DOI: 10.1002/cbic.200700019 Nucleophile Selectivity of Chorismate-Utilizing Enzymes Olivier Kerbarh, [a] Alessio Ciulli, [a] Dimitri Y. Chirgadze, [b] Tom L. Blundell, [b] and Chris Abell* [a] Chorismate (1) is the substrate for several related enzymes in- volved in the formation of amino- and hydroxybenzoates (Scheme 1). Isochorismate synthase (e.g., PchA in Pseudomonas aeruginosa [1] ) catalyzes the conversion of chorismate to iso- chorismate (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 [2] ). Very recently we described a bifunctional sali- cylate synthase (Irp9 from Yersinia enterocolitica) that converts chorismate into salicylate via isochorismate. [3] This two-step transformation is analogous to the formation of anthranilate from chorismate catalyzed by the TrpE subunit of anthranilate synthase. [4] The TrpE mechanism involves initial addition of am- monia to the C2 of chorismate to form 2-amino-2-deoxyiso- chorismate (ADIC; 4), followed by elimination of the enol pyru- vate side-chain to form anthranilate (5). The factors that con- trol the identity of the nucleophile in the Irp9 and TrpE reac- tions are unknown, and are the focus of this Communication. Although there is only approximately 20 % amino acid se- quence identity between PchA, Irp9 and TrpE, examination of the crystal structures of TrpE, [4] and Irp9 [5] shows that they are structurally homologous. Active sites from Serratia marcescens TrpE structure (in which benzoate and pyruvate are bound) [4] and the recently solved Irp9 structure (with salicylate and pyru- vate bound) [5] are almost identical (Figure 1). The key catalytic residues are strictly conserved and superimposed. The only sig- nificant 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 cat- alyze 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 resi- due was mutated. Accordingly, the Irp9 K193Q and the TrpE Q262K /TrpG mutants as well as their respective ACHTUNGTRENNUNGalanine substitutions Irp9 K193A and TrpE Q262A /TrpG were constructed and over-expressed as hexahisti- dine-tagged enzymes in E. coli. The mutants were studied by 1 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 NH 4 Cl (Figure 2 D), and that anthrani- late synthase forms anthranilate in the presence of NH 4 Cl (Figure 2 G) but neither anthranilate or salicy- late in the absence of NH 4 Cl (Figure 2 H). The Irp9 K193Q mutant does not form salicylate (Fig- ure 2 E), but does form anthranilate in the presence of NH 4 Cl (Figure 2 F). By using a fluorescence assay to [a] Dr. O. Kerbarh, Dr. A. Ciulli, Prof. C. Abell Department of Chemistry, University of Cambridge Lensfield Road, Cambridge CB2 1EW (UK) Fax: (+ 44)1223 336362 E-mail: ca26@cam.ac.uk [b] Dr. D. Y. Chirgadze, Prof. T. L. Blundell Department of Biochemistry, University of Cambridge 80 Tennis Court Road, Cambridge CB2 1GA (UK) Scheme 1. Chorismate-dependent enzymatic reactions. Figure 1. Overlay of the main catalytic residues of the active sites of Y. enter- ocolitica Irp9 [5] (grey) and S. marcescens TrpE [4] (dark grey). 622 # 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2007, 8, 622 – 624