Mutagenesis and modelling of linoleate-binding to pea seed lipoxygenase.
We have produced a model to define the linoleate-binding pocket of pea 9/13-lipoxygenase and have validated it by the construction and characterization of eight point mutants. Three of the mutations reduced, to varying degrees, the catalytic centre activity (kcat) of the enzyme with linoleate. In two of the mutants, reductions in turnover were associated with changes in iron-coordination. Multiple sequence alignments of recombinant plant and mammalian lipoxygenases of known positional specificity, and the results from numerous other mutagenesis and modelling studies, have been combined to discuss the possible role of the mutated residues in pea 9/13-lipoxygenase catalysis. A new nomenclature for recombinant plant lipoxygenases based on positional specificity has subsequently been proposed. The null-effect of mutating pea 9/13-lipoxygenase at the equivalent residue to that which controlled dual positional specificity in cucumber 13/9-lipoxygenase, strongly suggests that the mechanisms controlling dual positional specificity in pea 9/13-lipoxygenase and cucumber 13/9-lipoxygenase are different. This was supported from modelling of another isoform of pea lipoxygenase, pea 13/9-lipoxygenase. Dual positional specificity in pea lipoxygenases is more likely to be determined by the degree of penetration of the methyl terminus of linoleate and the volume of the linoleate-binding pocket rather than substrate orientation. A single model for positional specificity, that has proved to be inappropriate for arachidonate-binding to mammalian 5-, 12- and 15-lipoxygenases, would appear to be true also for linoleate-binding to plant 9- and 13-lipoxygenases.
Hughes
R K
RK
John Innes Centre, Norwich Research Park, Norwich, UK. richard.hughes@bbsrc.ac.uk
Lawson
D M
DM
Hornostaj
A R
AR
Fairhurst
S A
SA
Casey
R
R
D000595:Amino Acid Sequence; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D015870:Gene Expression; D019787:Linoleic Acid / Q000378:metabolism*; D008084:Lipoxygenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D012639:Seeds / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D010940:Plant Proteins; D019787:Linoleic Acid; D008084:Lipoxygenase