Nucleophile Selectivity of Chorismate‐Utilizing Enzymes

Abstract

aeruginosa) catalyzes the conversion of chorismate to isochorismate (2) by nucleophilic addition of water to the C2 of chorismate, with loss of the C4 hydroxyl. This is then converted to salicylate (3) by isochorismate pyruvate lyase (e.g. , PchB in P. aeruginosa). Very recently we described a bifunctional salicylate synthase (Irp9 from Yersinia enterocolitica) that converts chorismate into salicylate via isochorismate. This two-step transformation is analogous to the formation of anthranilate from chorismate catalyzed by the TrpE subunit of anthranilate synthase. The TrpE mechanism involves initial addition of ammonia to the C2 of chorismate to form 2-amino-2-deoxyisochorismate (ADIC; 4), followed by elimination of the enol pyruvate side-chain to form anthranilate (5). The factors that control the identity of the nucleophile in the Irp9 and TrpE reactions are unknown, and are the focus of this Communication. Although there is only approximately 20% amino acid sequence identity between PchA, Irp9 and TrpE, examination of the crystal structures of TrpE, and Irp9 shows that they are structurally homologous. Active sites from Serratia marcescens TrpE structure (in which benzoate and pyruvate are bound) and the recently solved Irp9 structure (with salicylate and pyruvate bound) are almost identical (Figure 1). The key catalytic residues are strictly conserved and superimposed. The only significant difference is the identity of the residue 5.5 : away from the C2 of salicylate in the Irp9 products complex. This is lysine in Irp9 (K193) and PchA (K221), which both catalyze nucleophilic attack by water, but glutamine in TrpE (Q262), for which ammonia is the nucleophile. In the absence of any other structural clues as to the factors involved in nucleophile selection, this residue was mutated. Accordingly, the Irp9 and the TrpE/TrpG mutants as well as their respective ACHTUNGTRENNUNGalanine substitutions Irp9 and TrpE/TrpG were constructed and over-expressed as hexahistidine-tagged enzymes in E. coli. The mutants were studied by H NMR spectroscopy to determine the nature of the products formed (Table 1). Control experiments with wild-type enzymes showed that Irp9 forms salicylate, independent of the presence of NH4Cl (Figure 2D), and that anthranilate synthase forms anthranilate in the presence of NH4Cl (Figure 2G) but neither anthranilate or salicylate in the absence of NH4Cl (Figure 2H). The Irp9 mutant does not form salicylate (Figure 2E), but does form anthranilate in the presence of NH4Cl (Figure 2F). By using a fluorescence assay to