Structure-function properties of human platelet 12-lipoxygenase: chimeric enzyme and in vitro mutagenesis studies.

Mutant and chimeric lipoxygenases were expressed in human embryonal kidney 293 cells to assess the importance of amino acids and domains for catalytic activity and positional specificity of molecular oxygen insertion. Histidines 360, 365, and 540, when changed to glutamine residues, completely abolished human platelet 12-lipoxygenase activity. Altered histidines at positions 355, 383, and 392 retained enzymatic activity. The former three histidines could possibly serve as ligands for the catalytically essential non-heme iron atom. Amino acids adjacent (residues 398-417) to the five centrally located histidines conserved among all plant and animal lipoxygenases controlled to a limited extent the positional specificity of oxygenation of 12-lipoxygenase. Variant A417I and the triple variant K416Q/A417I/V418M, designed to introduce 15-lipoxygenase substitutions, transformed the platelet 12-lipoxygenase that synthesizes exclusively 12-hydro(pero)xy-eicosatetraenoic acid (12-H(P)ETE) to an enzyme capable of 10-20% 15-lipoxygenation. When all amino acids between positions 398-429 of 12-lipoxygenase had the corresponding 15-lipoxygenase sequence, the enzyme made 66% 15-lipoxygenase products. The latter enzyme had markedly reduced enzyme activity, though, indicating an apparent shift in the optimal alignment of substrate at the active site for hydrogen atom abstraction. The platelet enzyme could not be altered to form 5-lipoxygenase products by similar manipulations of sequence within this region. Chimeric enzymes consisting of an NH2-terminal segment from one lipoxygenase and the COOH terminus from another lipoxygenase or large substitutions resulted in nonfunctional enzymes. NH2-terminal extensions, but not short deletions, could be tolerated functionally. These studies provide some new insights into lipoxygenase structure-function in the absence of an unresolved three-dimensional structure.