Defining the arachidonic acid binding site of human 15-lipoxygenase. Molecular modeling and mutagenesis.
Mammalian lipoxygenases have been implicated in the pathogenesis of several inflammatory disorders and are, therefore, important targets for drug discovery. Both plant and mammalian lipoxygenases catalyze the dioxygenation of polyunsaturated fatty acids, which contain one or more 1,4-cis,cis-pentadiene units to yield hydroperoxide products. At the time this study was initiated, soybean lipoxygenase-1 was the only lipoxygenase for which an atomic resolution structure had been determined. No structure of lipoxygenase with substrate or inhibitor bound is currently available. A model of arachidonic acid docked into the proposed substrate binding site in the soybean structure is presented here. Analysis of this model suggested two residues, an aromatic residue and a positively charged residue, could be critical for substrate binding. Validation of this model is provided by site-directed mutagenesis of human 15-lipoxygenase, despite the low amino acid sequence identity between the soybean and mammalian enzymes. Both a positively charged amino acid residue (Arg402) and an aromatic amino acid residue (Phe414) are identified as critical for the binding of fatty acid substrates in human 15-lipoxygenase. Thus, binding determinants shown to be characteristic of non-enzymatic fatty acid-binding proteins are now implicated in the substrate binding pocket of lipoxygenases.
Gan
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QF
Roche Bioscience, Palo Alto, California 94304, USA.
Browner
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MF
Sloane
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DL
Sigal
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E
D000595:Amino Acid Sequence; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000737:chemistry; D001093:Arachidonate 15-Lipoxygenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism*; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D008322:Mammals; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000201:enzymology
D011994:Recombinant Proteins; D016718:Arachidonic Acid; D010649:Phenylalanine; D001120:Arginine; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase