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1.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 12): 2524-33, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24311593

ABSTRACT

The use of controlled dehydration for improvement of protein crystal diffraction quality is increasing in popularity, although there are still relatively few documented examples of success. A study has been carried out to establish whether controlled dehydration could be used to improve the anisotropy of crystals of the core protein of the human proteoglycan glypican-1. Crystals were subjected to controlled dehydration using the HC1 device. The optimal protocol for dehydration was developed by careful investigation of the following parameters: dehydration rate, final relative humidity and total incubation time Tinc. Of these, the most important was shown to be Tinc. After dehydration using the optimal protocol the crystals showed significantly reduced anisotropy and improved electron density, allowing the building of previously disordered parts of the structure.


Subject(s)
Crystallization/methods , Glypicans/chemistry , Water/chemistry , Anisotropy , Crystallography, X-Ray/methods , Electrons , Humans , Models, Molecular , Protein Conformation
2.
J Synchrotron Radiat ; 20(Pt 4): 648-53, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23765310

ABSTRACT

The macromolecular crystallography beamline I911-3, part of the Cassiopeia/I911 suite of beamlines, is based on a superconducting wiggler at the MAX II ring of the MAX IV Laboratory in Lund, Sweden. The beamline is energy-tunable within a range between 6 and 18 keV. I911-3 opened for users in 2005. In 2010-2011 the experimental station was completely rebuilt and refurbished such that it has become a state-of-the-art experimental station with better possibilities for rapid throughput, crystal screening and work with smaller samples. This paper describes the complete I911-3 beamline and how it is embedded in the Cassiopeia suite of beamlines.

3.
J Mol Biol ; 406(1): 120-34, 2011 Feb 11.
Article in English | MEDLINE | ID: mdl-21146536

ABSTRACT

Vertebrate leukotriene A(4) hydrolases are bifunctional zinc metalloenzymes with an epoxide hydrolase and an aminopeptidase activity. In contrast, highly homologous enzymes from lower organisms only have the aminopeptidase activity. From sequence comparisons, it is not clear why this difference occurs. In order to obtain more information on the evolutionary relationship between these enzymes and their activities, the structure of a closely related leucine aminopeptidase from Saccharomyces cerevisiae that only shows a very low epoxide hydrolase activity was determined. To investigate the molecular architecture of the active site, the structures of both the native protein and the protein in complex with the aminopeptidase inhibitor bestatin were solved. These structures show a more spacious active site, and the protected cavity in which the labile substrate leukotriene A(4) is bound in the human enzyme is partially obstructed and in other parts is more solvent accessible. Furthermore, the enzyme undergoes induced fit upon binding of the inhibitor bestatin, leading to a movement of the C-terminal domain. The main triggers for the domain movement are a conformational change of Tyr312 and a subtle change in backbone conformation of the PYGAMEN fingerprint region for peptide substrate recognition. This leads to a change in the hydrogen-bonding network pulling the C-terminal domain into a different position. Inasmuch as bestatin is a structural analogue of a leucyl dipeptide and may be regarded as a transition state mimic, our results imply that the enzyme undergoes induced fit during substrate binding and turnover.


Subject(s)
Epoxide Hydrolases/chemistry , Leucine/analogs & derivatives , Protease Inhibitors/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Conserved Sequence , Epoxide Hydrolases/antagonists & inhibitors , Humans , Leucine/chemistry , Molecular Sequence Data , Protein Binding , Protein Conformation , Sequence Homology, Amino Acid , Substrate Specificity
4.
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
5.
Arch Biochem Biophys ; 497(1-2): 21-7, 2010 May.
Article in English | MEDLINE | ID: mdl-20193655

ABSTRACT

The zinc finger transcription factor Wilms tumour protein (WT 1) is known for its essential involvement in the development of the genitourinary system as well as of other organs and tissues. WT 1 is capable of selectively binding either DNA or mRNA targets. A KTS insertion due to alternative splicing between the zinc fingers 3 and 4 and an unconventional zinc finger 1 are the unique features that distinguish WT 1 from classical DNA-binding C(2)H(2)-type zinc finger proteins. The DNA binding characteristics of WT 1 are well studied. Due to lack of information about its native RNA targets, no extensive research has been directed at how WT 1 binds RNA. Using surface plasmon resonance, this study attempts to understand the binding behaviour of WT 1 zinc fingers with its recently reported and first putative mRNA target, ACT 34, whose stem-loop structure is believed to be critical for the interactions with WT 1. We have analysed the interactions of five WT 1 zinc finger truncations with wild-type ACT 34 and four variants. Our results indicate that WT 1 zinc fingers bind ACT 34 in a specific manner, and that this occurs as interplay of all four zinc fingers. We also report that a sensitive kinetic balance, which is equilibrated by both zinc finger 1 and KTS, regulates the interaction with ACT 34. The stem-loop and the flanking nucleotides are important elements for specific recognition by WT 1 zinc fingers.


Subject(s)
Actinin/metabolism , RNA, Messenger/metabolism , Transcription Factors/metabolism , WT1 Proteins/metabolism , Kinetics , Protein Binding/genetics , Protein Structure, Tertiary/genetics , RNA/metabolism , Surface Plasmon Resonance , Transcription Factors/genetics , WT1 Proteins/genetics , Zinc Fingers/genetics
6.
Biophys Chem ; 145(2-3): 116-25, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19853363

ABSTRACT

Wilms Tumor suppressor protein (WT1) is a transcription factor that is involved in a variety of developmental functions during organ development. It is also implicated in the pathology of several different cancer forms. The protein contains four C(2)H(2)-type zinc fingers and it specifically binds GC-rich sequences in the promoter regions of its target genes, which are either up or down regulated. Two properties make WT1 a more unusual transcription factor - an unconventional amino acid composition for zinc finger 1, and the insertion of a tri-peptide KTS in some of the splice isoforms of WT1. Using six WT1 constructs in which zinc fingers are systematically deleted, a dual study based on a bacterial 1-hybrid system and surface plasmon resonance measurements is performed. The experiments show that the effect of zinc finger 1 is not significant in terms of overall DNA-binding kinetics, however it influences both the specificity of target recognition and stability of interaction in presence of KTS. The KTS insertion, however, only mildly retards binding affinity, mainly by affecting the on-rate. We suggest that the insertion disturbs zinc finger 4 from its binding frame, thus weakening the rate of target recognition. Finally, for the construct in which both zinc fingers 1 and 4 were deleted, the two middle fingers 2-3 still could function as a 'minimal DNA-recognition domain' for WT1, however the formation of a stable protein-DNA complex is impaired since the overall affinity was dramatically reduced mainly since the off-rate was severely affected.


Subject(s)
DNA/metabolism , WT1 Proteins/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Cloning, Molecular , Gene Deletion , Kinetics , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Surface Plasmon Resonance , WT1 Proteins/chemistry , WT1 Proteins/genetics , Zinc Fingers
7.
J Exp Med ; 206(2): 449-62, 2009 Feb 16.
Article in English | MEDLINE | ID: mdl-19204106

ABSTRACT

Antibodies to citrulline-modified proteins have a high diagnostic value in rheumatoid arthritis (RA). However, their biological role in disease development is still unclear. To obtain insight into this question, a panel of mouse monoclonal antibodies was generated against a major triple helical collagen type II (CII) epitope (position 359-369; ARGLTGRPGDA) with or without arginines modified by citrullination. These antibodies bind cartilage and synovial tissue, and mediate arthritis in mice. Detection of citrullinated CII from RA patients' synovial fluid demonstrates that cartilage-derived CII is indeed citrullinated in vivo. The structure determination of a Fab fragment of one of these antibodies in complex with a citrullinated peptide showed a surprising beta-turn conformation of the peptide and provided information on citrulline recognition. Based on these findings, we propose that autoimmunity to CII, leading to the production of antibodies specific for both native and citrullinated CII, is an important pathogenic factor in the development of RA.


Subject(s)
Antibodies, Monoclonal/genetics , Arthritis, Experimental/immunology , Autoimmunity/immunology , Citrulline/immunology , Collagen Type II/immunology , Models, Molecular , Animals , Autoimmunity/genetics , Base Sequence , Citrulline/metabolism , Collagen Type II/metabolism , Enzyme-Linked Immunosorbent Assay , Immunoglobulin Fab Fragments/genetics , Immunohistochemistry , Immunoprecipitation , Mice , Molecular Sequence Data , Protein Conformation , Sequence Analysis, DNA , Synovial Fluid/immunology
8.
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
9.
Mol Immunol ; 45(8): 2196-204, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18241923

ABSTRACT

Monoclonal anti-collagen type II antibody CIIC1 is an arthritogenic autoantibody, which induces arthritis in mice. We crystallized and solved the structure of CIIC1 Fab molecule. Analysis of structure revealed an interaction between the CDR regions of one Fab to the CH1 domain of another Fab, which resembles an antibody-antigen interaction. ELISA experiments confirmed the cross-reactivity of both the full CIIC1 antibody and a single chain Fv fragment to other anti-collagen antibodies which are of different isotypes and epitope specificity. The rheumatoid factor like reactivity of CIIC1 antibody together with its collagen type II specificity may explain the pathogenicity of this antibody.


Subject(s)
Antibodies, Monoclonal/chemistry , Autoantibodies/chemistry , Collagen Type II/chemistry , Immunoglobulin Fab Fragments/chemistry , Amino Acid Sequence , Animals , Complementarity Determining Regions/chemistry , Crystallography, X-Ray , Enzyme-Linked Immunosorbent Assay , Epitopes/chemistry , Hydrogen Bonding , Immunoglobulin G/chemistry , Immunoglobulin Variable Region/chemistry , Mice , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Sequence Alignment , Sequence Analysis, Protein , Static Electricity , Structure-Activity Relationship
10.
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
11.
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
12.
Acta Crystallogr D Biol Crystallogr ; 59(Pt 6): 1093-5, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12777785

ABSTRACT

The Saccharomyces cerevisiae leukotriene A4 (LTA4) hydrolase (scLTA4 hydrolase) has been crystallized in order to study the two activities of LTA4 hydrolase in an evolutionary perspective. Single well diffracting crystals are obtained after switching from the hanging-drop method to liquid-liquid diffusion in capillaries using PEG 8000 as precipitant. These crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.8, b = 98.1, c = 99.2 A. Intensity data to 2.3 A resolution were collected from a native scLTA4 hydrolase crystal using synchrotron radiation. A molecular-replacement solution was obtained using the human LTA4 hydrolase structure and the program BEAST.


Subject(s)
Epoxide Hydrolases/chemistry , Saccharomyces cerevisiae/enzymology , Crystallization , Crystallography, X-Ray , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Epoxide Hydrolases/genetics , X-Ray Diffraction
13.
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
14.
FASEB J ; 16(12): 1648-50, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12207002

ABSTRACT

Leukotriene (LT) A4 hydrolase/aminopeptidase is a bifunctional zinc enzyme that catalyzes the final step in the biosynthesis of LTB4, a potent chemoattractant and immune modulating lipid mediator. Here, we report a high-resolution crystal structure of LTA4 hydrolase in complex with captopril, a classical inhibitor of the zinc peptidase angiotensin-converting enzyme. Captopril makes few interactions with the protein, but its free thiol group is bound to the zinc, apparently accounting for most of its inhibitory action on LTA4 hydrolase. In addition, we have determined the structures of LTA4 hydrolase in complex with two selective tight-binding inhibitors, a thioamine and a hydroxamic acid. Their common benzyloxyphenyl tail, designed to mimic the carbon backbone of LTA4, binds into a narrow hydrophobic cavity in the protein. The free hydroxyl group of the hydroxamic acid makes a suboptimal, monodentate complex with the zinc, and strategies for improved inhibitor design can be deduced from the structure. Taken together, the three crystal structures provide the molecular basis for the divergent pharmacological profiles of LTA4 hydrolase inhibitors. Moreover, they help define the binding pocket for the fatty acid-derived epoxide LTA4 as well as the subsites for a tripeptide substrate, which in turn have important implications for the molecular mechanisms of enzyme catalyses.


Subject(s)
Captopril/metabolism , Enzyme Inhibitors/metabolism , Epoxide Hydrolases/chemistry , Amines/chemistry , Amines/metabolism , Binding Sites , Binding, Competitive , Captopril/chemistry , Catalysis , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Epoxide Hydrolases/metabolism , Hydroxamic Acids/chemistry , Hydroxamic Acids/metabolism , Models, Molecular , Molecular Structure , Protein Binding , Protein Conformation , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/metabolism , Zinc/chemistry , Zinc/metabolism
15.
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
16.
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
17.
ScientificWorldJournal ; 2: 1734-49, 2002 Jun 26.
Article in English | MEDLINE | ID: mdl-12806167

ABSTRACT

The leukotrienes 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 nM 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 bifunctional zinc metalloenzyme with an anion-dependent aminopeptidase activity. Here we describe the most recent developments regarding our understanding of the function and molecular architecture of leukotriene A4 hydrolase.


Subject(s)
Chemotactic Factors/biosynthesis , Epoxide Hydrolases/chemistry , Epoxide Hydrolases/physiology , Leukotrienes/biosynthesis , Catalysis , Models, Molecular , Protein Structure, Quaternary/physiology , Structure-Activity Relationship
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