Pseudophosphorylation of Arabidopsis jasmonate biosynthesis enzyme lipoxygenase 2 via mutation of Ser(600) inhibits enzyme activity

Jasmonates are oxylipin phytohormones critical for plant resistance against necrotrophic pathogens and chewing herbivores. An early step in their biosynthesis is catalyzed by non-heme iron lipoxygenases (LOX; EC 1.13.11.12). In Arabidopsis thaliana, phosphorylation of Ser600 of AtLOX2 was previously reported, but whether phosphorylation regulates AtLOX2 activity is unclear. Here, we characterize the kinetic (alpha-LeA, jasmonate precursor), linoleic acid (LA), and arachidonic acid (AA) as substrates. Enzyme velocity with endogenous substrates alpha-LeA and LA increased with pH. For alpha-LeA, this increase was accompanied by a decrease in substrate affinity at alkaline pH; thus, the catalytic efficiency for alpha-LeA was not affected over the pH range tested. Analysis of Ser600 phosphovariants demonstrated that pseudophosphorylation inhibits enzyme activity. AtLOX2 activity was not detected in AA were used as substrates. In contrast, phosphonull mutant Atlox2S600A exhibited strong activity with all three substrates, alpha-LeA, LA, and AA. Structural comparison between the AtLOX2 AlphaFold model and a complex between 8R-LOX and a 20C polyunsaturated fatty acid suggests a close proximity the polyunsaturated fatty acid. This analysis indicates that structure and highlights how Ser600 phosphorylation could affect AtLOX2 catalytic activity. Overall, we propose that for the regulation of AtLOX2 activity and, thus, the jasmonate biosynthesis pathway and plant resistance.

Automated summary

Jasmonates play a critical role in plant resistance against pathogens and herbivores. The biosynthesis of jasmonates involves the catalytic activity of iron lipoxygenases, specifically AtLOX2 in Arabidopsis thaliana. The phosphorylation of Ser600 in AtLOX2 inhibits enzyme activity, while a mutant with abnormal phosphorylation exhibits strong activity. This suggests that Ser600 phosphorylation may regulate AtLOX2 activity and, consequently, the jasmonate biosynthesis pathway and plant resistance.