Isochorismate Synthase Enzymes in Arabidopsis.

摘要

Herein, I describe the overexpression and purification of both wild type and mutant forms of recombinant AtICS1 and AtICS2 proteins. I also describe a thorough biochemical characterization of these various proteins.;The pathogen-induced accumulation of the salicylic acid involved in plant defense requires AtICS1. AtICS1 acts as a monofunctional isochorismate synthase enzyme instead of a bifunctional salicylic acid synthase, and the reaction that it catalyzes operates near equilibrium (apparent Keq = 0.89). Using an irreversible coupled spectrophotometric assay, AtICS1 was found to have an apparent KM of 41.5 microM and kcat of 38.7 min-1 for chorismate. This affinity for chorismate would allow AtICS1 to successfully compete with other pathogen-induced chorismate-utilizing enzymes. Furthermore, the biochemical properties of AtICS1 indicate that its activity is not regulated by light-dependent changes in stromal pH, Mg 2+ ion concentration, or redox balance, and that it is remarkably active at 4 °C, consistent with a role for AtICS1 in salicylic acid production for the mediation of cold-tolerant growth. Finally, these analyses support plastidic synthesis of stress-induced salicylic acid by AtICS1 -- with the requirement for one or more additional enzymes responsible for the conversion of isochorismate to salicylic acid -- as non-enzymatic conversion of isochorismate to salicylic acid under physiological conditions was negligible.;The similarity between the pattern of expression of AtICS2 and that of other genes in the phylloquinone biosynthetic pathway strongly suggests that AtICS2 is involved in phylloquinone biosynthesis associated with plastid development during the dark-light transition. AtICS2 is also a monofunctional isochorismate synthase that catalyzes a reaction that operates near equilibrium (apparent Keq = 0.76). This is consistent with its proposed role in phylloquinone biosynthesis, and it rules out a role for AtICS2 as an isochorismate pyruvate lyase in salicylic acid biosynthesis. Using an irreversible coupled spectrophotometric assay, AtICS2 was found to have an apparent KM of 17.2 microM and kcat of 18.0 min-1 for chorismate -- these reaction parameters would allow AtICS2 to efficiently channel chorismate into the phylloquinone biosynthetic pathway. The biochemical properties of AtICS2 indicate that the enzyme is active within a broad range of stromal pH values, Mg2+ ion concentrations, and temperatures, consistent with the need for AtICS2 to operate under a variety of conditions during induced phylloquinone biosynthesis.;An examination of the amino acid sequence of various chorismate-utilizing enzymes revealed that one of the residues located within 6 A of the putative substrate binding site is an alanine in virtually all known monofunctional isochorismate synthase enzymes, but a threonine in most bifunctional anthranilate synthase and salicylic acid synthase enzymes. Furthermore, structural studies of several of these enzymes indicate that this residue is likely forming a hydrogen bond to the substrate. These factors suggest that the identity of this residue may determine whether the given enzyme catalyzes a secondary aromatization reaction after the initial substitution reaction common to all of these enzymes. Several mutants of AtICS1 and AtICS2 were overexpressed and purified, all possessing residues altered at this and adjacent positions. In every case, the mutant protein no longer possessed any isochorismate synthase activity, acting instead as a bifunctional chorismate mutase-prephenate dehydratase enzyme. This suggests that the active sites of isochorismate synthase enzymes require a highly organized amino acid configuration, and that even minor perturbations in it will abolish all activity. It further suggests that the default activity of this class of enzymes may be chorismate mutase activity.;Experiments were undertaken to determine whether any of the three known Arabidopsis chorismate mutase enzymes could be acting as an isochorismate pyruvate lyase enzymes in vivo, as they possess homology to Pseudomonas aeruginosa PchB, an enzyme known to possess this activity. These enzymes were overexpressed and purified in recombinant form, and exposed to isochorismate. AtCM2 and AtCM3 produced no salicylic acid, but AtCM1 possessed a very weak isochorismate pyruvate lyase specific activity of 0.864 nmoles min-1 mg protein-1. This suggests the possibility that AtCM1 is operating as an isochorismate pyruvate lyase in planta, where other factors could enable AtCM1 to catalyze this reaction more efficiently. (Abstract shortened by UMI.).