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1.
J Biol Chem ; 295(52): 17935-17949, 2020 12 25.
Article in English | MEDLINE | ID: mdl-32900849

ABSTRACT

The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.


Subject(s)
Acetanilides/pharmacology , Enzyme Inhibitors/pharmacology , Neoplasms/drug therapy , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Polypharmacology , Sirtuin 1/antagonists & inhibitors , Thiourea/analogs & derivatives , Tumor Suppressor Protein p53/metabolism , Autophagy , Cell Proliferation , Dihydroorotate Dehydrogenase , Humans , Neoplasms/metabolism , Neoplasms/pathology , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Thiourea/pharmacology , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics
2.
J Biol Inorg Chem ; 24(6): 841-848, 2019 09.
Article in English | MEDLINE | ID: mdl-31218442

ABSTRACT

Ribonucleotide reductase (RNR) has been extensively probed as a target enzyme in the search for selective antibiotics. Here we report on the mechanism of inhibition of nine compounds, serving as representative examples of three different inhibitor classes previously identified by us to efficiently inhibit RNR. The interaction between the inhibitors and Pseudomonas aeruginosa RNR was elucidated using a combination of electron paramagnetic resonance spectroscopy and thermal shift analysis. All nine inhibitors were found to efficiently quench the tyrosyl radical present in RNR, required for catalysis. Three different mechanisms of radical quenching were identified, and shown to depend on reduction potential of the assay solution and quaternary structure of the protein complex. These results form a good foundation for further development of P. aeruginosa selective antibiotics. Moreover, this study underscores the complex nature of RNR inhibition and the need for detailed spectroscopic studies to unravel the mechanism of RNR inhibitors.


Subject(s)
Free Radicals/chemistry , Free Radicals/metabolism , Pseudomonas aeruginosa/enzymology , Ribonucleotide Reductases/metabolism , Tyrosine/chemistry , Tyrosine/metabolism , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , Ribonucleotide Reductases/genetics
3.
PLoS One ; 13(4): e0195956, 2018.
Article in English | MEDLINE | ID: mdl-29684045

ABSTRACT

Tenovin-6 is the most studied member of a family of small molecules with antitumour activity in vivo. Previously, it has been determined that part of the effects of tenovin-6 associate with its ability to inhibit SirT1 and activate p53. However, tenovin-6 has also been shown to modulate autophagic flux. Here we show that blockage of autophagic flux occurs in a variety of cell lines in response to certain tenovins, that autophagy blockage occurs regardless of the effect of tenovins on SirT1 or p53, and that this blockage is dependent on the aliphatic tertiary amine side chain of these molecules. Additionally, we evaluate the contribution of this tertiary amine to the elimination of proliferating melanoma cells in culture. We also demonstrate that the presence of the tertiary amine is sufficient to lead to death of tumour cells arrested in G1 phase following vemurafenib treatment. We conclude that blockage of autophagic flux by tenovins is necessary to eliminate melanoma cells that survive B-Raf inhibition and achieve total tumour cell kill and that autophagy blockage can be achieved at a lower concentration than by chloroquine. This observation is of great relevance as relapse and resistance are frequently observed in cancer patients treated with B-Raf inhibitors.


Subject(s)
Antineoplastic Agents/pharmacology , Autophagy/drug effects , Benzamides/pharmacology , Indoles/pharmacology , Melanoma/genetics , Proto-Oncogene Proteins B-raf/genetics , Sulfonamides/pharmacology , Antineoplastic Agents/chemistry , Benzamides/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Resistance, Neoplasm/drug effects , Drug Synergism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Melanoma/drug therapy , Molecular Structure , Mutation , Sirtuins/genetics , Tumor Suppressor Protein p53/genetics , Vemurafenib
4.
Mol Oncol ; 10(9): 1375-1386, 2016 11.
Article in English | MEDLINE | ID: mdl-27511871

ABSTRACT

Ribonucleotide Reductase (RNR) is the sole enzyme that catalyzes the reduction of ribonucleotides into deoxyribonucleotides. Even though RNR is a recognized target for antiproliferative molecules, and the main target of the approved drug hydroxyurea, few new leads targeted to this enzyme have been developed. We have evaluated a recently identified set of RNR inhibitors with respect to inhibition of the human enzyme and cellular toxicity. One compound, NSC73735, is particularly interesting; it is specific for leukemia cells and is the first identified compound that hinders oligomerization of the mammalian large RNR subunit. Similar to hydroxyurea, it caused a disruption of the cell cycle distribution of cultured HL-60 cells. In contrast to hydroxyurea, the disruption was reversible, indicating higher specificity. NSC73735 thus defines a potential lead candidate for RNR-targeted anticancer drugs, as well as a chemical probe with better selectivity for RNR inhibition than hydroxyurea.


Subject(s)
Deoxyribonucleosides/pharmacology , Enzyme Inhibitors/pharmacology , Ribonucleotide Reductases/antagonists & inhibitors , Biological Assay , Cell Cycle/drug effects , Cell Death/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Flow Cytometry , Gene Expression Regulation/drug effects , HL-60 Cells , Humans , Hydroxyurea/pharmacology , Protein Structure, Quaternary , Protein Subunits/antagonists & inhibitors , Protein Subunits/chemistry , Protein Subunits/metabolism , Ribonucleotide Reductases/chemistry , Ribonucleotide Reductases/metabolism , Temperature
5.
J Biol Chem ; 291(35): 18410-8, 2016 08 26.
Article in English | MEDLINE | ID: mdl-27365393

ABSTRACT

Leukotriene C4 synthase (LTC4S) catalyzes the formation of the proinflammatory lipid mediator leukotriene C4 (LTC4). LTC4 is the parent molecule of the cysteinyl leukotrienes, which are recognized for their pathogenic role in asthma and allergic diseases. Cellular LTC4S activity is suppressed by PKC-mediated phosphorylation, and recently a downstream p70S6k was shown to play an important role in this process. Here, we identified Ser(36) as the major p70S6k phosphorylation site, along with a low frequency site at Thr(40), using an in vitro phosphorylation assay combined with mass spectrometry. The functional consequences of p70S6k phosphorylation were tested with the phosphomimetic mutant S36E, which displayed only about 20% (20 µmol/min/mg) of the activity of WT enzyme (95 µmol/min/mg), whereas the enzyme activity of T40E was not significantly affected. The enzyme activity of S36E increased linearly with increasing LTA4 concentrations during the steady-state kinetics analysis, indicating poor lipid substrate binding. The Ser(36) is located in a loop region close to the entrance of the proposed substrate binding pocket. Comparative molecular dynamics indicated that Ser(36) upon phosphorylation will pull the first luminal loop of LTC4S toward the neighboring subunit of the functional homotrimer, thereby forming hydrogen bonds with Arg(104) in the adjacent subunit. Because Arg(104) is a key catalytic residue responsible for stabilization of the glutathione thiolate anion, this phosphorylation-induced interaction leads to a reduction of the catalytic activity. In addition, the positional shift of the loop and its interaction with the neighboring subunit affect active site access. Thus, our mutational and kinetic data, together with molecular simulations, suggest that phosphorylation of Ser(36) inhibits the catalytic function of LTC4S by interference with the catalytic machinery.


Subject(s)
Glutathione Transferase/chemistry , Amino Acid Substitution , Animals , Binding Sites , Catalysis , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Humans , Leukotriene A4/biosynthesis , Leukotriene A4/chemistry , Leukotriene A4/genetics , Mice , Mutation, Missense , Phosphorylation , Protein Structure, Secondary , Ribosomal Protein S6 Kinases, 70-kDa/chemistry , Ribosomal Protein S6 Kinases, 70-kDa/genetics , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Serine/chemistry , Serine/genetics , Serine/metabolism
6.
PLoS One ; 10(7): e0128199, 2015.
Article in English | MEDLINE | ID: mdl-26147435

ABSTRACT

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria.


Subject(s)
Cysteine/chemistry , Ribonucleotide Reductases/chemistry , Thermotoga maritima/enzymology , Catalytic Domain , Crystallography, X-Ray , Models, Molecular , Protein Conformation
7.
PLoS One ; 10(7): e0134293, 2015.
Article in English | MEDLINE | ID: mdl-26225432

ABSTRACT

The opportunistic pathogen Pseudomonas aeruginosa can grow under both aerobic and anaerobic conditions. Its flexibility with respect to oxygen load is reflected by the fact that its genome encodes all three existing classes of ribonucleotides reductase (RNR): the oxygen-dependent class I RNR, the oxygen-indifferent class II RNR, and the oxygen-sensitive class III RNR. The P. aeruginosa class II RNR is expressed as two separate polypeptides (NrdJa and NrdJb), a unique example of a split RNR enzyme in a free-living organism. A split class II RNR is also found in a few closely related γ-Proteobacteria. We have characterized the P. aeruginosa class II RNR and show that both subunits are required for formation of a biologically functional enzyme that can sustain vitamin B12-dependent growth. Binding of the B12 coenzyme as well as substrate and allosteric effectors resides in the NrdJa subunit, whereas the NrdJb subunit mediates efficient reductive dithiol exchange during catalysis. A combination of activity assays and activity-independent methods like surface plasmon resonance and gas phase electrophoretic macromolecule analysis suggests that the enzymatically active form of the enzyme is a (NrdJa-NrdJb)2 homodimer of heterodimers, and a combination of hydrogen-deuterium exchange experiments and molecular modeling suggests a plausible region in NrdJa that interacts with NrdJb. Our detailed characterization of the split NrdJ from P. aeruginosa provides insight into the biochemical function of a unique enzyme known to have central roles in biofilm formation and anaerobic growth.


Subject(s)
Pseudomonas aeruginosa/enzymology , Ribonucleotide Reductases/metabolism , Protein Binding
8.
Proc Natl Acad Sci U S A ; 112(22): E2900-9, 2015 Jun 02.
Article in English | MEDLINE | ID: mdl-25991856

ABSTRACT

The biological functions of VEGF-B in cancer progression remain poorly understood. Here, we report that VEGF-B promotes cancer metastasis through the remodeling of tumor microvasculature. Knockdown of VEGF-B in tumors resulted in increased perivascular cell coverage and impaired pulmonary metastasis of human melanomas. In contrast, the gain of VEGF-B function in tumors led to pseudonormalized tumor vasculatures that were highly leaky and poorly perfused. Tumors expressing high levels of VEGF-B were more metastatic, although primary tumor growth was largely impaired. Similarly, VEGF-B in a VEGF-A-null tumor resulted in attenuated primary tumor growth but substantial pulmonary metastases. VEGF-B also led to highly metastatic phenotypes in Vegfr1 tk(-/-) mice and mice treated with anti-VEGF-A. These data indicate that VEGF-B promotes cancer metastasis through a VEGF-A-independent mechanism. High expression levels of VEGF-B in two large-cohort studies of human patients with lung squamous cell carcinoma and melanoma correlated with poor survival. Taken together, our findings demonstrate that VEGF-B is a vascular remodeling factor promoting cancer metastasis and that targeting VEGF-B may be an important therapeutic approach for cancer metastasis.


Subject(s)
Biomarkers, Tumor/metabolism , Microvessels/drug effects , Neoplasm Metastasis/physiopathology , Neoplasms/blood supply , Vascular Endothelial Growth Factor B/metabolism , Vascular Endothelial Growth Factor B/pharmacology , Animals , Blotting, Western , Cell Hypoxia , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Fluorescent Antibody Technique , Immunohistochemistry , Injections, Subcutaneous , Kaplan-Meier Estimate , Mice , Polymerase Chain Reaction , Vascular Endothelial Growth Factor B/administration & dosage , Zebrafish
9.
Biochim Biophys Acta ; 1844(2): 439-46, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24333438

ABSTRACT

Leukotriene A4 hydrolase/aminopeptidase (LTA4H) (EC 3.3.2.6) is a bifunctional zinc metalloenzyme with both an epoxide hydrolase and an aminopeptidase activity. LTA4H from the African claw toad, Xenopus laevis (xlLTA4H) has been shown to, unlike the human enzyme, convert LTA4 to two enzymatic metabolites, LTB4 and another biologically active product Δ(6)-trans-Δ(8)-cis-LTB4 (5(S),12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid). In order to study the molecular aspect of the formation of this product we have characterized the structure and function of xlLTA4H. We solved the structure of xlLTA4H to a resolution of 2.3Å. It is a dimeric structure where each monomer has three domains with the active site in between the domains, similar as to the human structure. An important difference between the human and amphibian enzyme is the phenylalanine to tyrosine exchange at position 375. Our studies show that mutating F375 in xlLTA4H to tyrosine abolishes the formation of the LTB4 isomeric product Δ(6)-trans-Δ(8)-cis-LTB4. In an attempt to understand how one amino acid exchange leads to a new product profile as seen in the xlLTA4H, we performed a conformer analysis of the triene part of the substrate LTA4. Our results show that the Boltzmann distribution of substrate conformers correlates with the observed distribution of products. We suggest that the observed difference in product profile between the human and the xlLTA4H arises from different level of discrimination between substrate LTA4 conformers.


Subject(s)
Epoxide Hydrolases/chemistry , Hydroxyeicosatetraenoic Acids/metabolism , Leukotriene B4/metabolism , Xenopus Proteins/chemistry , Xenopus laevis/metabolism , Amino Acid Sequence , Animals , Catalytic Domain , Crystallography, X-Ray , Humans , Hydrolysis , Hydroxyeicosatetraenoic Acids/chemistry , Kinetics , Leukotriene B4/chemistry , Models, Molecular , Molecular Sequence Data , Protein Multimerization , Sequence Homology, Amino Acid , Substrate Specificity
10.
PLoS One ; 7(10): e46764, 2012.
Article in English | MEDLINE | ID: mdl-23071631

ABSTRACT

A difficulty associated with high throughput screening for enzyme inhibitors is to establish reaction conditions that maximize the sensitivity and resolution of the assay. Deduction of information from end-point assays at single concentrations requires a detailed understanding of the time progress of the enzymatic reaction, an essential but often difficult process to model. A tool to simulate the time progress of enzyme catalyzed reactions and allows adjustment of reactant concentrations and parameters (initial concentrations, K(m), k(cat), K(i) values, enzyme half-life, product•enzyme dissociation constant, and the rate constant for the reversed reaction) has been developed. This tool provides comparison of the progress of uninhibited versus inhibited reactions for common inhibitory mechanisms, and guides the tuning of reaction conditions. Possible applications include: analysis of substrate turnover, identification of the point of maximum difference in product concentration (Δ(max)[P]) between inhibited and uninhibited reactions, determination of an optimal observation window unbiased for inhibitor mechanisms or potency, and interpretation of observed inhibition in terms of true inhibition. An important observation that can be utilized to improve assay signal strength and resolution is that Δ(max)[P] occurs at a high degree of substrate consumption (commonly >75%) and that observation close to this point does not adversely affect observed inhibition or IC(50) values.


Subject(s)
Enzyme Assays , Enzyme Inhibitors/chemistry , Software , Computer Simulation , Drug Evaluation, Preclinical , Epoxide Hydrolases/chemistry , Kinetics , Models, Biological
11.
Proc Natl Acad Sci U S A ; 109(25): 9798-803, 2012 Jun 19.
Article in English | MEDLINE | ID: mdl-22665797

ABSTRACT

Ribonucleotide reductase (RNR) catalyzes reduction of the four different ribonucleotides to their corresponding deoxyribonucleotides and is the rate-limiting enzyme in DNA synthesis. RNR is a well-established target for the antiproliferative drugs Gemzar and Hydrea, for antisense therapy, and in combination chemotherapies. Surprisingly, few novel drugs that target RNR have emerged, partly because RNR activity assays are laboratory-intense and exclude high-throughput methodologies. Here, we present a previously undescribed PCR-based assay for RNR activity measurements in microplate format. We validated the approach by screening a diverse library of 1,364 compounds for inhibitors of class I RNR from the opportunistic pathogen Pseudomonas aeruginosa, and we identified 27 inhibitors with IC(50) values from ∼200 nM to 30 µM. Interestingly, a majority of the identified inhibitors have been found inactive in human cell lines as well as in anticancer and in vivo tumor tests as reported by the PubChem BioAssay database. Four of the RNR inhibitors inhibited growth of P. aeruginosa, and two were also found to affect the transcription of RNR genes and to decrease the cellular deoxyribonucleotide pools. This unique PCR-based assay works with any RNR enzyme and any substrate nucleotide, and thus opens the door to high-throughput screening for RNR inhibitors in drug discovery.


Subject(s)
Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/pharmacology , Ribonucleotide Reductases/antagonists & inhibitors , Inhibitory Concentration 50 , Pseudomonas aeruginosa/enzymology
12.
FEBS Lett ; 584(15): 3446-51, 2010 Aug 04.
Article in English | MEDLINE | ID: mdl-20609366

ABSTRACT

Leukotriene A4 hydrolase (LTA4H) is a key enzyme in the inflammatory process of mammals. It is an epoxide hydrolase and an aminopeptidase of the M1 family of the MA clan of Zn-metallopeptidases. We have solved the crystal structure of LTA4H in complex with N-[3(R)-[(hydroxyamino)carbonyl]-2-benzyl-1-oxopropyl]-L-alanine, a potent inhibitor of several Zn-metalloenzymes, both endopeptidases and aminopeptidases. The inhibitor binds along the sequence signature for M1 aminopeptidases, GXMEN. It exhibits bidentate chelation of the catalytic zinc and binds to LTA4H's enzymatically essential carboxylate recognition site. The structure gives clues to the binding of this inhibitor to related enzymes and thereby identifies residues of their S1' sub sites as well as strategies for design of inhibitors.


Subject(s)
Dipeptides/chemistry , Drug Design , Epoxide Hydrolases/chemistry , Metalloproteases/antagonists & inhibitors , Protease Inhibitors/chemistry , Zinc/metabolism , Amino Acid Sequence , Conserved Sequence , Crystallography, X-Ray , Enzyme Assays , Epoxide Hydrolases/isolation & purification , Models, Molecular , Molecular Sequence Data , Protease Inhibitors/pharmacology , Protein Binding/drug effects , Sequence Alignment
13.
Chem Biol ; 15(9): 920-9, 2008 Sep 22.
Article in English | MEDLINE | ID: mdl-18804029

ABSTRACT

M1 aminopeptidases comprise a large family of biologically important zinc enzymes. We show that peptide turnover by the M1 prototype, leukotriene A4 hydrolase/aminopeptidase, involves a shift in substrate position associated with exchange of zinc coordinating groups, while maintaining the overall coordination geometry. The transition state is stabilized by residues conserved among M1 members and in the final reaction step, Glu-296 of the canonical zinc binding HEXXH motif shuffles a proton from the hydrolytic water to the leaving group. Tripeptide substrates bind along the conserved GXMEN motif, precisely occupying the distance between Glu-271 and Arg-563, whereas the Arg specificity is governed by a narrow S1 pocket capped with Asp-375. Our data provide detailed insights to the active site chemistry of M1 aminopeptidases and will aid in the development of novel enzyme inhibitors.


Subject(s)
Aminopeptidases/antagonists & inhibitors , Aminopeptidases/metabolism , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Epoxide Hydrolases/antagonists & inhibitors , Epoxide Hydrolases/metabolism , Amines/chemistry , Amines/metabolism , Aminopeptidases/chemistry , Binding Sites , Catalysis , Cations , Epoxide Hydrolases/chemistry , Hydrolysis , Kinetics , Models, Molecular , Oxidation-Reduction/drug effects , Structure-Activity Relationship , Substrate Specificity , Zinc/pharmacology
14.
Prostaglandins Other Lipid Mediat ; 83(3): 198-202, 2007 May.
Article in English | MEDLINE | ID: mdl-17481555

ABSTRACT

Leukotriene A4 hydrolase catalyzes the final and committed step in the biosynthesis of leukotriene B4, a potent chemotactic agent for neutrophils, eosinophils, monocytes, and T-cells that play key roles in the innate immune response. Recent data strongly implicates leukotriene B4 in the pathogenesis of cardiovascular diseases, in particular arteriosclerosis, myocardial infarction and stroke. Here, we highlight the most salient features of leukotriene A4 hydrolase with emphasis on its biochemistry and structure biology.


Subject(s)
Epoxide Hydrolases/chemistry , Animals , Catalysis , Humans , Models, Biological , Molecular Structure
15.
Proteins ; 67(4): 1113-8, 2007 Jun 01.
Article in English | MEDLINE | ID: mdl-17357161

ABSTRACT

Leukotriene A4 hydrolase is a bifunctional zinc metalloenzyme with an epoxide hydrolase activity as well as an arginyl tri-peptidase activity. Detailed enzymological and mechanistic investigations of the latter activity have been hampered by the lack of a rapid and convenient enzyme assay. Here we have developed a new method allowing direct spectrophotometric assessment of the tri-peptide cleaving activity of leukotriene A4 hydrolase, as well as other peptidases. The method utilizes two competing substrates, one chromogenic reference substrate together with the tri-peptide substrate of interest, and relies on computer-assisted analysis of progress curves. The chromogenic reference substrate serves to disclose the "invisible" tri-peptide substrate for kinetic analysis. The method is fast and simple and will allow detailed kinetic studies and screening for natural peptide substrates of leukotriene A4 hydrolase as well as other members of the M1 family of aminopeptidases.


Subject(s)
Epoxide Hydrolases/analysis , Epoxide Hydrolases/metabolism , Aniline Compounds/metabolism , Arginine/metabolism , Kinetics , Peptides/metabolism , Software Design , Substrate Specificity , Time Factors
16.
J Biol Chem ; 280(39): 33477-86, 2005 Sep 30.
Article in English | MEDLINE | ID: mdl-16024909

ABSTRACT

Mammalian leukotriene A4 (LTA4) hydrolase is a bifunctional zinc metalloenzyme possessing an Arg/Ala aminopeptidase and an epoxide hydrolase activity, which converts LTA4 into the chemoattractant LTB4. We have previously cloned an LTA4 hydrolase from Saccharomyces cerevisiae with a primitive epoxide hydrolase activity and a Leu aminopeptidase activity, which is stimulated by LTA4. Here we used a modeled structure of S. cerevisiae LTA4 hydrolase, mutational analysis, and binding studies to show that Glu-316 and Arg-627 are critical for catalysis, allowing us to a propose a mechanism for the epoxide hydrolase activity. Guided by the structure, we engineered S. cerevisiae LTA4 hydrolase to attain catalytic properties resembling those of human LTA4 hydrolase. Thus, six consecutive point mutations gradually introduced a novel Arg aminopeptidase activity and caused the specific Ala and Pro aminopeptidase activities to increase 24 and 63 times, respectively. In contrast to the wild type enzyme, the hexuple mutant was inhibited by LTA4 for all tested substrates and to the same extent as for the human enzyme. In addition, these mutations improved binding of LTA4 and increased the relative formation of LTB4, whereas the turnover of this substrate was only weakly affected. Our results suggest that during evolution, the active site of an ancestral eukaryotic zinc aminopeptidase has been reshaped to accommodate lipid substrates while using already existing catalytic residues for a novel, gradually evolving, epoxide hydrolase activity. Moreover, the unique ability to catalyze LTB4 synthesis appears to be the result of multiple and subtle structural rearrangements at the catalytic center rather than a limited set of specific amino acid substitutions.


Subject(s)
Epoxide Hydrolases/genetics , Epoxide Hydrolases/metabolism , Evolution, Molecular , Models, Molecular , Saccharomyces cerevisiae/enzymology , Amino Acid Substitution , Arginine/chemistry , Arginine/metabolism , Binding Sites , Catalysis , DNA Mutational Analysis , Epoxide Hydrolases/chemistry , Epoxide Hydrolases/isolation & purification , Glutamine/chemistry , Glutamine/metabolism , Humans , Kinetics , Models, Biological , Mutagenesis, Site-Directed , Point Mutation , Protein Binding , Protein Engineering , Substrate Specificity , Surface Plasmon Resonance
17.
J Biol Chem ; 279(26): 27376-82, 2004 Jun 25.
Article in English | MEDLINE | ID: mdl-15078870

ABSTRACT

Leukotriene (LT) A(4) hydrolase is a bifunctional zinc metalloenzyme, which converts LTA(4) into the neutrophil chemoattractant LTB(4) and also exhibits an anion-dependent aminopeptidase activity. In the x-ray crystal structure of LTA(4) hydrolase, Arg(563) and Lys(565) are found at the entrance of the active center. Here we report that replacement of Arg(563), but not Lys(565), leads to complete abrogation of the epoxide hydrolase activity. However, mutations of Arg(563) do not seem to affect substrate binding strength, because values of K(i) for LTA(4) are almost identical for wild type and (R563K)LTA(4) hydrolase. These results are supported by the 2.3-A crystal structure of (R563A)LTA(4) hydrolase, which does not reveal structural changes that can explain the complete loss of enzyme function. For the aminopeptidase reaction, mutations of Arg(563) reduce the catalytic activity (V(max) = 0.3-20%), whereas mutations of Lys(565) have limited effect on catalysis (V(max) = 58-108%). However, in (K565A)- and (K565M)LTA(4) hydrolase, i.e. mutants lacking a positive charge, values of the Michaelis constant for alanine-p-nitroanilide increase significantly (K(m) = 480-640%). Together, our data indicate that Arg(563) plays an unexpected, critical role in the epoxide hydrolase reaction, presumably in the positioning of the carboxylate tail to ensure perfect substrate alignment along the catalytic elements of the active site. In the aminopeptidase reaction, Arg(563) and Lys(565) seem to cooperate to provide sufficient binding strength and productive alignment of the substrate. In conclusion, Arg(563) and Lys(565) possess distinct roles as carboxylate recognition sites for two chemically different substrates, each of which is turned over in separate enzymatic reactions catalyzed by LTA(4) hydrolase.


Subject(s)
Aminopeptidases/metabolism , Carboxylic Acids/metabolism , Epoxide Hydrolases/metabolism , Amino Acid Sequence , Arginine/genetics , Arginine/metabolism , Binding Sites , Catalysis , Crystallography, X-Ray , Enzyme Inhibitors/pharmacology , Epoxide Hydrolases/antagonists & inhibitors , Epoxide Hydrolases/chemistry , Epoxide Hydrolases/genetics , Escherichia coli/metabolism , Humans , Hydroxamic Acids/pharmacology , Leukotriene A4/pharmacology , Lysine/genetics , Lysine/metabolism , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Static Electricity
18.
Prostaglandins Other Lipid Mediat ; 68-69: 495-510, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12432939

ABSTRACT

The leukotrienes (LTs) are a family of lipid mediators involved in inflammation and allergy. Leukotriene B4 is a classical chemoattractant, which triggers adherence and aggregation of leukocytes to the endothelium at only nanomolar concentrations. In addition, leukotriene B4 modulates immune responses, participates in the host-defense against infections, and is a key mediator of PAF-induced lethal shock. Because of these powerful biological effects, leukotriene B4 is implicated in a variety of acute and chronic inflammatory diseases, e.g. nephritis, arthritis, dermatitis, and chronic obstructive pulmonary disease. The final step in the biosynthesis of leukotriene B4 is catalyzed by leukotriene A4 hydrolase, a unique bi-functional zinc metalloenzyme with an anion-dependent aminopeptidase activity. Here we describe the most recent developments regarding our understanding of the structure, function, and catalytic mechanisms of leukotriene A4 hydrolase.


Subject(s)
Epoxide Hydrolases/metabolism , Leukotrienes/metabolism , Amino Acids/genetics , Amino Acids/metabolism , Animals , Catalytic Domain , Enzyme Inhibitors/metabolism , Epoxide Hydrolases/chemistry , Epoxide Hydrolases/classification , Epoxide Hydrolases/genetics , Humans , Ligands , Molecular Structure , Phylogeny , Protein Binding , Protein Structure, Tertiary
19.
Proc Natl Acad Sci U S A ; 99(7): 4215-20, 2002 Apr 02.
Article in English | MEDLINE | ID: mdl-11917124

ABSTRACT

Leukotriene A4 (LTA4, 5S-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid) hydrolase (LTA4H)/aminopeptidase is a bifunctional zinc metalloenzyme that catalyzes the final and rate-limiting step in the biosynthesis of leukotriene B4 (LTB4, 5S,12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid), a classical chemoattractant and immune modulating lipid mediator. Two chemical features are key to the bioactivity of LTB4, namely, the chirality of the 12R-hydroxyl group and the cis-trans-trans geometry of the conjugated triene structure. From the crystal structure of LTA4H, a hydrophilic patch composed of Gln-134, Tyr-267, and Asp-375 was identified in a narrow and otherwise hydrophobic pocket, believed to bind LTA4. In addition, Asp-375 belongs to peptide K21, a previously characterized 21-residue active site-peptide to which LTA4 binds during suicide inactivation. In the present report we used site-directed mutagenesis and x-ray crystallography to show that Asp-375, but none of the other candidate residues, is specifically required for the epoxide hydrolase activity of LTA4H. Thus, mutation of Asp-375 leads to a selective loss of the enzyme's ability to generate LTB4 whereas the aminopeptidase activity is preserved. We propose that Asp-375, possibly assisted by Gln-134, acts as a critical determinant for the stereoselective introduction of the 12R-hydroxyl group and thus the biological activity of LTB4.


Subject(s)
Epoxide Hydrolases/chemistry , Leukotriene B4/biosynthesis , Amino Acid Sequence , Aspartic Acid , Catalytic Domain , Epoxide Hydrolases/physiology , Humans , Molecular Sequence Data , Mutation , Recombinant Proteins/isolation & purification , Structure-Activity Relationship
20.
J Biol Chem ; 277(2): 1398-404, 2002 Jan 11.
Article in English | MEDLINE | ID: mdl-11675384

ABSTRACT

Leukotriene A(4) hydrolase/aminopeptidase is a bifunctional zinc metalloenzyme that converts the fatty acid epoxide leukotriene A(4) into leukotriene B(4), a potent chemoattractant and immune-modulating lipid mediator. Recently, the structure of leukotriene A(4) hydrolase revealed that Glu-271, which belongs to a conserved GXMEN motif in the M1 family of zinc peptidases, and Gln-136 are located at the active site. Here we report that mutagenetic replacements of Glu-271, but not Gln-136, abrogate both catalytic activities of leukotriene A(4) hydrolase. Furthermore, the 2.1 A crystal structure of [E271Q]leukotriene A(4) hydrolase revealed minimal conformational changes that could not explain the loss of enzyme function. We propose that the carboxylate of Glu-271 participates in an acid-induced opening of the epoxide moiety of leukotriene A(4) and formation of a carbocation intermediate. Moreover, Glu-271 appears to act as an N-terminal recognition site and may potentially stabilize the transition-state during turnover of peptides, a property that most likely pertains to all members of the M1 family of zinc aminopeptidases. Hence, Glu-271 is a unique example of an amino acid, which has dual and separate functions in two different catalytic reactions, involving lipid and peptide substrates, respectively.


Subject(s)
Epoxide Hydrolases/metabolism , Epoxide Hydrolases/chemistry , Epoxide Hydrolases/genetics , Humans , Models, Molecular , Molecular Structure , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
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