Front Matter

(14R,15S)-DiH(P)ETE by the soybean LOXs does not exclude an AA-like substrate

orientation. It may be possible that only the inversely aligned substrate is oxygenated

by the enzyme. If the hydrogen abstraction is sterically hindered, for instance by a

large distance of the doubly allylic methylene from the nonheme iron, the share of

15-HETE which is bound in an AA-like way, may not be oxygenated although it was

bound at the active site. Thus, the lack of (14R,15S)-DiH(P)ETE formation by the

soybean LOXs does not exclude that a share of the substrate may be bound in an AA-

like manner. Similarly, the formation of (5S,15S)-DiH(P)ETE from 15-HETE methyl

ester by the reticulocyte enzyme does not exclude an AA-like substrate alignment,

because abortive substrate binding may interfere with product formation.

Alteration of positional specificity by targeted site-directed mutagenesis

After sequence information on various mammalian LOXs became available (Dixon

et al., 1988; Fleming et al., 1989; Funk et al., 1990; Yoshimoto et al., 1990), scientists

began to investigate LOX/substrate interaction by site-directed mutagenesis. In order

to identify suitable targets for site-directed mutagenesis, multiple sequence align-

ments of various LOXs were required which would provide information on con-

served sequence differences between various LOX subfamilies.

Conversion of arachidonate 15-LOX to 12-lipoxygenating enzyme species

In 1991, Sloane and colleagues carried out a multiple alignment of 12- and 15-LOX

sequences and found four conserved differences between the two families (Sloane et

al., 1991). These amino acids of the human 15-LOX were mutated to their counter-

parts present in 12-LOXs. An enzyme species resulted which converted AA to 12-

and 15-HETE in almost equal amounts. Separate mutation of these four amino acids

indicated that the alterations in the product pattern were due exclusively to M419V

exchange (Sloane et al., 1991). In a follow-up study (Sloane et al., 1995), the authors

carried out simultaneous mutations of I418 and M419 by changing them to the re-

sidues present in the bovine and porcine 12-LOX. In doing this, the human 15-LOX

was converted completely to a 12-lipoxygenating species (12-/15-HETE ratio 20 : 1).

These data indicated that I418 and M419 may constitute sequence determinants for

the positional specificity of the human 15-LOX. Later experiments with the rabbit

reticulocyte 15-LOX (Ku ̈hn et al., unpublished data), with the human platelet 12-

LOXs (Chen and Funk, 1993), and with the porcine leukocyte 12-LOX (Suzuki

et al., 1994) confirmed this conclusion. However, a similar strategy was not success-

ful to alter the positional specificity of the leukocyte-type 12-LOXs from rats (Wa-

tanabe and Haeggstrom, 1993) and mice (Ku ̈hn et al., unpublished data). Thus, there

must be differences in the mechanism of the positional specificity between the por-

cine and the murine leukocyte-type 12-LOXs. In order to obtain more detailed in-

formation about these differences, we created chimeric LOX species combining

cDNA fragments of the rabbit reticulocyte 15-LOX cDNA with pieces of the two

above-mentioned enzymes (Borngra ̈ber et al., 1996). As first step, a chimeric

LOX was created in which a large amino acid fragment (301 amino acids) of the

rabbit reticulocyte 15-LOX was inserted into the porcine leukocyte-type 15-

324 15 Application of Lipoxygenases and Related Enzymes