Abstract
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.
Highlights
Lipoxygenases (LOXs)2 catalyze the stereo-specific peroxidation of free and/or esterified polyunsaturated fatty acids to corresponding hydroperoxy compounds and contribute to lipid signaling in mammalian cells [1, 2]
Human 12/15-LOX oxygenates arachidonic acid to a mixture of (12S)- and (15S)-H(p)ETE [30], and we confirmed this finding for the pure His tag fusion protein (Fig. 2)
On the basis of the triad concept, a similar reaction specificity was predicted for the P. pygmaeus LOX because the positions of the three sequence determinants were occupied by amino acids with rather bulky side chains
Summary
Lipoxygenases (LOXs) catalyze the stereo-specific peroxidation of free and/or esterified polyunsaturated fatty acids to corresponding hydroperoxy compounds and contribute to lipid signaling in mammalian cells [1, 2]. Similar results have since been obtained for the rabbit ortholog [19], but corresponding mutations on the rat enzyme were not consistent with these results [23] These data and additional mutagenesis studies on various 12/15-LOX isoforms prompted the development of the “triad concept” explaining the reaction specificity of the rabbit 12/15-LOX [18, 19]. This hypothesis emphasized that a triad of amino acids (Phe-353, Ile-418, and Ile-593) is important for the positional specificity. The Escherichia coli strain XL-1 Blue was purchased from Stratagene (La Jolla, CA)
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