoyu; Liu, Guoan; Wu, Ningfeng; Fan, Yunliu

    2011-10-01

    The thermostability of the methyl parathion hydrolase (MPH_OCH) from Ochrobactrum sp. M231 was improved using site-directed mutagenesis. Two prolines (Pro76 and Pro78) located on the protein surface were selected for mutations after inspection of the sequence alignment of MPH_OCH and OPHC2, a thermostable organophosphorus hydrolase from Pseudomonas pseudoalcaligenes C2-1. The temperature of the double-point mutant (P76D/P78K) at which the mutant lost 50% of its activity (T50) was approximately 68 °C, which is higher than that of WT enzyme (64 °C), P76D (67 °C), and P78K (59 °C). Structural analysis of P76D/P78K indicated that the substituted residues (Asp76 and Lys78) could generate an ionic bond and increase the structural electrostatic energy, which could then increase the stability of the protein. These results also suggest that the thermal stability of proteins could be improved by adding the ionic bond on protein surface.

  6. Hepatic Retinyl Ester Hydrolases and the Mobilization of Retinyl Ester Stores.

    PubMed

    Grumet, Lukas; Taschler, Ulrike; Lass, Achim

    2016-12-27

    For mammals, vitamin A (retinol and metabolites) is an essential micronutrient that is required for the maintenance of life. Mammals cannot synthesize vitamin A but have to obtain it from their diet. Resorbed dietary vitamin A is stored in large quantities in the form of retinyl esters (REs) in cytosolic lipid droplets of cells to ensure a constant supply of the body. The largest quantities of REs are stored in the liver, comprising around 80% of the body's total vitamin A content. These hepatic vitamin A stores are known to be mobilized under times of insufficient dietary vitamin A intake but also under pathological conditions such as chronic alcohol consumption and different forms of liver diseases. The mobilization of REs requires the activity of RE hydrolases. It is astounding that despite their physiological significance little is known about their identities as well as about factors or stimuli which lead to their activation and consequently to the mobilization of hepatic RE stores. In this review, we focus on the recent advances for the understanding of hepatic RE hydrolases and discuss pathological conditions which lead to the mobilization of hepatic RE stores.

  7. Substrate recognition and catalysis by LytB, a pneumococcal peptidoglycan hydrolase involved in virulence

    PubMed Central

    Rico-Lastres, Palma; Díez-Martínez, Roberto; Iglesias-Bexiga, Manuel; Bustamante, Noemí; Aldridge, Christine; Hesek, Dusan; Lee, Mijoon; Mobashery, Shahriar; Gray, Joe; Vollmer, Waldemar; García, Pedro; Menéndez, Margarita

    2015-01-01

    Streptococcus pneumoniae is a major cause of life-threatening diseases worldwide. Here we provide an in-depth functional characterization of LytB, the peptidoglycan hydrolase responsible for physical separation of daughter cells. Identified herein as an N-acetylglucosaminidase, LytB is involved also in colonization and invasion of the nasopharynx, biofilm formation and evasion of host immunity as previously demonstrated. We have shown that LytB cleaves the GlcNAc-β-(1,4)-MurNAc glycosidic bond of peptidoglycan building units. The hydrolysis occurs at sites with fully acetylated GlcNAc moieties, with preference for uncross-linked muropeptides. The necessity of GlcN acetylation and the presence of a single acidic moiety (Glu585) essential for catalysis strongly suggest a substrate-assisted mechanism with anchimeric assistance of the acetamido group of GlcNAc moieties. Additionally, modelling of the catalytic region bound to a hexasaccharide tripentapeptide provided insights into substrate-binding subsites and peptidoglycan recognition. Besides, cell-wall digestion products and solubilisation rates might indicate a tight control of LytB activity to prevent unrestrained breakdown of the cell wall. Choline-independent localization at the poles of the cell, mediated by the choline-binding domain, peptidoglycan modification, and choline-mediated (lipo)teichoic-acid attachment contribute to the high selectivity of LytB. Moreover, so far unknown chitin hydrolase and glycosyltransferase activities were detected using GlcNAc oligomers as substrate. PMID:26537571

  8. Differential Recognition and Hydrolysis of Host Carbohydrate Antigens by Streptococcus pneumoniae Family 98 Glycoside Hydrolases

    SciTech Connect

    Higgins, M.; Whitworth, G; El Warry, N; Randriantsoa, M; Samain, E; Burke, R; Vocadlo, D; Boraston, A

    2009-01-01

    The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-{beta}-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-{beta}-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

  9. Beta-glucuronidase of family-2 glycosyl hydrolase: a missing member in plants.

    PubMed

    Arul, Loganathan; Benita, George; Sudhakar, Duraialagaraja; Thayumanavan, Balsamy; Balasubramanian, Ponnusamy

    2008-01-01

    Glycosyl hydrolases hydrolyze the glycosidic bond in carbohydrates or between a carbohydrate and a non-carbohydrate moiety. beta-glucuronidase (GUS) is classified under two glycosyl hydrolase families (2 and 79) and the family-2 beta-glucuronidase is reported in a wide range of organisms, but not in plants. The family-79 endo-beta-glucuronidase (heparanase) is reported in microorganisms, vertebrates and plants. The E. coli family-2 beta-glucuronidase (uidA) had been successfully devised as a reporter gene in plant transformation on the basis that plants do not have homologous GUS activity. On the contrary, histochemical staining with X-Gluc was reported in wild type (non-transgenic) plants. Data shows that, family-2 beta-glucuronidase homologous sequence is not found in plants. Further, beta-glucuronidases of family-2 and 79 lack appreciable sequence similarity. However, the catalytic site residues, glutamic acid and tyrosine of the family-2 beta-glucuronidase are found to be conserved in family-79 beta-glucuronidase of plants. This led to propose that the GUS staining reported in wild type plants is largely because of the broad substrate specificity of family-79 beta-glucuronidase on X-Gluc and not due to the family-2 beta-glucuronidase, as the latter has been found to be missing in plants.

  10. Structural insights into the reaction mechanism of S-adenosyl-L-homocysteine hydrolase

    PubMed Central

    Kusakabe, Yoshio; Ishihara, Masaaki; Umeda, Tomonobu; Kuroda, Daisuke; Nakanishi, Masayuki; Kitade, Yukio; Gouda, Hiroaki; Nakamura, Kazuo T.; Tanaka, Nobutada

    2015-01-01

    S-adenosyl-L-homocysteine hydrolase (SAH hydrolase or SAHH) is a highly conserved enzyme that catalyses the reversible hydrolysis of SAH to L-homocysteine (HCY) and adenosine (ADO). High-resolution crystal structures have been reported for bacterial and plant SAHHs, but not mammalian SAHHs. Here, we report the first high-resolution crystal structure of mammalian SAHH (mouse SAHH) in complex with a reaction product (ADO) and with two reaction intermediate analogues—3’-keto-aristeromycin (3KA) and noraristeromycin (NRN)—at resolutions of 1.55, 1.55, and 1.65 Å. Each of the three structures constitutes a structural snapshot of one of the last three steps of the five-step process of SAH hydrolysis by SAHH. In the NRN complex, a water molecule, which is an essential substrate for ADO formation, is structurally identified for the first time as the candidate donor in a Michael addition by SAHH to the 3’-keto-4’,5’-didehydroadenosine reaction intermediate. The presence of the water molecule is consistent with the reaction mechanism proposed by Palmer & Abeles in 1979. These results provide insights into the reaction mechanism of the SAHH enzyme. PMID:26573329

  11. Probing the mechanisms for the selectivity and promiscuity of methyl parathion hydrolase.

    PubMed

    Purg, Miha; Pabis, Anna; Baier, Florian; Tokuriki, Nobuhiko; Jackson, Colin; Kamerlin, Shina Caroline Lynn

    2016-11-13

    Diverse organophosphate hydrolases have convergently evolved the ability to hydrolyse man-made organophosphates. Thus, these enzymes are attractive model systems for studying the factors shaping enzyme functional evolution. Methyl parathion hydrolase (MPH) is an enzyme from the metallo-β-lactamase superfamily, which hydrolyses a wide range of organophosphate, aryl ester and lactone substrates. In addition, MPH demonstrates metal-ion-dependent selectivity patterns. The origins of this remain unclear, but are linked to open questions about the more general role of metal ions in functional evolution and divergence within enzyme superfamilies. Here, we present detailed mechanistic studies of the paraoxonase and arylesterase activities of MPH complexed with five different transition metal ions, and demonstrate that the hydrolysis reactions proceed via similar pathways and transition states. However, while it is possible to discern a clear structural origin for the selectivity between different substrates, the selectivity between different metal ions appears to lie instead in the distinct electrostatic properties of the metal ions themselves, which causes subtle changes in transition state geometries and metal-metal distances at the transition state rather than significant structural changes in the active site. While subtle, these differences can be significant for shaping the metal-ion-dependent activity patterns observed for this enzyme.This article is part of the themed issue 'Multiscale modelling at the physics-chemistry-biology interface'.

  12. Mfge8 regulates enterocyte lipid storage by promoting enterocyte triglyceride hydrolase activity

    PubMed Central

    Khalifeh-Soltani, Amin; Gupta, Deepti; Ha, Arnold; Iqbal, Jahangir; Hussain, Mahmood; Podolsky, Michael J.

    2016-01-01

    The small intestine has an underappreciated role as a lipid storage organ. Under conditions of high dietary fat intake, enterocytes can minimize the extent of postprandial lipemia by storing newly absorbed dietary fat in cytoplasmic lipid droplets. Lipid droplets can be subsequently mobilized for the production of chylomicrons. The mechanisms that regulate this process are poorly understood. We report here that the milk protein Mfge8 regulates hydrolysis of cytoplasmic lipid droplets in enterocytes after interacting with the αvβ3 and αvβ5 integrins. Mice deficient in Mfge8 or the αvβ3 and αvβ5 integrins accumulate excess cytoplasmic lipid droplets after a fat challenge. Mechanistically, interruption of the Mfge8-integrin axis leads to impaired enterocyte intracellular triglyceride hydrolase activity in vitro and in vivo. Furthermore, Mfge8 increases triglyceride hydrolase activity through a PI3 kinase/mTORC2–dependent signaling pathway. These data identify a key role for Mfge8 and the αvβ3 and αvβ5 integrins in regulating enterocyte lipid processing. PMID:27812539

  13. Cotranslocation of Methyl Parathion Hydrolase to the Periplasm and of Organophosphorus Hydrolase to the Cell Surface of Escherichia coli by the Tat Pathway and Ice Nucleation Protein Display System ▿

    PubMed Central

    Yang, Chao; Freudl, Roland; Qiao, Chuanling; Mulchandani, Ashok

    2010-01-01

    A genetically engineered Escherichia coli strain coexpressing organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH) was constructed for the first time by cotransforming two compatible plasmids. Since these two enzymes have different substrate specificities, the coexpression strain showed a broader substrate range than strains expressing either one of the hydrolases. To reduce the mass transport limitation of organophosphates (OPs) across the cell membrane, MPH and OPH were simultaneously translocated to the periplasm and cell surface of E. coli, respectively, by employing the twin-arginine translocation (Tat) pathway and ice nucleation protein (INP) display system. The resulting recombinant strain showed sixfold-higher whole-cell activity than the control strain expressing cytosolic OP hydrolases. The correct localization of MPH and OPH was demonstrated by cell fractionation, immunoblotting, and enzyme activity assays. No growth inhibition was observed for the recombinant E. coli strain, and suspended cultures retained almost 100% of the activity over a period of 2 weeks. Owing to its high level of activity and superior stability, the recombinant E. coli strain could be employed as a whole-cell biocatalyst for detoxification of OPs. This strategy of utilizing dual translocation pathways should open up new avenues for cotranslocating multiple functional moieties to different extracytosolic compartments of a bacterial cell. PMID:19933341

  14. Inhibitors of Pyruvate Carboxylase

    PubMed Central

    Zeczycki, Tonya N.; Maurice, Martin St.; Attwood, Paul V.

    2010-01-01

    This review aims to discuss the varied types of inhibitors of biotin-dependent carboxylases, with an emphasis on the inhibitors of pyruvate carboxylase. Some of these inhibitors are physiologically relevant, in that they provide ways of regulating the cellular activities of the enzymes e.g. aspartate and prohibitin inhibition of pyruvate carboxylase. Most of the inhibitors that will be discussed have been used to probe various aspects of the structure and function of these enzymes. They target particular parts of the structure e.g. avidin – biotin, FTP – ATP binding site, oxamate – pyruvate binding site, phosphonoacetate – binding site of the putative carboxyphosphate intermediate. PMID:22180764

  15. Acquired Factor V Inhibitor

    PubMed Central

    Hirai, Daisuke; Yamashita, Yugo; Masunaga, Nobutoyo; Katsura, Toshiaki; Akao, Masaharu; Okuno, Yoshiaki; Koyama, Hiroshi

    2016-01-01

    Inhibitors directed against factor V rarely occur, and the clinical symptoms vary. We herein report the case of a patient who presented with a decreased factor V activity that had decreased to <3 %. We administered vitamin K and 6 units of fresh frozen plasma, but she thereafter developed an intracerebral hemorrhage. It is unclear whether surgery >10 years earlier might have caused the development of a factor V inhibitor. The treatment of acquired factor V inhibitors is mainly the transfusion of platelet concentrates and corticosteroids. Both early detection and the early initiation of the treatment of factor V inhibitor are thus considered to be important. PMID:27746446

  16. Algerian pearl millet ( Pennisetum glaucum L.) contains XIP but not TAXI and TLXI type xylanase inhibitors.

    PubMed

    Mokrane, Hind; Gebruers, Kurt; Beaugrand, Johnny; Proost, Paul; Nadjemi, Boubekeur; Belhanèche-Bensemra, Naima; Courtin, Christophe M; Delcour, Jan A

    2009-06-24

    An XIP (xylanase inhibiting protein) type xylanase inhibitor was purified from Algerian pearl millet ( Pennisetum glaucum L.) grains and characterized for the first time. Cation exchange and affinity chromatography with immobilized Trichoderma longibrachiatum glycoside hydrolase (GH) family 11 xylanase resulted in electrophoretically pure protein with a molecular mass of 27-29 kDa and a pI value of 6.7. The experimentally determined N-terminal amino acid sequence of the purified XIP protein is 87.5%, identical to that of sorghum ( Sorghum bicolor L.) XIP and 79.2% identical to that of wheat ( Triticum aestivum L.) XIP-I. The biochemical properties of pearl millet XIP are comparable to those described earlier for sorghum XIP, except for the higher specific activity toward a T. longibrachiatum GH family 11 xylanase. On the basis of immunoblot neither TAXI nor TLXI type xylanase inhibitors were detected in pearl millet grains.

  17. [Controlling arachidonic acid metabolic network: from single- to multi-target inhibitors of key enzymes].

    PubMed

    Liu, Ying; Chen, Zheng; Shang, Er-chang; Yang, Kun; Wei, Deng-guo; Zhou, Lu; Jiang, Xiao-lu; He, Chong; Lai, Lu-hua

    2009-03-01

    Inflammatory diseases are common medical conditions seen in disorders of human immune system. There is a great demand for anti-inflammatory drugs. There are major inflammatory mediators in arachidonic acid metabolic network. Several enzymes in this network have been used as key targets for the development of anti-inflammatory drugs. However, specific single-target inhibitors can not sufficiently control the network balance and may cause side effects at the same time. Most inflammation induced diseases come from the complicated coupling of inflammatory cascades involving multiple targets. In order to treat these complicated diseases, drugs that can intervene multi-targets at the same time attracted much attention. The goal of this review is mainly focused on the key enzymes in arachidonic acid metabolic network, such as phospholipase A2, cyclooxygenase, 5-lipoxygenase and eukotriene A4 hydrolase. Advance in single target and multi-targe inhibitors is summarized.

  18. Structural insights into chitinolytic enzymes and inhibition mechanisms of selective inhibitors.

    PubMed

    Liu, Tian; Chen, Lei; Ma, Qiang; Shen, Xu; Yang, Qing

    2014-01-01

    Chitin biodegradation is linked to fungi cell differentiation, nematode egg hatching, arthropods morphogenesis and human defense against malaria and other pathogens infection as well. Two classes of enzymes for chitin degradation include glycosyl hydrolase (GH) family 18 chitinases and family 20 β-N-acetyl-D-hexosaminidases. However, more and more research papers have revealed that either GH 18 family chitinases or GH 20 family β-N-acetyl-D-hexosaminidases are a family composed of a number of isoforms, each of which plays an exclusive role in different life processes. The development of novel and specific inhibitors towards chitinolytic enzymes is of great importance in the investigation of or interference with chitin biodegradation. This review focuses on identified enzymes that are specifically involved in chitin degradation. And the latest progresses on crystal structures and specific inhibitors are summarized within the realm of this field.

  19. Development of inhibitors as research tools for carbohydrate-processing enzymes.

    PubMed

    Gloster, Tracey M

    2012-10-01

    Carbohydrates, which are present in all domains of life, play important roles in a host of cellular processes. These ubiquitous biomolecules form highly diverse and often complex glycan structures without the aid of a template. The carbohydrate structures are regulated solely by the location and specificity of the enzymes responsible for their synthesis and degradation. These enzymes, glycosyltransferases and glycoside hydrolases, need to be functionally well characterized in order to investigate the structure and function of glycans. The use of enzyme inhibitors, which target a particular enzyme, can significantly aid this understanding, and may also provide insights into therapeutic applications. The present article describes some of the approaches used to design and develop enzyme inhibitors as tools for investigating carbohydrate-processing enzymes.

  20. AMPEROMETRIC THICK-FILM STRIP ELECTRODES FOR MONITORING ORGANOPHOSPHATE NERVE AGENTS BASED ON IMMOBILIZED ORGANOPHOSPHORUS HYDROLASE. (R823663)

    EPA Science Inventory

    An amperometric biosensor based on the immobilization of organophosphorus hydrolase
    (OPH) onto screen-printed carbon electrodes is shown useful for the rapid, sensitive, and low-cost
    detection of organophosphate (OP) nerve agents. The sensor relies upon the sensitive and ra...

  1. Structural analysis of a glycosides hydrolase family 42 cold-adapted ß-galactosidase from Rahnella sp. R3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ß-galactosidase isolated from a psychrotrophic bacterium, Rahnella sp. R3 (R-ß-Gal), exhibits high activity at low temperature. R-ß-Gal is a member of the glycoside hydrolases family 42 (GH42), and forms a 225 kDa trimeric structure in solution. The X-ray crystal structure of R-ß-Gal was determi...

  2. Hydrolysis of filter-paper cellulose to glucose by two recombinant endogenous glycosyl hydrolases of Coptotermes formosanus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genes encoding for glycosyl hydrolases in multiple families were recovered from an EST library of Coptotermes formosanus, a wood-eating insect. Functional analyses of these genes not only shed light on the mechanisms the insect employs to successfully use cellulosic materials as energy sources, whic...

  3. 3-D QSAR ANALYSIS OF INHIBITION OF MURINE SOLUBLE EPOXIDE HYDROLASE (MSEH) BY BENZOYLUREAS, ARYLUREAS, AND THEIR ANALOGUES. (R825433)

    EPA Science Inventory

    Two hundred and seventy-one compounds including benzoylureas, arylureas and related compounds were assayed using recombinant murine soluble epoxide hydrolase (MsEH) produced from a baculovirus expression system. Among all the insect growth regulators assayed, 18 benzoylphenylu...

  4. Targeted gene inactivation in Clostridium phytofermentans shows that cellulose degradation requires the family 9 hydrolase Cphy3367.

    PubMed

    Tolonen, Andrew C; Chilaka, Amanda C; Church, George M

    2009-12-01

    Summary Microbial cellulose degradation is a central part of the global carbon cycle and has great potential for the development of inexpensive, carbon-neutral biofuels from non-food crops. Clostridium phytofermentans has a repertoire of 108 putative glycoside hydrolases to break down cellulose and hemicellulose into sugars, which this organism then ferments primarily to ethanol. An understanding of cellulose degradation at the molecular level requires learning the different roles of these hydrolases. In this study, we show that interspecific conjugation with Escherichia coli can be used to transfer a plasmid into C. phytofermentans that has a resistance marker, an origin of replication that can be selectively lost, and a designed group II intron for efficient, targeted chromosomal insertions without selection. We applied these methods to disrupt the cphy3367 gene, which encodes the sole family 9 glycoside hydrolase (GH9) in the C. phytofermentans genome. The GH9-deficient strain grew normally on some carbon sources such as glucose, but had lost the ability to degrade cellulose. Although C. phytofermentans upregulates the expression of numerous enzymes to break down cellulose, this process thus relies upon a single, key hydrolase, Cphy3367.

  5. A modified expression of the major hydrolase activator in Hypocrea jecorina (Trichoderma reesei) changes enzymatic catalysis of biopolymer degradation.

    PubMed

    Pucher, Marion E; Steiger, Matthias G; Mach, Robert L; Mach-Aigner, Astrid R

    2011-06-10

    Hypocrea jecorina (anamorph Trichoderma reesei) is a saprophytic fungus that produces hydrolases, which are applied in different types of industries and used for the production of biofuel. A recombinant Hypocrea strain, which constantly expresses the main transcription activator of hydrolases (Xylanase regulator 1), was found to grow faster on xylan and its monomeric backbone molecule d-xylose. This strain also showed improved ability of clearing xylan medium on plates. Furthermore, this strain has a changed transcription profile concerning genes encoding for hydrolases and enzymes associated with degradation of (hemi)celluloses. We demonstrated that enzymes of this strain from a xylan cultivation favoured break down of hemicelluloses to the monomer d-xylose compared to the parental strain, while the enzymes of the latter one formed more xylobiose. Applying supernatants from cultivation on carboxymethylcellulose in enzymatic conversion of hemicelluloses, the enzymes of the recombinant strain were clearly producing more of both, d-xylose and xylobiose, compared to the parental strain. Altogether, these results point to a changed hydrolase expression profile, an enhanced capability to form the xylan-monomer d-xylose and the assumption that there is a disordered induction pattern if the Xylanase regulator 1 is de-regulated in Hypocrea.

  6. Potential of the virion-associated peptidoglycan hydrolase HydH5 and its derivative fusion proteins in milk biopreservation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [(a virion-associated peptidoglycan hydrolase (VAPGH) encoded by the Staphylococcus aureus bacteriophage vB_SauS-ph...

  7. Isolation of Methyl Parathion-Degrading Strain M6 and Cloning of the Methyl Parathion Hydrolase Gene

    PubMed Central

    Zhongli, Cui; Shunpeng, Li; Guoping, Fu

    2001-01-01

    A degradative bacterium, M6, was isolated and presumptively identified as Plesiomonas sp. strain M6 was able to hydrolyze methyl parathion to p-nitrophenol. A novel organophosphate hydrolase gene designated mpd was selected from its genomic library prepared by shotgun cloning. The nucleotide sequence of the mpd gene was determined. The gene could be effectively expressed in Esherichia coli. PMID:11571204

  8. A dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate films.

    PubMed

    Barth, Markus; Honak, Annett; Oeser, Thorsten; Wei, Ren; Belisário-Ferrari, Matheus R; Then, Johannes; Schmidt, Juliane; Zimmermann, Wolfgang

    2016-08-01

    TfCut2 from Thermobifida fusca KW3 and the metagenome-derived LC-cutinase are bacterial polyester hydrolases capable of efficiently degrading polyethylene terephthalate (PET) films. Since the enzymatic PET hydrolysis is inhibited by the degradation intermediate mono-(2-hydroxyethyl) terephthalate (MHET), a dual enzyme system consisting of a polyester hydrolase and the immobilized carboxylesterase TfCa from Thermobifida fusca KW3 was employed for the hydrolysis of PET films at 60°C. HPLC analysis of the reaction products obtained after 24 h of hydrolysis showed an increased amount of soluble products with a lower proportion of MHET in the presence of the immobilized TfCa. The results indicated a continuous hydrolysis of the inhibitory MHET by the immobilized TfCa and demonstrated its advantage as a second biocatalyst in combination with a polyester hydrolase for an efficient degradation oft PET films. The dual enzyme system with LC-cutinase produced a 2.4-fold higher amount of degradation products compared to TfCut2 after a reaction time of 24 h confirming the superior activity of his polyester hydrolase against PET films.

  9. Targeted gene inactivation in Clostridium phytofermentans shows that cellulose degradation requires the family 9 hydrolase Cphy3367

    PubMed Central

    Tolonen, Andrew C; Chilaka, Amanda C; Church, George M

    2009-01-01

    Microbial cellulose degradation is a central part of the global carbon cycle and has great potential for the development of inexpensive, carbon-neutral biofuels from non-food crops. Clostridium phytofermentans has a repertoire of 108 putative glycoside hydrolases to break down cellulose and hemicellulose into sugars, which this organism then ferments primarily to ethanol. An understanding of cellulose degradation at the molecular level requires learning the different roles of these hydrolases. In this study, we show that interspecific conjugation with Escherichia coli can be used to transfer a plasmid into C. phytofermentans that has a resistance marker, an origin of replication that can be selectively lost, and a designed group II intron for efficient, targeted chromosomal insertions without selection. We applied these methods to disrupt the cphy3367 gene, which encodes the sole family 9 glycoside hydrolase (GH9) in the C. phytofermentans genome. The GH9-deficient strain grew normally on some carbon sources such as glucose, but had lost the ability to degrade cellulose. Although C. phytofermentans upregulates the expression of numerous enzymes to break down cellulose, this process thus relies upon a single, key hydrolase, Cphy3367. PMID:19775243

  10. Cloning, crystallization and preliminary X-ray study of XC1258, a CN-hydrolase superfamily protein from Xanthomonas campestris

    SciTech Connect

    Tsai, Ying-Der; Chin, Ko-Hsin; Shr, Hui-Lin; Gao, Fei Philip; Lyu, Ping-Chiang; Wang, Andrew H.-J.; Chou, Shan-Ho

    2006-10-01

    A CN-hydrolase superfamily protein from the plant pathogen X. campestris has been overexpressed in E. coli, purified and crystallized. CN-hydrolase superfamily proteins are involved in a wide variety of non-peptide carbon–nitrogen hydrolysis reactions, producing some important natural products such as auxin, biotin, precursors of antibiotics etc. These reactions all involve attack on a cyano or carbonyl carbon by a conserved novel catalytic triad Glu-Lys-Cys through a thiol acylenzyme intermediate. However, classification into the CN-hydrolase superfamily based on sequence similarity alone is not straightforward and further structural data are necessary to improve this categorization. Here, the cloning, expression, crystallization and preliminary X-ray analysis of XC1258, a CN-hydrolase superfamily protein from the plant pathogen Xanthomonas campestris (Xcc), are reported. The SeMet-substituted XC1258 crystals diffracted to a resolution of 1.73 Å. They are orthorhombic and belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 143.8, b = 154.63, c = 51.3 Å, respectively.

  11. In vivo inhibition of acylpeptide hydrolase by carbapenem antibiotics causes the decrease of plasma concentration of valproic acid in dogs.

    PubMed

    Suzuki, Eiko; Nakai, Daisuke; Ikenaga, Hidenori; Fusegawa, Keiichi; Goda, Ryoya; Kobayashi, Nobuhiro; Kuga, Hiroshi; Izumi, Takashi

    2016-01-01

    1. Our previous in vitro studies suggest that inhibition of the acylpeptide hydrolase (APEH) activity as valproic acid glucuronide (VPA-G) hydrolase by carbapenems in human liver cytosol is a key process for clinical drug-drug interaction (DDI) of valproic acid (VPA) with carbapenems. Here, we investigated whether in vivo DDI of VPA with meropenem (MEPM) was caused via inhibition of APEH in dogs. 2. More rapid decrease of plasma VPA levels and increased urinary excretion of VPA-G were observed after co-administration with MEPM compared with those after without co-administration, whereas the plasma level and bile excretion of VPA-G showed no change. 3. Dog VPA-G hydrolase activity, inhibited by carbapenems, was mainly located in cytosol from both the liver and kidney. APEH-immunodepleted cytosols lacked VPA-G hydrolase activity. Hepatic and renal APEH activity was negligible even at 24 h after dosing of MEPM to a dog. 4. In conclusion, DDI of VPA with carbapenems in dogs is caused by long-lasting inhibition of APEH-mediated VPA-G hydrolysis by carbapenems, which could explain the delayed recovery of plasma VPA levels to the therapeutic window even after discontinuation of carbapenems in humans.

  12. Development of organophosphate hydrolase activity in a bacterial homolog of human cholinesterase

    PubMed Central

    Legler, Patricia M.; Boisvert, Susanne M.; Compton, Jaimee R.; Millard, Charles B.

    2014-01-01

    We applied a combination of rational design and directed evolution (DE) to Bacillus subtilis p-nitrobenzyl esterase (pNBE) with the goal of enhancing organophosphorus acid anhydride hydrolase (OPAAH) activity. DE started with a designed variant, pNBE A107H, carrying a histidine homologous with human butyrylcholinesterase G117H to find complementary mutations that further enhance its OPAAH activity. Five sites were selected (G105, G106, A107, A190, and A400) within a 6.7 Å radius of the nucleophilic serine Oγ. All 95 variants were screened for esterase activity with a set of five substrates: pNP-acetate, pNP-butyrate, acetylthiocholine, butyrylthiocholine, or benzoylthiocholine. A microscale assay for OPAAH activity was developed for screening DE libraries. Reductions in esterase activity were generally concomitant with enhancements in OPAAH activity. One variant, A107K, showed an unexpected 7-fold increase in its kcat/Km for benzoylthiocholine, demonstrating that it is also possible to enhance the cholinesterase activity of pNBE. Moreover, DE resulted in at least three variants with modestly enhanced OPAAH activity compared to wild type pNBE. A107H/A190C showed a 50-fold increase in paraoxonase activity and underwent a slow time- and temperature-dependent change affecting the hydrolysis of OPAA and ester substrates. Structural analysis suggests that pNBE may represent a precursor leading to human cholinesterase and carboxylesterase 1 through extension of two vestigial specificity loops; a preliminary attempt to transfer the Ω-loop of BChE into pNBE is described. Unlike butyrylcholinesterase and pNBE, introducing a G143H mutation (equivalent to G117H) did not confer detectable OP hydrolase activity on human carboxylesterase 1 (hCE1). We discuss the use of pNBE as a surrogate scaffold for the mammalian esterases, and the importance of the oxyanion-hole residues for enhancing the OPAAH activity of selected serine hydrolases. PMID:25077141

  13. Purification and Characterization of a Novel Chlorpyrifos Hydrolase from Cladosporium cladosporioides Hu-01

    PubMed Central

    Chen, Shaohua; Hu, Meiying; Luo, Jianjun; Li, Yanan

    2012-01-01

    Chlorpyrifos is of great environmental concern due to its widespread use in the past several decades and its potential toxic effects on human health. Thus, the degradation study of chlorpyrifos has become increasing important in recent years. A fungus capable of using chlorpyrifos as the sole carbon source was isolated from organophosphate-contaminated soil and characterized as Cladosporium cladosporioides Hu-01 (collection number: CCTCC M 20711). A novel chlorpyrifos hydrolase from cell extract was purified 35.6-fold to apparent homogeneity with 38.5% overall recovery by ammoniumsulfate precipitation, gel filtration chromatography and anion-exchange chromatography. It is a monomeric structure with a molecular mass of 38.3 kDa. The pI value was estimated to be 5.2. The optimal pH and temperature of the purified enzyme were 6.5 and 40°C, respectively. No cofactors were required for the chlorpyrifos-hydrolysis activity. The enzyme was strongly inhibited by Hg2+, Fe3+, DTT, β-mercaptoethanol and SDS, whereas slight inhibitory effects (5–10% inhibition) were observed in the presence of Mn2+, Zn2+, Cu2+, Mg2+, and EDTA. The purified enzyme hydrolyzed various organophosphorus insecticides with P-O and P-S bond. Chlorpyrifos was the preferred substrate. The Km and Vmax values of the enzyme for chlorpyrifos were 6.7974 μM and 2.6473 μmol·min−1, respectively. Both NH2-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometer (MALDI-TOF-MS) identified an amino acid sequence MEPDGELSALTQGANS, which shared no similarity with any reported organophosphate-hydrolyzing enzymes. These results suggested that the purified enzyme was a novel hydrolase and might conceivably be developed to fulfill the practical requirements to enable its use in situ for detoxification of chlorpyrifos. Finally, this is the first described chlorpyrifos hydrolase from fungus. PMID:22693630

  14. Three-dimensional Structure of Nylon Hydrolase and Mechanism of Nylon-6 Hydrolysis*

    PubMed Central

    Negoro, Seiji; Shibata, Naoki; Tanaka, Yusuke; Yasuhira, Kengo; Shibata, Hiroshi; Hashimoto, Haruka; Lee, Young-Ho; Oshima, Shohei; Santa, Ryuji; Oshima, Shohei; Mochiji, Kozo; Goto, Yuji; Ikegami, Takahisa; Nagai, Keisuke; Kato, Dai-ichiro; Takeo, Masahiro; Higuchi, Yoshiki

    2012-01-01

    We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αββα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of Tm = 52 °C, enhanced the protein thermostability by 36 °C (Tm = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000–25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500–3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed. PMID:22187439

  15. TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi.

    PubMed

    Li, Youshan; Zhao, Ping; Liu, Huawei; Guo, Xiaomeng; He, Huawei; Zhu, Rui; Xiang, Zhonghuai; Xia, Qingyou

    2015-02-01

    Entomopathogenic fungi penetrate the insect cuticle using their abundant hydrolases. These hydrolases, which include cuticle-degrading proteases and chitinases, are important virulence factors. Our recent findings suggest that many serine protease inhibitors, especially TIL-type protease inhibitors, are involved in insect resistance to pathogenic microorganisms. To clarify the molecular mechanism underlying this resistance to entomopathogenic fungi and identify novel genes to improve the silkworm antifungal capacity, we conducted an in-depth study of serine protease inhibitors. Here, we cloned and expressed a novel silkworm TIL-type protease inhibitor, BmSPI39. In activity assays, BmSPI39 potently inhibited the virulence protease CDEP-1 of Beauveria bassiana, suggesting that it might suppress the fungal penetration of the silkworm integument by inhibiting the cuticle-degrading proteases secreted by the fungus. Phenol oxidase activation studies showed that melanization is involved in the insect immune response to fungal invasion, and that fungus-induced excessive melanization is suppressed by BmSPI39 by inhibiting the fungal cuticle-degrading proteases. To better understand the mechanism involved in the inhibition of fungal virulence by protease inhibitors, their effects on the germination of B. bassiana conidia was examined. BmSPI38 and BmSPI39 significantly inhibited the germination of B. bassiana conidia. Survival assays showed that BmSPI38 and BmSPI39 markedly improved the survival rates of silkworms, and can therefore be used as targeted resistance proteins in the silkworm. These results provided new insight into the molecular mechanisms whereby insect protease inhibitors confer resistance against entomopathogenic fungi, suggesting their potential application in medicinal or agricultural fields.

  16. Isolation and characterization of an extracellular glycosylated protein complex from Clostridium thermosaccharolyticum with pectin methylesterase and polygalacturonate hydrolase activity.

    PubMed Central

    Van Rijssel, M; Gerwig, G J; Hansen, T A

    1993-01-01

    An extracellular protein complex was isolated from the supernatant of a pectin-limited continuous culture of Clostridium thermosaccharolyticum Haren. The complex possessed both pectin methylesterase (EC 3.1.1.11) and exo-poly-alpha-galacturonate hydrolase (EC 3.2.1.82) activity and produced digalacturonate from the nonreducing end of the pectin chain. The protein consisted of 230- and 25-kDa subunits. The large subunit contained 10% (wt/wt) sugars (N-acetylgalactosamine and galactose). Under physiological conditions both activities acted in a coordinated manner: the ratio between methanol and digalacturonate released during degradation was constant and equal to the degree of esterification of the pectin used. Prolonged incubation of the enzyme with pectin led to a nondialyzable fraction that was enriched in neutral sugars, such as arabinose, rhamnose, and galactose; the high rhamnose/galacturonic acid ratio was indicative of hairy region-like structures. The smallest substrate utilized by the hydrolase was a tetragalacturonate. Vmax with oligogalacturonates increased with increasing chain length. The Km and Vmax for the polygalacturonate hydrolase with citrus pectate as a substrate were 0.8 g liter-1 and 180 mumol min-1 mg of protein-1, respectively. The Km and Vmax for the esterase with citrus pectin as a substrate were 1.2 g liter-1 and 440 mumol min-1 mg of protein-1, respectively. The temperature optima for the hydrolase and esterase were 70 and 60 degrees C, respectively. Both enzyme activities were stable for more than 1 h at 70 degrees C. The exo-polygalacturonate hydrolase of Clostridium thermosulfurogenes was partially purified while the methylesterase was also copurified. Images PMID:8481009

  17. Novel corrosion inhibitor technology

    SciTech Connect

    Van de Ven, P.; Fritz, P.; Pellet, R.

    1999-11-01

    A novel, patented corrosion inhibitor technology has been identified for use in heat transfer applications such as automotive and heavy-duty coolant. The new technology is based on a low-toxic, virtually depletion-free carboxylic acid corrosion inhibitor package that performs equally well in mono ethylene glycol and in less toxic propylene glycol coolants. An aqueous inhibitor concentrate is available to provide corrosion protection where freezing protection is not an issue. In the present paper, this inhibitor package is evaluated in the different base fluids: mono ethylene glycol, mono propylene glycol and water. Results are obtained in both standardized and specific corrosion tests as well as in selected field trials. These results indicate that the inhibitor package remains effective and retains the benefits previously identified in automotive engine coolant applications: excellent corrosion protection under localized conditions, general corrosion conditions as well as at high temperature.

  18. Structural and kinetic insights into the mechanism of 5-hydroxyisourate hydrolase from Klebsiella pneumoniae

    SciTech Connect

    French, Jarrod B.; Ealick, Steven E.

    2011-07-19

    The stereospecific oxidative degradation of uric acid to (S)-allantoin has recently been demonstrated to proceed via two unstable intermediates and requires three separate enzymatic reactions. The second step of this reaction, the conversion of 5-hydroxyisourate (HIU) to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, is catalyzed by HIU hydrolase (HIUH). The high-resolution crystal structure of HIUH from the opportunistic pathogen Klebsiella pneumoniae (KpHIUH) has been determined. KpHIUH is a homotetrameric protein that, based on sequence and structural similarity, belongs to the transthyretin-related protein family. In addition, the steady-state kinetic parameters for this enzyme and four active-site mutants have been measured. These data provide valuable insight into the functional roles of the active-site residues. Based upon the structural and kinetic data, a mechanism is proposed for the KpHIUH-catalyzed reaction.

  19. Cytochemical localization of some hydrolases in the pollen and pollen tubes of Amaryllis vittata Ait.

    PubMed

    Sharma, D

    1982-01-01

    Some hydrolases are localized cytochemically in the pollen and pollen tubes of Amaryllis vittata Ait. The function of different enzymes is discussed in relation to pollen tubes morphogenesis. Activity of most of the enzymes was confined to colpus region, pollen wall and general cytoplasm of pollen and pollen tube. The activity of hydrolytic enzymes like acid monophosphoesterase and lipase and was nil in the exine of both germinated and ungerminated pollen, whereas intense reaction for esterase was observed in exine. Enzyme activity increased after germination which suggest the hydrolysis of stored metabolites and synthesis of proteins and other metabolites for the active growth of pollen tube. Intense reaction for enzymes like alkaline phosphomonoesterase, ATP-ase, 5-nucleotidase etc. at the tip region of pollen tube suggest their role in physiological processes associated with exchange of materials through intercellular transport during tube wall polysaccharide biogenesis.

  20. Mutagenesis of organophosphorus hydrolase to enhance hydrolysis of the nerve agent VX.

    PubMed

    Gopal, S; Rastogi, V; Ashman, W; Mulbry, W

    2000-12-20

    Organophosphorus hydrolase (OPH) is capable of hydrolyzing a wide variety of organophosphorus pesticides and chemical warfare agents. However, the hydrolytic activity of OPH against the warfare agent VX is less than 0.1% relative to its activity against parathion and paraoxon. Based on the crystal structure of OPH and the similarities it shares with acetylcholinesterase, eight OPH mutants were constructed with the goal of increasing OPH activity toward VX. The activities of crude extracts from these mutants were measured using VX, demeton-S methyl, diisopropylfluoro-phosphate, ethyl parathion, paraoxon, and EPN as substrates. One mutant (L136Y) displayed a 33% increase in the relative VX hydrolysis rate compared to wild type enzyme. The other seven mutations resulted in 55-76% decreases in the relative rates of VX hydrolysis. There was no apparent relationship between the hydrolysis rates of VX and the rates of the other organophosphorus compounds tested.

  1. Detoxication strategy of epoxide hydrolase-the basis for a novel threshold for definable genotoxic carcinogens.

    PubMed

    Oesch, Franz; Hengstler, Jan Georg; Arand, Michael

    2004-01-01

    From our recent work on the three-dimensional structure of epoxide hydrolases we theoretically deduced the likelihood of a two-step catalytic mechanism that we and others have subsequently experimentally confirmed. Analysis of the rate of the two steps by us and by others show that the first step-responsible for removal of the reactive epoxide from the system-works extraordinarily fast (typically three orders of magnitude faster than the second step), sucking up the epoxide like a sponge. Regeneration of the free enzyme (the second step of the catalytic mechanism) is slow. This becomes a toxicological problem only at doses of the epoxide that titrate the enzyme out. Our genotoxicity work shows that indeed this generates a practical threshold below which no genotoxicity is observed. This shows that-contrary to old dogma-practical thresholds exist for definable genotoxic carcinogens.

  2. Chitosanases from Family 46 of Glycoside Hydrolases: From Proteins to Phenotypes.

    PubMed

    Viens, Pascal; Lacombe-Harvey, Marie-Ève; Brzezinski, Ryszard

    2015-10-28

    Chitosanases, enzymes that catalyze the endo-hydrolysis of glycolytic links in chitosan, are the subject of numerous studies as biotechnological tools to generate low molecular weight chitosan (LMWC) or chitosan oligosaccharides (CHOS) from native, high molecular weight chitosan. Glycoside hydrolases belonging to family GH46 are among the best-studied chitosanases, with four crystallography-derived structures available and more than forty enzymes studied at the biochemical level. They were also subjected to numerous site-directed mutagenesis studies, unraveling the molecular mechanisms of hydrolysis. This review is focused on the taxonomic distribution of GH46 proteins, their multi-modular character, the structure-function relationships and their biological functions in the host organisms.

  3. A classification of glycosyl hydrolases based on amino acid sequence similarities.

    PubMed Central

    Henrissat, B

    1991-01-01

    The amino acid sequences of 301 glycosyl hydrolases and related enzymes have been compared. A total of 291 sequences corresponding to 39 EC entries could be classified into 35 families. Only ten sequences (less than 5% of the sample) could not be assigned to any family. With the sequences available for this analysis, 18 families were found to be monospecific (containing only one EC number) and 17 were found to be polyspecific (containing at least two EC numbers). Implications on the folding characteristics and mechanism of action of these enzymes and on the evolution of carbohydrate metabolism are discussed. With the steady increase in sequence and structural data, it is suggested that the enzyme classification system should perhaps be revised. PMID:1747104

  4. Potential anti-obesity effects of a long-acting cocaine hydrolase.

    PubMed

    Zheng, Xirong; Deng, Jing; Zhang, Ting; Yao, Jianzhuang; Zheng, Fang; Zhan, Chang-Guo

    2016-11-25

    A long-acting cocaine hydrolase, known as CocH3-Fc(M3), engineered from human butyrylcholinesterase (BChE) was tested, in this study, for its potential anti-obesity effects. Mice on a high-fat diet gained significantly less body weight when treated weekly with 1 mg/kg CocH3-Fc(M3) compared to control mice, though their food intake was similar. There is no correlation between the average body weight and the average food intake, which is consistent with the previously reported observation in BChE knockout mice. In addition, molecular modeling was carried out to understand how ghrelin binds with CocH3, showing that ghrelin binds with CocH3 in a similar mode as ghrelin binding with wild-type human BChE. The similar binding structures explains why CocH3 and BChE have similar catalytic activity against ghrelin.

  5. A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast

    PubMed Central

    Dudin, Omaya; Bendezú, Felipe O.; Groux, Raphael; Laroche, Thierry; Seitz, Arne

    2015-01-01

    Cell–cell fusion is essential for fertilization. For fusion of walled cells, the cell wall must be degraded at a precise location but maintained in surrounding regions to protect against lysis. In fission yeast cells, the formin Fus1, which nucleates linear actin filaments, is essential for this process. In this paper, we show that this formin organizes a specific actin structure—the actin fusion focus. Structured illumination microscopy and live-cell imaging of Fus1, actin, and type V myosins revealed an aster of actin filaments whose barbed ends are focalized near the plasma membrane. Focalization requires Fus1 and type V myosins and happens asynchronously always in the M cell first. Type V myosins are essential for fusion and concentrate cell wall hydrolases, but not cell wall synthases, at the fusion focus. Thus, the fusion focus focalizes cell wall dissolution within a broader cell wall synthesis zone to shift from cell growth to cell fusion. PMID:25825517

  6. Secretory expression of organophosphorus hydrolase OPHC2 in Yarrowia lipolytica Polg.

    PubMed

    Li, Meng; Yu, Xiaolan; Wang, Fei; Zhai, Chao; Shen, Wei; Yu, Xianhong; Wang, Xiaojuan; Ma, Lixin

    2015-01-01

    In the present study, recombinant organophosphorus hydrolase OPHC2 was successfully produced by Yarrowia lipolytica and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses showed a major polypeptide band of 36 kDa. The purified enzyme was optimally active at 65°C and pH 8.5 and also displayed good thermal and pH stability using methyl parathion (O,O-dimethyl-O-4-p-nitrophenyl phosphorothioate) as a substrate. Moreover, as Y. lipolytica is a non-pathogenic, generally regarded as safe (GRAS) yeast, the cell culture supernatant can be used directly on vegetables and fruits that are contaminated by organophosphorus pesticides.

  7. Extensive hydrolysis of phosphonates as unexpected behaviour of the known His6-organophosphorus hydrolase.

    PubMed

    Lyagin, Ilya V; Andrianova, Mariia S; Efremenko, Elena N

    2016-07-01

    The catalytic activity of hexahistidine-tagged organophosphorus hydrolase (His6-OPH) in hydrolytic reactions of methylphosphonic acid (MPA) and its monoesters and diesters being decomposition products of R-VX was demonstrated for the first time. The catalytic constants of enzyme in such reactions were determined. The mechanism of C-P bond cleavage in the MPA by His6-OPH was proposed. Such reaction was estimated to be carried out with the soluble and nanocapsulated forms of His6-OPH. His6-OPH was demonstrated to be capable of degrading the key organophosphorus components of reaction masses (RMs) that are produced by the chemical detoxification of R-VX and RMs are multi-substrate mixtures for this enzyme. The kinetic model describing the behaviour of His6-OPH in RMs was proposed and was shown to adequately fit experimental points during degradation of the real samples of RMs.

  8. Gene duplication event in family 12 glycosyl hydrolase from Phytophthora spp.

    PubMed

    Costanzo, Stefano; Ospina-Giraldo, M D; Deahl, K L; Baker, C J; Jones, Richard W

    2006-10-01

    A total of 18 paralogs of xyloglucan-specific endoglucanases (EGLs) from the glycosyl hydrolase family 12 were identified and characterized in Phytophthora sojae and Phytophthora ramorum. These genes encode predicted extracellular enzymes, with sizes ranging from 189 to 435 amino acid residues, that would be capable of hydrolyzing the xyloglucan component of the host cell wall. In two cases, four and six functional copies of these genes were found in tight succession within a region of 5 and 18 kb, respectively. The overall gene copy number and relative organization appeared well conserved between P. sojae and P. ramorum, with apparent synteny in this region of their respective genomes. Phylogenetic analyses of Phytophthora endoglucanases of family 12 and other known members of EGL 12, revealed a close relatedness with a fairly conserved gene sub-family containing, among others, sequences from the fungi Emericella desertorum and Aspergillus aculeatus. This is the first report of family 12 EGLs present in plant pathogenic eukaryotes.

  9. Recombinant S-adenosylhomocysteine hydrolase from Thermotoga maritima: cloning, overexpression, characterization, and thermal purification studies.

    PubMed

    Lozada-Ramírez, J D; Sánchez-Ferrer, A; García-Carmona, F

    2013-06-01

    S-Adenosylhomocysteine hydrolase (SAHase) encoded by sahase gene is a determinant when catalyzing the reversible conversion of adenosine and homocysteine to S-adenosylhomocysteine in most living organisms. The sahase gene was isolated from the genome of the highly thermostable anaerobic bacteria Thermotoga maritima, and then it was cloned, characterized, overexpressed using Escherichia coli, and partially purified by thermal precipitation. The thermal purification of the recombinant SAHase resulted in changes in the circular dichroism spectra. As a result of this analysis, it was possible to determine the structural changes in the composition of the α-helix and β-sheet content of the recombinant enzyme after purification. Moreover, a predicted secondary structure and 3D structural model was rendered by comparative molecular modeling to further understand the molecular function of this protein including its attractive biotechnological use.

  10. Improvement of corn stover bioconversion efficiency by using plant glycoside hydrolase.

    PubMed

    Han, Yejun; Chen, Hongzhang

    2011-04-01

    Plant cell wall is the most abundant substrate for bioethanol production, and plants also represent a key resource for glycoside hydrolase (GH). To exploit efficient way for bioethanol production with lower cellulase loading, the potential of plant GH for lignocellulose bioconversion was evaluated. The GH activity for cell wall proteins (CWPs) was detected from fresh corn stover (FCS), and the synergism of which with Trichoderma reesei cellulase was also observed. The properties for the GH of FCS make it a promising enzyme additive for lignocellulose biodegradation. To make use of the plant GH, novel technology for hydrolysis and ethanol fermentation was developed with corn stover as substrate. Taking steam-exploded corn stover as substrate for hydrolysis and ethanol fermentation, compared with T. reesei cellulase loaded alone, the final glucose and ethanol accumulation increased by 60% and 63% respectively with GH of FCS as an addition.

  11. Ubiquitin C-terminal hydrolase L1 deficiency decreases bone mineralization.

    PubMed

    Shim, Sehwan; Kwon, Young-Bae; Yoshikawa, Yasuhiro; Kwon, Jungkee

    2008-06-01

    Ubiquitin C-terminal hydrolase L1 is a component of the ubiquitin proteasome system, which evidences unique biological activities. In this study, we report the pattern of UCH-L1 expression, and show that it regulates bone mineralization in osteogenesis. UCH-L1 was expressed in osteoblasts, osteoclasts, and hematopoietic precursor cells of bone marrow in the metaphysis and diaphysis of the femora. To further assess the involvement of UCH-L1 in the regulation of bone mineralization, we evaluated the bone mineral density (BMD) rate of gad mice, using the Latheta computed tomography system. Male gad mice evidenced a significantly decreased BMD rate in the metaphysis and diaphysis of the femora. These findings of decreased BMD rate in the bones of gad mice may suggest that UCH-L1 function regulates bone mineralization during osteogenesis.

  12. The CHAP domain: a large family of amidases including GSP amidase and peptidoglycan hydrolases.

    PubMed

    Bateman, Alex; Rawlings, Neil D

    2003-05-01

    Cleavage of peptidoglycan plays an important role in bacterial cell division, cell growth and cell lysis. Here, we reveal that several known peptidoglycan amidases fall into a family, which includes many proteins of previously unknown function. The family includes two different peptidoglycan cleavage activities: L-muramoyl-L-alanine amidase and D-alanyl-glycyl endopeptidase activity. The family includes the amidase portion of the bifunctional glutathionylspermidine synthase/amidase enzyme from bacteria and pathogenic trypanosomes. The glutathionylspermidine synthase is thought to be a key component of the alternative pathway in trypanosomes for protection from oxygen-radical damage and has been proposed as a potential drug target. The CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain is often found in association with other domains that cleave peptidoglycan. The large number of multifunctional hydrolases suggests that they might act in a cooperative manner to cleave specialized substrates.

  13. Membrane lipids are key modulators of the endocannabinoid-hydrolase FAAH.

    PubMed

    Dainese, Enrico; De Fabritiis, Gianni; Sabatucci, Annalaura; Oddi, Sergio; Angelucci, Clotilde Beatrice; Di Pancrazio, Chiara; Giorgino, Toni; Stanley, Nathaniel; Del Carlo, Michele; Cravatt, Benjamin F; Maccarrone, Mauro

    2014-02-01

    Lipid composition is expected to play an important role in modulating membrane enzyme activity, in particular if the substrates are themselves lipid molecules. A paradigmatic case is FAAH (fatty acid amide hydrolase), an enzyme critical in terminating endocannabinoid signalling and an important therapeutic target. In the present study, using a combined experimental and computational approach, we show that membrane lipids modulate the structure, subcellular localization and activity of FAAH. We report that the FAAH dimer is stabilized by the lipid bilayer and shows a higher membrane-binding affinity and enzymatic activity within membranes containing both cholesterol and the natural FAAH substrate AEA (anandamide). Additionally, co-localization of cholesterol, AEA and FAAH in mouse neuroblastoma cells suggests a mechanism through which cholesterol increases the substrate accessibility of FAAH.

  14. Use of Nanostructure-Initiator Mass Spectrometry to Deduce Selectivity of Reaction in Glycoside Hydrolases

    PubMed Central

    Deng, Kai; Takasuka, Taichi E.; Bianchetti, Christopher M.; Bergeman, Lai F.; Adams, Paul D.; Northen, Trent R.; Fox, Brian G.

    2015-01-01

    Chemically synthesized nanostructure-initiator mass spectrometry (NIMS) probes derivatized with tetrasaccharides were used to study the reactivity of representative Clostridium thermocellum β-glucosidase, endoglucanases, and cellobiohydrolase. Diagnostic patterns for reactions of these different classes of enzymes were observed. Results show sequential removal of glucose by the β-glucosidase and a progressive increase in specificity of reaction from endoglucanases to cellobiohydrolase. Time-dependent reactions of these polysaccharide-selective enzymes were modeled by numerical integration, which provides a quantitative basis to make functional distinctions among a continuum of naturally evolved catalytic properties. Consequently, our method, which combines automated protein translation with high-sensitivity and time-dependent detection of multiple products, provides a new approach to annotate glycoside hydrolase phylogenetic trees with functional measurements. PMID:26579511

  15. Excess α-synuclein worsens disease in mice lacking ubiquitin