Front Matter

be favored (Figure 3B, right-hand side). In addition, the stereochemistry of the LOX

reaction can be explained perfectly with this concept. However, an inverse head-to-

tail substrate orientation should be inhibited by an energy barrier associated with

burying the polar carboxylate in the hydrophobic environment of the substrate-bind-

ing cage (Browner et al., 1998; Gillmor et al., 1998). This energy barrier would be

reduced strongly, if a polar amino acid were to be located at the surface of the active

site (residue B in Figure 3B). Similarly, demasking of a polar residue as consequence

of site-directed mutagenesis may also reduce this energy barrier.

15.3.1 Alteration of the positional specificity by site-directed

mutagenesis of LOX preferring C 18 fatty acids as substrates

In the past, site-directed mutagenesis studies have mainly been carried out with

mammalian LOXs. In these experiments two regions have been identified in the

primary structure containing sequence determinants for the positional specificity.

An alignment of these determinants within selected plant LOXs is shown in Table

1. Amino acids aligning with the Sloane-determinants (Sloane et al., 1991) are highly

conserved among plant LOXs, irrespective of their positional specificity. In contrast,

there is an amino acid heterogeneity among plant LOXs at the position which aligns

with P353 of the rabbit reticulocyte 15-LOX (Borngra ̈ber-determinants) (Borngra ̈ber

et al., 1996). For a more comprehensive understanding of the mechanistic reasons for

the positional specificity of LA oxygenation by plant LOXs, structural modeling of

the active site of plant LOXs was carried out. For this purpose the X-ray coordinates

of soybean LOXs-1 and -3 were utilized (Boyington et al., 1993; Minor et al., 1996;

Skrzypczak-Jankun et al., 1997). These modeling studies revealed that an arginine

residue is localized in the vicinity of the putative substrate-binding pocket of plant

LOXs which may interact with the carboxy group of the substrates when there is an

inverse head-to-tail substrate orientation (Figure 3B, residue B). Moreover, in wild-

type plant LOXs there is a phenylalanine or a histidine located at the position, which

aligns with the second Sloane-determinants of mammalian LOXs (Table 1).

These amino acids may shield the positive charge of the arginine and thus, the

substrate may penetrate the active site with its methyl end since there is no counter-

part to neutralize the charge of the carboxy group (Hornung et al., 1999). Mutagen-

esis studies of the Sloane-determinants with the lipid body LOX of cucumber seed-

lings indicate for the first time the possibility to convert a plant LOX catalyzing a

[+2] radical rearrangement to a LOX catalyzing a [-2] rearrangement (Hornung et al.,

1999) (Figure 4). In this case a single point mutation (H608V) converted the wild-

type linoleate 13-LOX to a 9-lipoxygenating mutant species. From these results it is

hypothesized that the exchange of the bulky phenylalanine to a less space-filling

valine may inverse the orientation of the substrate at the active site (Figure 3B).

However, when more complex substrates such as methyl linoleate (LAMe),a-

LeA,c-LeA or trilinolein (TL, see below) were used the situation becomes more

complex, and more structural determinants within the active site may be required

for a perfect alignment of the lipid substrate (Table 2).

316 15 Application of Lipoxygenases and Related Enzymes