Direct Detection and Kinetic Analysis of Covalent Intermediate Formation in the 4-Amino-4-deoxychorismate Synthase Catalyzed Reaction

Abstract

Chorismate-utilizing enzymes catalyze diverse reactions, providing critical physiological functions unique to plants, bacteria, fungi, and some parasites. Their absence in animals makes them excellent targets for antimicrobials and herbicides. 4-Amino-4-deoxychorismate synthase (ADCS) catalyzes the first step in folate biosynthsis and shares a common core mechanism with isochorismate synthase (IS) and anthranilate synthase (AS), in which nucleophile addition at C2 initiates these reactions. Evidence was presented previously [He, Z., Stigers Lavoie, K. D., Bartlett, P. A., and Toney, M. D. (2004) J. Am. Chem. Soc. 126, 2378-2385] that K274 is the nucleophile in ADCS, implying formation of a covalent intermediate. Herein, we report the direct detection of this covalent intermediate formed in ADCS-catalyzed reactions by ESI-MS. Difference spectra show the covalent intermediate has an absorption maximum at 310 nm. This was used to study the pre-steady-state kinetics of covalent intermediate formation under various conditions. Additionally, E258 in ADCS was shown to be critical to formation of the covalent intermediate by acting as a general acid catalyst for loss of the C4 hydroxyl group. The E258A/D mutants both exhibit very low activity. Acetate is a poor chemical rescue agent for E258D but an excellent one for E258A, with a 20000-fold and 3000-fold rate increase for Gln-dependent and NH(4)(+)-dependent activities, respectively. Lastly, A213 in IS (structurally homologous to K274 in ADCS) was changed to lysine in an attempt to convert IS to an ADCS-like enzyme. HPLC studies support the formation of a covalent intermediate with this mutant.

Biosynthesis of the enediyne antitumor antibiotic C-1027 involves a new branching point in chorismate metabolism

“…6 and SI Table 1), including the His residue proposed to participate in pyruvate elimination. A different mutagenesis study, using E. coli ADC synthase, was also successful in generating an artificial ADIC synthase (27,42). Based on structural analysis of ADC synthase (25,27), it was predicted that the -amino group of an active site Lys is the nucleophile analogous to the free amine for ASI and SgcD that attacks C2 of chorismate, which, in the case of ADC synthase, then undergoes a second S N 2Љ addition at C4 with concomitant release of Lys to yield ADC.…”

The Structure of MbtI from Mycobacterium tuberculosis , the First Enzyme in the Biosynthesis of the Siderophore Mycobactin, Reveals It To Be a Salicylate Synthase

“…However, His334 is on the mobile ␤16-␤17 loop and Thr271 on the adjacent ␤14 strand, so are both swung away from the active site in this "open" form; a ligand-binding role for these residues may be the major factor in loop closure. Adjacent to this part of the active site is Ala269, which is conserved as alanine in the Irp9 and TrpE enzymes but corresponds to Lys274 in PabB, where it has been proposed to discriminate between C-2 and C-4 substitution on chorismate substrates (39) and has recently been shown to be of critical importance for catalytic activity, as it forms a covalent intermediate during the reaction (3,4,27).…”

Structure of isochorismate synthase DhbC fromBacillus anthracis

“…1) and its dependence on Mg 2+ ions. The assay monitors the formation of isochorismate by measuring the increase in absorbance at 278 nm (He & Toney, 2006). The kinetic assay was performed using a PHERAstar FS microplate reader at 303 K for 10 min.…”

“…(Li et al, 2011) 16 vation of both of these residues in DhbC, EntC and MenF indicates their importance in sustaining isochorismate synthase activity. Lys147 of E. coli (Lys142* in B. anthracis) is thought to act as a catalytic base by activating a nucleophilic water molecule which is also hydrogenbonded to the C2 hydroxyl group of isochorismate (He & Toney, 2006). This residue is substituted by Glu182 in the ADC synthase from C. hutchinsonii (PDB entry 3h9m; New York SGX Research Center for Structural Genomics, unpublished work) and by Gln210 in that from E. coli (PDB entry 1k0e; Parsons et al, 2002).…”