Sequence Determinants for the Positional Specificity of Mammalian and Plant Lipoxygenases

Abstract

Lipoxygenases (LO’s), which catalyze the conversion of polyunsaturated fatty acids to their corresponding hydroperoxy derivatives are widely distributed in plants and animals. They contain one gram-atom non-heme iron per mole enzyme and this iron appears to be involved in the rate limiting step of the oxygenase reaction, the initial hydrogen removal. In mammalian cells, mainly linoleic acid and arachidonic acid serve as substrates for the LO reaction whereas in plants linolenic acid may constitute the major LO substrate. According to the currently used nomenclature, LO’s are categorized with respect to their positional specificity of arachidonic acid oxygenation into 5-, 8-, 11-, 12- and 15-LO’s. Several studies have been carried out in the past on the structural reasons for the positional specificity of LO’s. Comparing the sequences of various mammalian 12- and 15-LO’s, Sloane and colleagues identified conserved differences in the primary structure of these enzymes. When the amino acids ile418 and met419 of the human 15-LO were mutated to their counterparts present in the human platelet-type 12-LO (ala and val), this enzyme was converted to a 12-lipoxygenating species (Sloane et al., 1991). It was hypothesized that these residues form the bottom of the substrate binding pocket so that their bulkiness and the geometry of their side-chains may determine the substrate alignment at the active site. Later on, these findings were confirmed for the human platelet 12-LO (Chen and Funk, 1993) and for the porcine leukocyte-type 12-LO (Suzuki et al., 1994). However, similar attempts to alter the positional specificity of the rat leukocyte-type 12-LO failed (Watanabe and Haeggstrom, 1993; Hada et al., 1994) and the authors suggested that additional “structural features” may be of importance for the positional specificity of these LO isoforms. To obtain more detailed information on these “structural features”, we constructed chimeric LO species combining different parts of the cDNA of various LO isoforms and identified amino acid 353 as sequence determinant for the positional specificity of 12- and 15-LO’s (Borngräber et al., 1996). These data were used to establish a structural model for the alignment of substrate fatty acids at the active site of mammalian 12- and 15-LO’s.