Earlier attempts to alter the direction of radical rearrangement during LOX reac-
tion did not lead to active LOX mutants. However, recently a linoleate 13-LOX from
cucumber seedlings was completely converted to a 9-lipoxygenating species by mu-
tation of a single amino acid (Hornung et al., 1999).
For the time being there is no comprehensive theory which explains the positional
specificity of various LOX isoforms. However, there are two hypotheses which may
rationalize the effects of substrate binding on the oxygenation specificity. The space-
related hypothesis was developed for mammalian LOXs after the X-ray structures of
plant and mammalian LOXs became available (Minor et al., 1996; Skrzypczak-Jan-
kun et al., 1997; Gillmor et al., 1998). It postulates that the volume of the substrate-
binding pocket may be decisive for the positional specificity of the LOX reaction.
Mammalian arachidonate 15-LOXs have a smaller substrate-binding cavity, and
since fatty acids may penetrate the active site with their methyl terminus first
they are dioxygenated close to this end of the molecule (Figure 3A, left-hand
side). In contrast, more space appears to be available in the substrate-binding cleft
of 12-LOXs. In that case, the fatty acid substrate may slide in farther into the sub-
strate-binding pocket and thus, it is oxygenated closer to the carboxy terminus (Fig-
ure 3A, right-hand side). For 5-LOXs an even deeper active site was postulated so
that the doubly allylic C-7 approaches the nonheme iron. This model explains the
different sites of hydrogen abstraction, but the reversal of direction of radical rear-
rangement is hard to fit into this concept. The second hypothesis to explain the
positional specificity of LOXs is the orientation-related theory. This concept postu-
lates that substrate fatty acids are inversely bound at the active site of 5- and 15-
LOXs (Egmond et al., 1973; Ku ̈hn et al., 1986; Gardner, 1989; Lehmann, 1994;
Prigge et al., 1998) (Figure 3B). For linoleate 15-lipoxygenation the substrate ap-
pears to penetrate the active site with its methyl terminus, favoring a [+2] radical
rearrangement (Figure 3B, left-hand side). In contrast, when the substrate slides
into the active site with its carboxy terminus a [-2] radical rearrangement would
15.3 The structural bases of the positional specificity of LOXs 315
Figure 3. Comparison of the two different models explaining the positional specificity of LOXs. (A)
Straight substrate alignment at the active site of LOXs for the space-related theory. (B) Orientation-de-
pendent theory: straight- and inverse-substrate orientation at the active site of LOXs.