Comparative analysis of plant isochorismate synthases reveals structural mechanisms underlying their distinct biochemical properties

Abstract

Isochorismate synthase (ICS) converts chorismate into isochorismate, a precursor of primary and secondary metabolites including salicylic acid (SA). SA plays important roles in responses to stress conditions in plants. Many studies have suggested that the function of plant ICSs is regulated at the transcriptional level. In <i>Arabidopsis thaliana</i>, the expression of <i>AtICS1</i> is induced by stress conditions in parallel with SA synthesis, and <i>AtICS1</i> is required for SA synthesis. In contrast, the expression of <i>NtICS</i> is not induced when SA synthesis is activated in tobacco, and it is unlikely to be involved in SA synthesis. Studies on the biochemical properties of plant ICSs are limited, compared with those on transcriptional regulation. We analyzed the biochemical properties of four plant ICSs: AtICS1, NtICS, NbICS from <i>Nicotiana benthamiana</i>, and OsICS from rice. Multiple sequence alignment analysis revealed that their primary structures were well conserved, and predicted key residues for ICS activity were almost completely conserved. However, AtICS1 showed much higher activity than the other ICSs when expressed in <i>Escherichia coli</i> and <i>N. benthamiana</i> leaves. Moreover, the levels of AtICS1 protein expression in <i>N. benthamiana</i> leaves were higher than the other ICSs. Construction and analysis of chimeras between AtICS1 and OsICS revealed that the putative chloroplast transit peptides (TPs) significantly affected the levels of protein accumulation in <i>N. benthamiana</i> leaves. Chimeric and point-mutation analyses revealed that Thr⁵³¹, Ser⁵³⁷, and Ile⁵⁵⁰ of AtICS1 are essential for its high activity. These distinct biochemical properties of plant ICSs may suggest different roles in their respective plant species.