CDNA CLONING, EXPRESSION, MUTAGENESIS OF C-TERMINAL ISOLEUCINE, GENOMIC STRUCTURE, AND CHROMOSOMAL LOCALIZATIONS OF MURINE 12-LIPOXYGENASES

Two types of 12-lipoxygenase that catalyze the transformation of arach idonic acid to 12(S)-hydroperoxyeicosatetraenoic acid (12-HPETE) have been previously classified into platelet-type and leukocyte-type categ ories. Here, we document, for the first time, a molecular characteriza tion of both forms within the same species. The amino acid sequence of the murine platelet 12-lipoxygenase deduced from its cDNA is 58% iden tical to the murine spleen/leukocyte 12-lipoxygenase. Expression const ructs carrying the cDNAs for the two 12-lipoxygenase forms were introd uced into human embryonic kidney 293 cells. The platelet-type enzyme m etabolized arachidonic acid exclusively to 12-HPETE, whereas the leuko cyte-type enzyme formed both 12-HPETE and 15-hydro(pero)xyeicosatetrae noic acid in a ratio of approximate to 3:1. Linoleic acid was metaboli zed to a similar extent by the latter enzyme to 13-hydro(pero)xyoctade cadienoic acid but not by the platelet enzyme. Mutagenesis and deletio n of the highly conserved lipoxygenase C-terminal isoleucine (Ile(663) ), a residue believed to be involved in the non-heme iron atom coordin ation of all lipoxygenases, was performed. Deletion of Ile(663) and su bstitution with most amino acids abolished enzyme activity. Only a val ine substitution retained significant activity. These findings would t end to indicate a stringent requirement for the proper spatial alignme nt and folding of the C-terminal chain back into the core of the enzym e to interact with the iron atom by analogy with the recently determin ed crystal structure of a soybean lipoxygenase (Boyington, J. C., Gaff ney, B. J., and Amzel, L. M. (1993) Science 260, 1482-1486). The plate let-type and leukocyte-type 12-lipoxygenase genes were cloned from a m urine 129 Sv genomic library. Both genes are divided into a similar 14 -exon/13-intron format, with the platelet-type gene being approximatel y twice the size of the leukocyte-type gene (13 versus 7.5 kilobases). A segment of a third gene was also isolated and probably represents a pseudogene derivative of either of these 12-lipoxygenase genes. All t hree genes were mapped to the central region of mouse chromosome 11 in a region of homology with human chromosome 17. Antibodies prepared ag ainst the two forms of 12-lipoxygenase revealed the differential distr ibution of the two enzymes throughout the mouse.