Probing a novel potato lipoxygenase with dual positional specificity reveals primary determinants of substrate binding and requirements for a surfacehydrophobic loop and has implications for the role of lipoxygenases in tubers

A new potato tuber lipoxygenase full-length cDNA sequence (lox1: St:2) has been isolated from potato tubers and used to express in Escherichia coli an d characterize a novel recombinant lipoxygenase (potato 13/9-lipoxygenase), Like most plant lipoxygenases it produced carbonyl compounds from linoleat e (the preferred substrate) and was purified in the Fe(II) (ferrous) state. Typical of other potato tuber lipoxygenases, it produced 5-HPETE [5(S)-hyd roperoxy-(6E, 8Z, 11Z, 14Z)-eicosatetraenoic acid] from arachidonate. In co ntrast to any other potato tuber lipoxygenase, it exhibited dual positional specificity and produced roughly equimolar amounts of 13- and 9-hydroperox ides (or only a slight molar excess of 9-hydroperoxides) from linoleate, We have used a homology model of pea 9/13-lipoxygenase to superimpose and com pare the linoleate-binding pockets of different potato lipoxygenases of kno wn positional specificity. We then tested this model by using site-directed mutagenesis to identify some primary determinants of linoleate binding to potato 13/9-lipoxygenase and concluded that the mechanism determining posit ional specificity described for a cucumber lipoxygenase does not apply to p otato 13/9-lipoxygenase. This supports our previous studies on pea seed lip oxygenases for the role of pocket volume rather than inverse orientation as a determinant of dual positional specificity in plant lipoxygenases. We ha ve also used deletion mutagenesis to identify a critical role in catalysis for a surface hydrophobic loop in potato 13/9-lipoxygenase and speculate th at this may control substrate access. Although potato 13/9-lipoxygenase rep resents only a minor isoform in tubers, such evidence for a single lipoxyge nase species with dual positional specificity in tubers has implications fo r the proposed role of potato lipoxygenases in the plant.