Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis.
Multiple lipoxygenase sequence alignments and structural modeling of the enzyme/substrate interaction of the cucumber lipid body lipoxygenase suggested histidine 608 as the primary determinant of positional specificity. Replacement of this amino acid by a less-space-filling valine altered the positional specificity of this linoleate 13-lipoxygenase in favor of 9-lipoxygenation. These alterations may be explained by the fact that H608V mutation may demask the positively charged guanidino group of R758, which, in turn, may force an inverse head-to-tail orientation of the fatty acid substrate. The R758L+H608V double mutant exhibited a strongly reduced reaction rate and a random positional specificity. Trilinolein, which lacks free carboxylic groups, was oxygenated to the corresponding (13S)-hydro(pero)xy derivatives by both the wild-type enzyme and the linoleate 9-lipoxygenating H608V mutant. These data indicate the complete conversion of a linoleate 13-lipoxygenase to a 9-lipoxygenating species by a single point mutation. It is hypothesized that H608V exchange may alter the orientation of the substrate at the active site and/or its steric configuration in such a way that a stereospecific dioxygen insertion at C-9 may exclusively take place.
Hornung
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Institute of Plant Biochemistry, D-06120 Halle, Germany.
Walther
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M
Kühn
H
H
Feussner
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I
D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology; D001665:Binding Sites; D003198:Computer Simulation; D018553:Cucumis sativus / Q000201:enzymology* / Q000235:genetics; D006639:Histidine; D007700:Kinetics; D008084:Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D011198:Solanum tuberosum / Q000201:enzymology; D013379:Substrate Specificity; D014633:Valine
D011994:Recombinant Proteins; D006639:Histidine; C117433:13-lipoxygenase; D008084:Lipoxygenase; D014633:Valine