catalytic mechanism, the two carboxyl groups of chorismate form a H-bond network with residues R228, Y215, Y155, and R162. These H-bonds are maintained during the whole catalytic process, which may play a role in stabilizing the negative charged substrate. The protonated E338 builds a strong H-bond with the 4-OH of chorismate. The distance between HA of E338 and C3' of chorismate is 4.00 A. Two water molecules enter the reactive center, which may function as mediators in the catalytic reaction. The reaction steps are protonation of methylene group, followed by nucleophilic attack of carbocation by (activated) water molecule W1, and cleavage of C2'-O8 bond
for the CH-FkbO subfamily, an isochorismatase-like hydrolysis mechanism is suggested where the acidic active site residue E338FkbO initially protonates the C3' of chorismate. This resulting carbocation intermediate is nucleophilically attacked by (activated) water leading to the formation of an unstable tetrahedral intermediate (hemiketal), which decomposes spontaneously into the products 3,4-trans-dihydroxy-cyclohexa-1,5-dienecarboxylate and pyruvate. Hydrolysis reaction mechanism analysis, overview
reaction steps are: 1. the protonation of the methylene group of chorismate, 2. nucleophilic attack by activated water at the C2' carbocation, and 3. the elimination of pyruvate
three subfamilies of chorismatases are described that convert chorismate into different (dihydro-)benzoate derivatives (CH-FkbO, CH-Hyg5, and CH-XanB2). The CH-FkbO and CH-Hyg5 subfamilies share the same protein fold, but employ fundamentally different reaction mechanisms, comparisons of the reaction mechanism of CH-FkbO and CH-Hyg5 and structure-function analysis, overview
analysis of catalytic mechanisms of chorismatases EC 3.3.2.13 and EC 4.1.3.45 by molecular dynamics simulations and hybrid quantum mechanical/molecular mechanical (QM/MM) calculations of the Michaelis complexes of two wild-type models (FkbO and Hyg5) and four mutant models with chorismate as substrate, comparison of the catalytic mechanisms between FkbO and Hyg5, overview. The A/G residue group (A244FkbO/G240Hyg5) causes changes in the binding states of the substrate and the orientation of the catalytic glutamate, but only these changes affect the product selectivity in chorismatases limitedly. The distal V/Q residue group, which determines the internal water self-regulating ability at the active site, has significant impact on the selectivity of the catalytic mechanisms. The V/Q residue group is suggested to be an important factor to control the catalytic activities in chorismatases
structure of Fkbo-substrate complex, the solvated model of CH-Fkbo is optimized to calculate the catalytic mechanism. In the reactant structure, the substrate chorismate takes the similar binding model as inhibitor 3-(2-carboxyethyl)benzoate in crystal structure and adopts a trans-pseudodiaxial conformation. The structure of Streptomyces hygroscopicus chorismatase CH-Hyg5 is very similar to that of Streptomyces hygroscopicus chorismatase CH-Fkbo, all amino acid residues in the active site are the same except residues G240Hyg5 (A244Fkb8) and C327Hyg5 (A331Fkb8) are the same. The optimized active site structure of Hyg5 complex is superpositioned with that of Fkbo, overview. Two nonconserved active site residues are responsible for the different reaction mechanism of CH-Fkbo and CH-Hyg5. In CH-Hyg5, the lack of methyl in G240Hyg5 leads to the different conformation of the side chain of E334 (or E338) in Hyg5 and Fkbo. This small change eventually decreases the ESP charge of C3 atom of the substrate, which facilitates the cleavage of C3-O8 bond in Hyg5. Furthermore, C327Hyg5 is involved in the selective product formation in Hyg5 enzyme
structure of Fkbo-substrate complex, the solvated model of CH-Fkbo is optimized to calculate the catalytic mechanism. In the reactant structure, the substrate chorismate takes the similar binding model as inhibitor 3-(2-carboxyethyl)benzoate in crystal structure and adopts a trans-pseudodiaxial conformation. The structure of Streptomyces hygroscopicus chorismatase CH-Hyg5 is very similar to that of Streptomyces hygroscopicus chorismatase CH-Fkbo, all amino acid residues in the active site are the same except residues G240Hyg5 (A244Fkb8) and C327Hyg5 (A331Fkb8) are the same. The optimized active site structure of Hyg5 complex is superpositioned with that of Fkbo, overview. Two nonconserved active site residues are responsible for the different reaction mechanism of CH-Fkbo and CH-Hyg5. In CH-Hyg5, the lack of methyl in G240Hyg5 leads to the different conformation of the side chain of E334 (or E338) in Hyg5 and Fkbo. This small change eventually decreases the ESP charge of C3 atom of the substrate, which facilitates the cleavage of C3-O8 bond in Hyg5. Furthermore, C327Hyg5 is involved in the selective product formation in Hyg5 enzyme
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
in complex with 3-(2-carboxyethyl)benzoate, hanging drop vapor diffusion method, using 0.1 M 4-morpholineethanesulfonic acid (pH 6.5) and 20-25% (w/v) polyethylene glycol 4000
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