Front Matter

fatty acid isomers enzymatically since the diversity of LOX positional specificity is

not sufficient. For instance, currently it is not possible to prepare (5R)-hydroperoxy

eicosatetraenoic acid ((5R)-HPETE)) since no LOX with a (5R)-specificity has been

discovered so far. It may even be the case that such a LOX is not available in nature.

In this case it may be possible to create such enzymes by site-directed mutagenesis.

In order to do this, one has to identify the structural basis of LOX specificity so that

certain amino acids can be targeted by mutagenesis.

A number of reviews have been published to date discussing the diversity of LOX-

derived products formed in biological systems (Feussner and Wasternack, 1998).

However, the use of isolated LOXs in organic solvents and their immobilization

for biotechnological applications is less well investigated. The interested reader

is referred to reviews by Gardner (1996; 1997) and Piazza (1996). This chapter

will mainly describe our recent advances in creating LOX mutants which exhibit

an altered product specificity.

15.2 LOXs are versatile catalysts

LOXs are multifunctional enzymes, which catalyze at least three different types of

reactions: (i) dioxygenation of lipid substrates (dioxygenase reaction); (ii) secondary

conversion of hydroperoxy lipids (hydroperoxidase reaction); and (iii) formation of

epoxy leukotrienes (leukotriene synthase reaction).

15.2.1 Dioxygenase reaction

Although much effort has been put into the investigation of the dioxygenase reaction,

its detailed mechanism remains a matter of discussion (Glickman and Klinman,

1996a,b; Hwang and Grissom, 1996; Nelson, 1996; Sloane, 1996; Prigge et al.,

1997). Kinetic isotope effects appear to indicate that the rate-limiting step of the

overall reaction is the stereoselective hydrogen removal from a doubly allylic methy-

lene (Glickman and Klinman, 1996b). LOXs contain one nonheme iron per mole

enzyme which is catalytically active and undergoes redox shuttling (Figure 1)

(De Groot et al., 1975). From natural sources a LOXs is usually prepared in its

ferrous (Fe(II)) ground state which is catalytically inactive and requires activation

to start the catalytic cycle. This activation can be achieved by the reaction with small

amounts of hydroperoxides. Such peroxides oxidize the inactive ferrous ground state

enzyme to an active ferric form (Fe(III)-LOX) (Figure 1, step a). After binding of the

substrate, a hydrogen is abstracted from a doubly allylic methylene group, resulting

in reduction of Fe(III) and the formation of a carbon centered pentadienenyl radical

(Figure 1, step b). Under certain conditions, such as reduced oxygen tension or in the

presence of excess substrate, this radical may dissociate from the active site (Figure

1, step f), leading to the formation of unspecific oxygenation products. Alternatively,

it is assumed that for some LOXs the substrate may not perfectly be aligned so that an

unspecific oxygenation may be possible. Such an imperfect fit in the active site and

310 15 Application of Lipoxygenases and Related Enzymes