Mechanistic Implications for the Chorismatase FkbO Based on the Crystal Structure

Abstract

Chorismate-converting enzymes are involved in many biosynthetic pathways leading to natural products and can often be used as tools for the synthesis of chemical building blocks. Chorismatases such as FkbO from Streptomyces species catalyse the hydrolysis of chorismate yielding (dihydro)benzoic acid derivatives. In contrast to many other chorismate-converting enzymes, the structure and catalytic mechanism of a chorismatase had not been previously elucidated. Here we present the crystal structure of the chorismatase FkbO in complex with a competitive inhibitor at 1.08Å resolution. FkbO is a monomer in solution and exhibits pseudo-3-fold symmetry; the structure of the individual domains indicates a possible connection to the trimeric RidA/YjgF family and related enzymes. The co-crystallised inhibitor led to the identification of FkbO's active site in the cleft between the central and the C-terminal domains. A mechanism for FkbO is proposed based on both interactions between the inhibitor and the surrounding amino acids and an FkbO structure with chorismate modelled in the active site. We suggest that the methylene group of the chorismate enol ether takes up a proton from an active-site glutamic acid residue, thereby initiating chorismate hydrolysis. A similar chemistry has been described for isochorismatases, albeit implemented in an entirely different protein scaffold. This reaction model is supported by kinetic data from active-site variants of FkbO derived by site-directed mutagenesis.

Comparative studies of the catalytic mechanisms of two chorismatases: CH‐fkbo and CH‐Hyg5

“…Based on the optimized reactant structure and earlier proposals, the calculated reaction path contains three distinct steps: the protonation of the methylene group of chorismate, the nucleophilic attack of carbocation and the C‐O bond cleavage of substrate, as depicted in Scheme . The reaction details will be discussed in the following subsections.…”

“…Up to now, two crystal structures of chorismatases have been determined from Streptomyces hygroscopicus by Andexer's laboratory . One is that of CH‐Fkbo in complex with its competitive inhibitor 3–(2‐carboxyethyl)‐benzoate at a resolution of 1.08 Å (PDB code 4BPS), and the other is that of CH‐Hyg5 with the same inhibitor at the resolution of 1.9 Å (PDB code 5AG3).…”

“…These three‐dimensional structures provide useful information for understanding the catalytic mechanism of chorismatases. Besides, mutagenesis and isotope analysis have also been conducted on CH‐Fkbo and CH‐Hyg5 enzymes . It was found that a glutamic residue (E338 FKB ° or E334 Hyg5 ) in the active site plays a crucial role in triggering the catalytic process in both chorismatases.…”

“…Based on the experimental results of chorismatases and other chorismate‐converting enzymes, Andexer et al proposed an isochorismatase‐like hydrolysis mechanism for CH‐Fkbo subfamily: firstly, the methylene group of chorismate is protonated by the carboxyl proton of E338 to initiate the catalytic reaction; then the generated carbocation intermediate is nucleophilically attacked by an activated water to form an unstable tetrahedral intermediate; finally, the intermediate decomposes to produce 3,4‐CHD and pyruvate. The 18 O labeled assays support this proposed mechanism.…”

“…Due to the lack of chorismate pathway in mammals, these enzymes have been exploited as attractive targets for many potential herbicides and antibiotic agents. [5][6][7] In recent years, a new class of bacterial chorismateconverting enzymes, named as chorismatases (CH), [8][9][10][11][12][13][14] have been recognized, which catalyze the hydrolysis of chorismate into pyruvate and (dihydro)-benzoic acid derivatives. According to the products of the catalytic reactions, as shown in Figure 1, these chorismatases can be divided into three subfamilies: CH-Fkbo, CH-Hyg5 and CH-XanB2.…”