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 Arabidopsis thaliana, the expression of AtICS1 is induced by stress conditions in parallel with SA synthesis, and AtICS1 is required for SA synthesis. In contrast, the expression of NtICS 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 Nicotiana benthamiana, 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 Escherichia coli and N. benthamiana leaves. Moreover, the levels of AtICS1 protein expression in N. benthamiana 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 N. benthamiana 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.

Keywords: isochorismate synthase; phytopathology; resistance; salicylic acid.

Figure 1. Metabolism of chorismate in plants and microorganisms

Chorismate is a branch-point compound for the production of primary and secondary compounds such as aromatic amino acids and vitamins. Enzymes present in both plants and microorganisms, and those found only in bacteria are shown in white and gray boxes, respectively. Abbreviations: ADC, 4-amino-4-deoxychorismate; ADCS, ADC synthase; AS, anthranilate synthase; CM, chorismate mutase; CPL, chorismate pyruvate lyase.

Figure 2. Comparison of ICSs expressed in E. coli

(A) Recombinant proteins of EntC (Ec), NtICS (Nt), OsICS (Os), AtICS1 (At), and NbICS (Nb) with His₆ tags at the N-terminus were expressed in E. coli. The crude protein fractions were prepared from the cells, and the production of recombinant proteins was confirmed by immunoblotting analyses using an anti-His₆ tag antibody (α-His). E. coli transformed with an empty vector were used as a control (V). As a loading control, parallel gels were stained with Coomassie Brilliant Blue (CBB). To verify that similar levels of the recombinant proteins were expressed, serially diluted (fold) crude protein fractions were analyzed. (B) ICS activity of the crude protein fractions was measured. Values are means with S.D. of three independently prepared crude protein fractions.

Figure 3. Comparison of ICSs expressed in N. benthamiana leaves

(A) Agrobacterium cells (A₆₀₀ = 0.1) carrying each ICS with three FLAG tags at the C-terminus were infiltrated into the leaves of N. benthamiana. Agrobacterium carrying an empty vector was used as a control (V). Two days later, the accumulation of ICS proteins was detected by immunoblotting analyses using an anti-FLAG tag antibody (α-FLAG). As a loading control, parallel gels were stained with Coomassie Brilliant Blue (CBB). (B) ICS activity of the crude protein fractions was measured. Values are means with S.D. (n=3–11). Abbreviations: At, AtICS1; Ec, TP^SS-EntC; Nb, NbICS; Nt, NtICS; Os, OsICS.

Figure 4. Comparison of ICSs in planta

(A) Agrobacterium cells (A₆₀₀ = 0.1) carrying each artificial SAS construct (TP^SS-IPL^PmsB-ICSΔTP) were infiltrated into the leaves of N. benthamiana. Agrobacterium carrying TP^SS-IPL^PmsB only was used as a control (none). Two days later, the levels of SA were determined. Values are means with S.D. (n=3). (B) Agrobacterium cells (A₆₀₀ = 0.1) carrying each ICS were mixed with 99 volumes (left) or 19 volumes (right) of Agrobacterium cells (A₆₀₀ = 0.1) carrying TP^SS-IPL^PmsB (IPL). Mixtures were infiltrated into the leaves of N. benthamiana. Two days later, the levels of total SA were determined. Values are means with S.D. (n=3). Abbreviations: At, AtICS1; Ec, EntC and TP^SS-EntC; Nb, NbICS; Nt, NtICS; Os, OsICS.

Figure 5. Chimeric analysis of AtICS1 and OsICS

(A) Schematic representation of AtICS1 and OsICS chimeras. Putative chloroplast TPs are shown as hatched boxes. Restriction enzyme sites used to create chimeric proteins are shown at the top. (B) Agrobacterium cells (A₆₀₀ = 0.1) carrying each ICS with three FLAG tags at the C-terminus were infiltrated into the leaves of N. benthamiana. Two days later, the accumulation of ICS proteins was detected by immunoblotting analyses using an anti-FLAG tag antibody (α-FLAG). As a loading control, parallel gels were stained with Coomassie Brilliant Blue (CBB). (C) Agrobacterium cells (A₆₀₀ = 0.1) carrying each ICS with three times FLAG tag at the C-terminus were mixed with Agrobacterium cells (A₆₀₀ = 0.1) carrying TP^SS-IPL^PmsB. Mixtures were infiltrated into the leaves of N. benthamiana. Two days later, the levels of SA were determined. Values are means with S.D. (n=3). Abbreviations: At, AtICS1; Os, OsICS.

Figure 6. Putative chloroplast TPs affect the levels of protein accumulation in N. benthamiana leaves

(A) Schematic representation of AtICS1 (gray) and OsICS (white) chimeras. Putative chloroplast TPs are shown as hatched boxes. Agrobacterium cells (A₆₀₀ = 0.1) carrying each ICS with three FLAG tags at the C-terminus were infiltrated into the leaves of N. benthamiana. Two days later, the accumulation of ICS proteins was detected by immunoblotting analyses using an anti-FLAG tag antibody. The protein levels of the chimeric ICSs are summarized on the right (α-FLAG). (B) Agrobacterium cells (A₆₀₀ = 0.1) carrying each ICS with three FLAG tags at the C-terminus were infiltrated into the leaves of N. benthamiana. Two days later, the accumulation of the ICS proteins was detected by immunoblotting analyses using an anti-FLAG tag antibody (α-FLAG). As a loading control, parallel gels were stained with Coomassie Brilliant Blue (CBB). (C) Agrobacterium cells (A₆₀₀ = 0.1) carrying each ICS were mixed with Agrobacterium cells (A₆₀₀ = 0.1) carrying TP^SS-IPL^PmsB. Mixtures were infiltrated into the leaves of N. benthamiana. Two days later, the levels of SA were determined. Values are means with S.D. (n=3). Abbreviations: A and At, AtICS1; b, NbICS; O and Os, OsICS; SS, tobacco ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit; t, NtICS.

Figure 7. Identification of amino acid residues required for ICS activity of AtICS1

(A) Schematic representation of AtICS1 (gray) and OsICS (white) chimeras. Recombinant proteins of chimeric ICSs with a His₆ tag (His) at the N-terminus were expressed in E. coli. The crude protein fractions were prepared from the cells, and their ICS activities was measured. The activities of the chimeric ICSs are summarized on the right (activity). (B) Alignment of the 529–555th amino acid sequence of AtICS1 with the corresponding region of OsICS. The asterisks (*) and colons (:) indicate identical and similar amino acid residues between AtICS1 and OsICS, respectively. Amino acid residues known to be essential for the anthranilate synthase activity of S. typhimurium TrpE are underlined. Each different amino acid of AtICS1 was replaced with the corresponding amino acid of OsICS, and the recombinant proteins of the AtICS1 mutants were expressed as an N-terminal His₆-tagged protein in E. coli. Crude protein fractions were prepared from the cells, and their ICS activities were measured. Effects of single amino acid substitutions on the ICS activity of AtICS1 are summarized below the sequence of OsICS. n, no significant effect; y, significant effect. (C) Recombinant proteins of AtICS1 mutants with a His₆ tag at the N-terminus were expressed in E. coli. The crude protein fractions were prepared from the cells, and their ICS activities were measured. Values are means with S.D. of three independently prepared crude protein fractions. The production of recombinant proteins was confirmed by immunoblotting analyses using an anti-His₆ tag antibody (α-His). As a loading control, parallel gels were stained with Coomassie Brilliant Blue (CBB). Abbreviations: At, AtICS1; Cho-BD, chorismate-binding domain; Os, OsICS; Triple, T531A/S537T/I550A.

Figure 8. Tertiary structure model of AtICS1 predicted by homology modeling

(A) Ribbon representation of the structure model of the chorismate-binding domain (279-558) of AtICS1. The region (529–555) responsible for the high activity of AtICS1 and the other regions are indicated in green and cyan, respectively. Important residues for enzymatic activity and structure are shown as stick models. Isochorismate is shown as a yellow stick model and the magnesium ion is a purple sphere. Critical mutation sites (Thr⁵³¹, Ser⁵³⁷, and Ile⁵⁵⁰) for the activity are shown as orange stick models. (B) Close-up view of the AtICS1 model at the Thr⁵³¹ and Ser⁵³⁷ sites. (C) Close-up view of the AtICS1 model at the Ile⁵⁵⁰ site.