To investigate the role of other sequence determinants for the positional specificity
of plant LOXs, we point-mutated the Borngra ̈ber-determinant of the cucumber lipid
body LOX (V542F). Introduction of the space-filling phenylalanine in addition to the
already existing bulky H608 led to an enzyme species which exhibited a so far un-
known positional specificity (Figure 5). The V542F mutant convertedc-LeA mainly
to (6S,7E,9Z,12Z)-6-hydroperoxy-7,9,12-octadecatrienoic acid. In contrast, the
wild-type enzyme produced mainly the corresponding (13S)-hydroperoxy deriva-
tive. Here again, the direction of radical rearrangement was inverted by the mutation.
The lipid body LOX from cucumber seedlings and the LOX-2 from barley grains
are capable of oxidizing unpolar lipids (Holtman et al., 1997; Feussner et al., 1997a).
When expressed inE. colior underin vivoconditions the cucumber lipid body LOX
oxygenates all three LA moieties of trilinolein (Figure 6, Cc-16-LOX) (Feussner et
al., 1998). At the pH-optimum of soybean LOX-1, TL is oxygenated only to 28 % of
the amount converted by the lipid body LOX (Figure 6, Gm-LOX versus Cs-lb-
LOX). Moreover, TL oxygenation by soybean LOX-1 lead mainly to mono-hydro-
peroxy derivatives (Figure 6, peak 1), whereas oxygenation by lipid body LOX lead
to a tri-hydroperoxy derivative (Figure 6, peak 3). Since triacylglycerols do not con-
tain free carboxylic groups, no major differences are expected when the patterns of
oxygenation products of the wild-type lipid body LOX and of their linoleate 9-li-
poxygenating mutants were compared. Indeed, the wild-type enzyme and all 9-li-
poxygenating mutants exhibited a trilinoleate 13-LOX activity (Hornung et al.,
1999). In addition, the rates of TL oxygenation by the 9-lipoxygenating mutants
were comparable to those measured for other plant LOXs with a substrate prefer-
ence against free polyenoic fatty acids. Moreover, TL oxygenation by the mutant
enzymes mainly led to mono-hydroperoxy derivatives (Hornung et al., 1999).
15.3 The structural bases of the positional specificity of LOXs 317
Table 1.Alignment of amino acid residues possibly determining the positional specificity of plant and
mammalian LOXs.
Enzyme Acc.-No. Amino acid residues
(Position of amino
acid residues)^1
Amino acid residues
(Position of amino
acid residue)^213-LOX
Cucumber lipid body LOX X92890 Thr/His (607/608) Val (542)
Soybean seed LOX-1 P08170 Thr/Phe (556/557) Ser (491)
Potato LOX-H1 X96405 Ser/Phe (614/615) Ser (551)
Arabidopsis LOX-2 P38418 Cys/Phe (611/612) Ala (548)
Rabbit reticulocyte LOX P12530 Ile/Met (418/419) Phe (353)9-LOX
Potato tuber LOX P37831 Thr/Val (579/580) Val (514)
Tobaccoelicitor-ind. LOX X84040 Thr/Val (580/581) Ser (515)
ArabidopsisLOX-1 Q06327 Thr/Val (577/578) Ser (522)
Barley grain LOX-A L35931 Thr/Val (574/575) Ser (511)
Human LOX P09917 Ala/Asn (424/425) Phe (359)(^1) According to Sloane et al. (1991).
(^2) According to Borngra ̈ber et al. (1996).
Acc. no., accession number.