RESUMO
Lipoxygenases are non-heme iron containing enzymes that catalyze oxygenation of poly-unsaturated fatty acids in different animal and plant species with extremely high regio- and stereospecificity. Nature employs 8-lipoxygenase to produce 8R-hydroperoxide from the oxygenation of arachidonic acid. A single-point L434F mutation of 8-lipoxygenase alters the regio- and stereospecificity of the final products, with a product ratio of 66 : 34 for 8R- and 12S-hydroperoxide, respectively. A molecular level explanation of this flipped regiospecificity is presented in this work on the basis of molecular dynamics simulations and transition network analysis of oxygen migration in the protein matrix. Phe434 is shown to exist in two conformations, the so-called open and closed conformations. In the closed conformation, the phenyl group of Phe434 shields the C8 site of the substrate, thereby preventing access of the oxygen molecule to this site, which leads to a quenching of the 8R-product. On the other hand, both closed and open conformations of Phe434 allow the oxygen molecule to approach the pro-S face of the C12 site of the substrate, which enhances the propensity of the 12S-hydroperoxide.
Assuntos
Araquidonato Lipoxigenases/genética , Animais , Araquidonato Lipoxigenases/química , Araquidonato Lipoxigenases/metabolismo , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Mutação , Conformação ProteicaRESUMO
Lipoxygenases (lox's) are a group of non-heme iron containing enzymes that catalyze oxygenation of polyunsaturated fatty acids with precise regio- and stereoselectivities. The origin of regio- and stereospecific catalysis by 8-lox is explored in its wild-type (wt) form and in three mutants (Arg185Ala, Ala592Met, and Ala623His). The catalytic action of this enzyme progresses in two steps, namely, hydrogen abstraction from one double allylic carbon atom of substrate followed by oxygen insertion at the resulting prochiral carbon radical of the substrate. It is shown that the positional specificity of the hydrogen abstraction is a result of conformational dynamics of the bound substrate. While the C10 atom of the substrate is found to be the most probable site of hydrogen abstraction in the wt-lox, hydrogen abstraction from C13 is more favorable in the mutants. The present study discovers the presence of an interconnected network of a three-channel migration pathway operating in the protein matrix for efficient oxygen transport. Each migration channel is bestowed with a pocket at the peripheral region of protein as an oxygen access site, which transfers the oxygen to the active site through a well-connected migration path on a time scale of a few hundred picoseconds. By a careful geometric analysis of the oxygen pockets near the substrate binding cleft, the present study identifies the launching sites for oxygenation at the prochiral carbon centers C8, C11, C12, and C15 and the stereochemistry (R/S) of the corresponding products. It is found that the dominating 8R product in the wt-lox is due to the presence of the aromatic ring pair of Tyr181 and Phe173 acting as a gatekeeper for efficient delivery of oxygen at the pro-R face of C8. The change in the stereochemistry of the products in mutants is explained in terms of dynamic interactions between substrate and the surrounding residues.
Assuntos
Araquidonato Lipoxigenases/metabolismo , Biocatálise , Simulação de Dinâmica Molecular , Araquidonato Lipoxigenases/química , Araquidonato Lipoxigenases/genética , Mutação , Conformação Proteica , Estereoisomerismo , Especificidade por SubstratoRESUMO
Methods are presented for the use of the coral 8R-lipoxygenase from the Caribbean sea whip coral Plexaura homomalla as a model enzyme for structural studies of animal lipoxygenases. The 8R-lipoxygenase is remarkably stable and can be stored at 4°C for 3 months with virtually no loss of activity. In addition, an engineered "pseudo wild-type" enzyme is soluble in the absence of detergents, which helps facilitate the preparation of enzyme:substrate complexes.
Assuntos
Antozoários/metabolismo , Araquidonato Lipoxigenases/isolamento & purificação , Ácido Araquidônico/metabolismo , Ensaios Enzimáticos/métodos , Domínios Proteicos/genética , Animais , Araquidonato Lipoxigenases/química , Araquidonato Lipoxigenases/genética , Araquidonato Lipoxigenases/metabolismo , Ácido Araquidônico/química , Sítios de Ligação/genética , Cristalografia por Raios X/métodos , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato/genéticaRESUMO
In this chapter, we discuss the influence of an anisotropic protein environment on the reaction mechanisms of saccharopine reductase and uroporphyrinogen decarboxylase, respectively, via the use of a quantum mechanical and molecular mechanical (QM/MM) approach. In addition, we discuss the importance of selecting a suitable DFT functional to be used in a QM/MM study of a key intermediate in the mechanism of 8R-lipoxygenase, a nonheme iron enzyme. In the case of saccharopine reductase, while the enzyme utilizes a substrate-assisted catalytic pathway, it was found that only through treating the polarizing effect of the active site, via the use of an electronic embedding formalism, was agreement with experimental kinetic data obtained. Similarly, in the case of uroporphyrinogen decarboxylase, the effect of the protein environment on the catalytic mechanism was found to be such that the calculated rate-limiting barrier is in good agreement with related experimentally determined values for the first decarboxylation of the substrate. For 8R-lipoxygenase, it was found that the geometries and energies of the multicentered open-shell intermediate complexes formed during the mechanism are quite sensitive to the choice of the density functional theory method. Thus, while density functional theory has become the method of choice in QM/MM studies, care must be taken in the selection of a particular high-level method.
Assuntos
Araquidonato Lipoxigenases/química , Simulação de Dinâmica Molecular , Sacaropina Desidrogenases/química , Uroporfirinogênio Descarboxilase/química , Animais , Anisotropia , Antozoários/química , Antozoários/enzimologia , Domínio Catalítico , Humanos , Cinética , Teoria Quântica , Especificidade por Substrato , TermodinâmicaRESUMO
Lipoxygenases (LOX) play critical roles in mammalian biology in the generation of potent lipid mediators of the inflammatory response; consequently, they are targets for the development of isoform-specific inhibitors. The regio- and stereo-specificity of the oxygenation of polyunsaturated fatty acids by the enzymes is understood in terms of the chemistry, but structural observation of the enzyme-substrate interactions is lacking. Although several LOX crystal structures are available, heretofore the rapid oxygenation of bound substrate has precluded capture of the enzyme-substrate complex, leaving a gap between chemical and structural insights. In this report, we describe the 2.0 Å resolution structure of 8R-LOX in complex with arachidonic acid obtained under anaerobic conditions. Subtle rearrangements, primarily in the side chains of three amino acids, allow binding of arachidonic acid in a catalytically competent conformation. Accompanying experimental work supports a model in which both substrate tethering and cavity depth contribute to positioning the appropriate carbon at the catalytic machinery.
Assuntos
Araquidonato Lipoxigenases/química , Animais , Ácido Araquidônico/química , Sítios de Ligação , Catálise , Cristalografia por Raios X , Humanos , Inflamação , Ferro/química , Lipídeos/química , Modelos Moleculares , Mutagênese , Mutação , Oxigênio/química , Ligação Proteica , Conformação Proteica , Coelhos , SuínosRESUMO
Lipoxygenases (LOXs) are ubiquitous in nature and catalyze a range of life-essential reactions within organisms. In particular they are critical to the formation of eicosanoids, which are critical for normal cell function. However, a number of important questions about the reactivity and mechanism of these enzymes still remain. Specifically, although the initial step in the mechanism of LOXs has been well studied, little is known of subsequent steps. Thus, with use of a quantum mechanical/molecular mechanical approach, the complete catalytic mechanism of (8R)-LOX was investigated. The results have provided a better understanding of the general chemistry of LOXs as a whole. In particular, from comparisons with soybean LOX-1, it appears that the initial proton-coupled electron transfer may be very similar among all LOXs. Furthermore, LOXs appear to undergo multistate reactivity where potential spin inversion of an electron may occur either in the attack of O(2) or in the regeneration of the active site. Lastly, it is shown that with the explicit modeling of the environment, the regeneration of the active center likely occurs via the rotation of the intermediate followed by an outer-sphere [Formula: see text] transfer as opposed to the formation of a "purple intermediate" complex.
Assuntos
Antozoários/enzimologia , Araquidonato Lipoxigenases/química , Araquidonato Lipoxigenases/metabolismo , Animais , Antozoários/química , Domínio Catalítico , Ativação Enzimática , Lipoxigenase/química , Lipoxigenase/metabolismo , Simulação de Acoplamento Molecular , Peróxidos/química , Peróxidos/metabolismo , Conformação Proteica , Teoria Quântica , Glycine max/enzimologiaRESUMO
Lipoxygenases (LOXs) contain a hydrophobic substrate channel with the conserved Gly/Ala determinant of regio- and stereospecificity and a conserved Leu residue near the catalytic non-heme iron. Our goal was to study the importance of this region (Gly(332), Leu(336), and Phe(337)) of a lipoxygenase with catalytic manganese (13R-MnLOX). Recombinant 13R-MnLOX oxidizes 18:2n-6 and 18:3n-3 to 13R-, 11(S or R)-, and 9S-hydroperoxy metabolites (â¼80-85, 15-20, and 2-3%, respectively) by suprafacial hydrogen abstraction and oxygenation. Replacement of Phe(337) with Ile changed the stereochemistry of the 13-hydroperoxy metabolites of 18:2n-6 and 18:3n-3 (from â¼100% R to 69-74% S) with little effect on regiospecificity. The abstraction of the pro-S hydrogen of 18:2n-6 was retained, suggesting antarafacial hydrogen abstraction and oxygenation. Replacement of Leu(336) with smaller hydrophobic residues (Val, Ala, and Gly) shifted the oxygenation from C-13 toward C-9 with formation of 9S- and 9R-hydroperoxy metabolites of 18:2n-6 and 18:3n-3. Replacement of Gly(332) and Leu(336) with larger hydrophobic residues (G332A and L336F) selectively augmented dehydration of 13R-hydroperoxyoctadeca-9Z,11E,15Z-trienoic acid and increased the oxidation at C-13 of 18:1n-6. We conclude that hydrophobic replacements of Leu(336) can modify the hydroperoxide configurations at C-9 with little effect on the R configuration at C-13 of the 18:2n-6 and 18:3n-3 metabolites. Replacement of Phe(337) with Ile changed the stereospecific oxidation of 18:2n-6 and 18:3n-3 with formation of 13S-hydroperoxides by hydrogen abstraction and oxygenation in analogy with soybean LOX-1.
Assuntos
Aminoácidos/química , Ferro/química , Lipoxigenase/química , Lipoxigenase/genética , Manganês/química , Sequência de Aminoácidos , Animais , Antozoários , Araquidonato Lipoxigenases/química , Catálise , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Hidrogênio/química , Cinética , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Oxigênio/química , Pichia/metabolismo , Homologia de Sequência de Aminoácidos , Glycine max/enzimologiaRESUMO
Lipoxygenases (LOXs) are a key part of several signaling pathways that lead to inflammation and cancer. Yet, the mechanisms of substrate binding and allosteric regulation by the various LOX isoforms remain speculative. Here we report the 2.47-Å resolution crystal structure of the arachidonate 11R-LOX from Gersemia fruticosa, which sheds new light on the mechanism of LOX catalysis. Our crystallographic and mutational studies suggest that the aliphatic tail of the fatty acid is bound in a hydrophobic pocket with two potential entrances. We speculate that LOXs share a common T-shaped substrate channel architecture that gives rise to the varying positional specificities. A general allosteric mechanism is proposed for transmitting the activity-inducing effect of calcium binding from the membrane-targeting PLAT (polycystin-1/lipoxygenase/α-toxin) domain to the active site via a conserved π-cation bridge.
Assuntos
Araquidonato Lipoxigenases/química , Cálcio/metabolismo , Regulação da Expressão Gênica , Sítio Alostérico , Animais , Antozoários , Proteínas de Ligação ao Cálcio/metabolismo , Domínio Catalítico , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Cristalografia por Raios X/métodos , Dimerização , Eicosanoides/química , Humanos , Cinética , Lipossomos/metabolismo , Espectrometria de Massas/métodos , Modelos Químicos , Conformação Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Lipoxygenases (LOX) play pivotal roles in the biosynthesis of leukotrienes and other biologically active potent signalling compounds. Developing inhibitors for LOX is of high interest to researchers. Modelling the interactions between LOX and its substrate arachidonic acid is critical for developing LOX specific inhibitors. Currently, there are no LOX-substrate structures. Recently, the structure of a coral LOX, 8R-LOX, which is 41% sequence identical to the human 5-LOX was solved to 1.85Å resolution. This structure provides a foundation for modelling enzyme-substrate interactions. METHODS: In this research, we applied a computational method, Internal Coordinate Mechanics (ICM), to model the interactions between 8R-LOX and its substrate arachidonic acid. Docking arachidonic acid to 8R-LOX was performed. The most favoured docked ligand conformations were retained. We compared the results of our simulation with a proposed model and concluded that the binding pocket identified in this study agrees with the proposed model partially. RESULTS: The results showed that the conformation of arachidonic acid docked into the ICM-identified docking site has less energy than that docked into the manually defined docking site for pseudo wild type 8R-LOX. The mutation at I805 resulted in no docking pocket found near Fe atom. The energy of the arachidonic acid conformation docked into the manually defined docking site is higher in mutant 8R-LOX than in wild type 8R-LOX. The arachidonic acid conformations are not productive conformations. CONCLUSIONS: We concluded that, for the wild type 8R-LOX, the conformation of arachidonic acid docked into the ICM-identified docking site is more stable than that docked into the manually defined docking site. Mutation affects the structure of the putative active site pocket of 8R-LOX, and leads no docking pockets around the catalytic Fe atom. The docking simulation in a mutant 8R-LOX demonstrated that the structural change due to the mutation impacts the enzyme activity. Further research and analysis is required to obtain the 8R-LOX-substrate model.
Assuntos
Araquidonato Lipoxigenases/química , Biologia Computacional/métodos , Modelos Moleculares , Ácido Araquidônico/química , Ácido Araquidônico/metabolismo , Sítios de Ligação , Mutação , Especificidade por SubstratoRESUMO
The nomenclature of lipoxygenases (LOXs) is partly based on the positional specificity of arachidonic acid oxygenation, but there is no unifying concept explaining the mechanistic basis of this enzyme property. According to the triad model, Phe-353, Ile-418, and Ile-593 of the rabbit 12/15-LOX form the bottom of the substrate-binding pocket, and introduction of less space-filling residues at either of these positions favors arachidonic acid 12-lipoxygenation. The present study was aimed at exploring the validity of the triad concept for two novel primate 12/15-LOX (Macaca mulatta and Pongo pygmaeus) and for five known members of the mammalian LOX family (human 12/15-LOX, mouse 12/15-LOX, human 15-LOX2, human platelet type 12-LOX, and mouse (12R)-LOX). The enzymes were expressed as N-terminal His tag fusion proteins in E. coli, the potential sequence determinants were mutated, and the specificity of arachidonic acid oxygenation was quantified. Taken together, our data indicate that the triad concept explains the positional specificity of all 12/15-LOXs tested (rabbit, human, M. mulatta, P. pygmaeus, and mouse). For the new enzymes of M. mulatta and P. pygmaeus, the concept had predictive value because the positional specificity predicted on the basis of the amino acid sequence was confirmed experimentally. The specificity of the platelet 12-LOX was partly explained by the triad hypothesis, but the concept was not applicable for 15-LOX2 and (12R)-LOX.
Assuntos
Araquidonato Lipoxigenases/química , Ácido Araquidônico/química , Modelos Moleculares , Animais , Araquidonato Lipoxigenases/classificação , Araquidonato Lipoxigenases/genética , Araquidonato Lipoxigenases/metabolismo , Ácido Araquidônico/metabolismo , Humanos , Macaca mulatta , Camundongos , Oxirredução , Pongo pygmaeus , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/classificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato/fisiologiaRESUMO
Lipoxygenases have been classified according to their specificity of fatty acid oxygenation and for several plant enzymes pH-dependent alterations in the product patterns have been reported. Assuming that the biological role of mammalian lipoxygenases is based on the formation of specific reaction products, pH-dependent alterations would impact enzymes' functionality. In this study we systematically investigated the pH-dependence of vertebrate lipoxygenases and observed a remarkable stability of the product pattern in the near physiological range for the wild-type enzyme species. Site-directed mutagenesis of selected amino acids and alterations in the substrate concentrations induced a more pronounced pH-dependence of the reaction specificity. For instance, for the V603H mutant of the human 15-lipoxygenase-2 8-lipoxygenation was dominant at acidic pH (65%) whereas 15-H(p)ETE was the major oxygenation product at pH 8. Similarly, the product pattern of the wild-type mouse 8-lipoxygenase was hardly altered in the near physiological pH range but H604F exchange induced strong pH-dependent alterations in the positional specificity. Taken together, our data suggest that the reaction specificities of wild-type vertebrate lipoxygenase isoforms are largely resistant towards pH alterations. However, we found that changes in the assay conditions (low substrate concentration) and introduction/removal of a critical histidine at the active site impact the pH-dependence of reaction specificity for some lipoxygenase isoforms.
Assuntos
Araquidonato 15-Lipoxigenase/química , Araquidonato 15-Lipoxigenase/metabolismo , Araquidonato Lipoxigenases/química , Araquidonato Lipoxigenases/metabolismo , Animais , Biocatálise , Humanos , Concentração de Íons de Hidrogênio , Isoenzimas , Camundongos , Modelos Biológicos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxigênio/metabolismo , Especificidade por SubstratoRESUMO
Lipoxygenases (LOXs) catalyze the regio- and stereospecific dioxygenation of polyunsaturated membrane-embedded fatty acids. We report here the 3.2 A resolution structure of 8R-LOX from the Caribbean sea whip coral Plexaura homomalla, a LOX isozyme with calcium dependence and the uncommon R chiral stereospecificity. Structural and spectroscopic analyses demonstrated calcium binding in a C2-like membrane-binding domain, illuminating the function of similar amino acids in calcium-activated mammalian 5-LOX, the key enzyme in the pathway to the pro-inflammatory leukotrienes. Mutation of Ca(2+)-ligating amino acids in 8R-LOX resulted not only in a diminished capacity to bind membranes, as monitored by fluorescence resonance energy transfer, but also in an associated loss of Ca(2+)-regulated enzyme activity. Moreover, a structural basis for R chiral specificity is also revealed; creation of a small oxygen pocket next to Gly(428) (Ala in all S-LOX isozymes) promoted C-8 oxygenation with R chirality on the activated fatty acid substrate.
Assuntos
Antozoários/enzimologia , Araquidonato Lipoxigenases/química , Sequência de Aminoácidos , Animais , Antozoários/genética , Araquidonato Lipoxigenases/genética , Araquidonato Lipoxigenases/metabolismo , Sítios de Ligação/genética , Cálcio/metabolismo , Domínio Catalítico/genética , Sequência Conservada , Cristalografia por Raios X , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Estereoisomerismo , Triptofano/químicaRESUMO
The recently identified mouse 8(S)-lipoxygenase almost exclusively directs oxygen insertion into the 8(S) position of arachidonic acid and, with lower efficiency, into the 9(S) position of linoleic acid. The protein of 677 amino acids displays 78% sequence identity to human 15(S)-lipoxygenase-2 which is considered to be its human orthologue. The 8(S)-lipoxygenase gene, Alox15b, consisting of 14 exons and spanning 14.5 kb is located within a gene cluster of related epidermis-type lipoxygenases at the central region of mouse chromosome 11. 8(S)-Lipoxygenase is predominantly expressed in stratifying epithelia of mice, constitutively in the hair follicle, forestomach, and foot-sole and inducible in the back skin with strain-dependent variations. The expression is restricted to terminally differentiating keratinocytes, in particular the stratum granulosum and 8(S)-lipoxygenase activity seems to be involved in terminal differentiation of mouse epidermis. Tumor-specific up-regulation of 8(S)-lipoxygenase expression and activity indicate a critical role of this enzyme in malignant progression during tumor development in mouse skin.
Assuntos
Araquidonato Lipoxigenases/metabolismo , Animais , Araquidonato Lipoxigenases/química , Araquidonato Lipoxigenases/classificação , Araquidonato Lipoxigenases/genética , Diferenciação Celular/fisiologia , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Queratinócitos/fisiologia , Neoplasias/metabolismo , Filogenia , Especificidade por Substrato , Distribuição TecidualRESUMO
The reported crystal structures of plant and animal lipoxygenases (LOX) show that the nonheme iron in the catalytic domain is ligated by three histidines, the C-terminal isoleucine, and in certain structures also by a fifth iron ligand, an asparagine or histidine residue. Mouse 8-LOX and its homologues (e.g., human 15-LOX-2) are unique in having a serine in place of the usual Asn or His in this fifth position. To investigate the importance of the residue in mouse 8-LOX structure-function, the serine-558 was replaced by asparagine, histidine, or alanine using oligonucleotide-directed mutagenesis. Wild-type mouse 8-LOX and the mutant cDNAs were expressed in HeLa cells infected with vaccinia virus encoding T7 RNA polymerase and their relative lipoxygenase activities assessed by incubation with [14C]arachidonic acid or [14C]linoleic acid followed by HPLC analysis of the products. The Ser558Asn and Ser558His mutants had equivalent or greater activity than wild-type 8-LOX. They also exhibited some 15-LOX activity, indicating that small structural perturbations (in this case to a residue identical in mouse 8-LOX and its 15-LOX-2 homologues) can interchange the positional specificity of these closely related enzymes. Remarkably, the Ser558Ala mutant exhibited significant 8-LOX activity, indicating that this position is not an essential iron ligand in the enzyme. We conclude that mouse 8-LOX is catalytically competent with only four amino acid iron ligands, and that Ser-558 of the wild-type enzyme does not play an essential role in catalysis.
Assuntos
Substituição de Aminoácidos/genética , Araquidonato Lipoxigenases/química , Araquidonato Lipoxigenases/metabolismo , Ferro/metabolismo , Ácidos Linoleicos Conjugados , Mutagênese Sítio-Dirigida/genética , Sequência de Aminoácidos , Animais , Araquidonato Lipoxigenases/genética , Sequência de Bases , Western Blotting , Catálise , Cromatografia Líquida de Alta Pressão , Sequência Conservada/genética , Células HeLa , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ligantes , Ácidos Linoleicos/metabolismo , Camundongos , Relação Estrutura-AtividadeRESUMO
The positional specificity of arachidonic acid oxygenation is currently the decisive parameter for classification of mammalian lipoxygenases but, unfortunately, the structural reasons for lipoxygenase specificity are not well understood. Although there are no direct structural data on lipoxygenase/substrate interaction, experiments with modified fatty acid substrates and mutagenesis studies suggest that for 12- and 15-lipoxygenases, arachidonic acid slides into the substrate-binding pocket with its methyl end ahead. For arachidonate 5- and/or 8-lipoxygenation two alternative models for the enzyme/substrate interaction have been developed: 1) The orientation-determined model and 2) the space-determined model. This review explores the experimental data available on the mechanistic reasons for lipoxygenase specificity and concludes that each of the above-mentioned hypotheses may be valid for arachidonate 5-lipoxygenation under certain circumstances.
Assuntos
Araquidonato Lipoxigenases/química , Ácidos Graxos/química , Lipoxigenase/química , Animais , Araquidonato Lipoxigenases/metabolismo , Sítios de Ligação , Hemeproteínas , Modelos Moleculares , Conformação Molecular , Especificidade por SubstratoRESUMO
Phorbol ester-inducible mouse 8S-lipoxygenase (8-LOX) and its human homologue, 15S-lipoxygenase-2 (15-LOX-2), share 78% identity in amino acid sequences, yet there is no overlap in their positional specificities. In this study, we investigated the determinants of positional specificity using a random chimeragenesis approach in combination with site-directed mutagenesis. Exchange of the C-terminal one-third of the 8-LOX with the corresponding portion of 15-LOX-2 yielded a chimeric enzyme with exclusively 15S-lipoxygenase activity. The critical region was narrowed down to a cluster of five amino acids by expression of multiple cDNAs obtained by in situ chimeragenesis in Escherichia coli. Finally, a pair of amino acids, Tyr(603) and His(604), was identified as the positional determinant by site-directed mutagenesis. Mutation of both of these amino acids to the corresponding amino acids in 15-LOX-2 (Asp and Val, respectively) converted the positional specificity from 8S to 90% 15S without yielding any other by-products. Mutation of the corresponding residues in 15-LOX-2 to the 8-LOX sequence changed specificity to 50% oxygenation at C-8 for one amino acid substitution and 70% at C-8 for the double mutant. Based on the crystal structure of the reticulocyte 15-LOX, these two amino acids lie opposite the open coordination position of the catalytic iron in a likely site for substrate binding. The change from 8 to 15 specificity entails a switch in the head to tail binding of substrate. Enzymes that react with substrate "head first" (5-LOX and 8-LOX) have a bulky aromatic amino acid and a histidine in these positions, whereas lipoxygenases that accept substrates "tail first" (12-LOX and 15-LOX) have an aliphatic residue with a glutamine or aspartate. Thus, this positional determinant of the 8-LOX and 15-LOX-2 may have significance for other lipoxygenases.
Assuntos
Araquidonato 15-Lipoxigenase/química , Araquidonato 15-Lipoxigenase/metabolismo , Araquidonato Lipoxigenases/química , Araquidonato Lipoxigenases/metabolismo , Sequência de Aminoácidos , Animais , Araquidonato 15-Lipoxigenase/genética , Araquidonato Lipoxigenases/genética , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , Primers do DNA , Escherichia coli , Humanos , Mamíferos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Using a combination of PCR cloning and conventional screening procedures, we isolated from phorbol ester-treated mouse epidermis two full length cDNA clones encoding novel lipoxygenases. One of the cDNAs turned out to be identical to the recently cloned 8-lipoxygenase [Jisaka et al., J. Biol. Chem. 272 (1997) 24 410-24 416], the open reading frame of the second one corresponded to a protein of 701 amino acids with a calculated molecular mass of 80.6 kDa. The amino acid sequence showed 50.8% identity to human 15-lipoxygenase 2, approximately 40% to 5-lipoxygenase and 35% to 12- and 15-lipoxygenases. A unique structural feature is the insertion of 31 amino acid residues in the amino-terminal part of the molecule. Based on these data, we conclude that this epidermis-derived cDNA encodes a novel lipoxygenase isoform termed provisionally epidermis-type lipoxygenase 2 (e-LOX 2).