Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase: the side-chain geometry of amino acids 417 and 418 determine positional specificity

Positional specificity determinants of human 15-lipoxygenasewere examined by site-directed mutagenesis and by kinetic analysisof the wild-type and variant enzymes. By comparing conserveddifferences among sequences of 12- and 15-lipoxygenases, a smallregion responsible for functional differences between 12- and15-lipoxygenases has been identified. Furthermore, the replacementof only two amino acids in 15-lipoxygenase (at 417 and 418 inthe primary sequence) by those found in certain 12-lipoxygenasesresults in an enzyme that has activity similar to 12-lipoxygenase.An examination of the activity of nine variants of lipoxygenasedemonstrated that the amino acid side-chain bulk and geometryof residues 417 and 418 are the key components of the positionalspecificity determinant of 15-lipoxygenase. Overexpression ofa variant (containing valines at positions 417 and 418) thatperforms predominantly 12-lipoxygenation was achieved in a baculovirus-insectcell culture system. This variant was purified to >90% homogeneityand its kinetics were compared with the wild-type 15-lipoxygenase.The variant enzyme has no change in its apparent K_M for arachidonicacid and a minor(3-fold) change in its V_max. For linoleic acid,the variant has no change in its K_M and a 10-fold reductionin its V_max, as expected for an enzyme performing predominantly12-lipoxygenation. The results are consistent with a model inwhich two amino acids of 15-lipoxygenase (isoleucine 417 andmethionine 418) constitute a structural element which contributesto the regiospecificity of the enzyme. Replacement of theseamino acids with those found in certain 12-lipoxygenases resultsin an enzyme which can bind arachidonic acid in a catalyticregister that prefers 12-lipoxygenation.