Probing a novel potato lipoxygenase with dual positional specificity reveals primary determinants of substrate binding and requirements for a surface hydrophobic 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 and characterize a novel recombinant lipoxygenase (potato 13/9-lipoxygenase). Like most plant lipoxygenases it produced carbonyl compounds from linoleate (the preferred substrate) and was purified in the Fe(II) (ferrous) state. Typical of other potato tuber lipoxygenases, it produced 5-HPETE [5(S)-hydroperoxy-(6E, 8Z, 11Z, 14Z)-eicosatetraenoic acid] from arachidonate. In contrast to any other potato tuber lipoxygenase, it exhibited dual positional specificity and produced roughly equimolar amounts of 13- and 9-hydroperoxides (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 compare the linoleate-binding pockets of different potato lipoxygenases of known 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 positional specificity described for a cucumber lipoxygenase does not apply to potato 13/9-lipoxygenase. This supports our previous studies on pea seed lipoxygenases for the role of pocket volume rather than inverse orientation as a determinant of dual positional specificity in plant lipoxygenases. We have also used deletion mutagenesis to identify a critical role in catalysis for a surface hydrophobic loop in potato 13/9-lipoxygenase and speculate that this may control substrate access. Although potato 13/9-lipoxygenase represents only a minor isoform in tubers, such evidence for a single lipoxygenase species with dual positional specificity in tubers has implications for the proposed role of potato lipoxygenases in the plant.
Hughes
R K
RK
John Innes Centre, Norwich Research Park, Norwich NR4 7UH, U.K.
West
S I
SI
Hornostaj
A R
AR
Lawson
D M
DM
Fairhurst
S A
SA
Sanchez
R O
RO
Hough
P
P
Robinson
B H
BH
Casey
R
R
D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008084:Lipoxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D010940:Plant Proteins / Q000378:metabolism*; D011198:Solanum tuberosum / Q000201:enzymology*; D013379:Substrate Specificity
D010940:Plant Proteins; D008084:Lipoxygenase