Modification of Residue 42 of the Active Site Loop with a Lysine-Mimetic Side Chain Rescues Isochorismate-Pyruvate Lyase Activity in Pseudomonas aeruginosa PchB

Abstract

PchB is an isochorismate-pyruvate lyase from Pseudomonas aeruginosa. A positively charged lysine residue is located in a flexible loop that behaves as a lid to the active site, and the lysine residue is required for efficient production of salicylate. A variant of PchB that lacks the lysine at residue 42 has a reduced catalytic free energy of activation of up to 4.4 kcal/mol. Construction of a lysine isosteric residue bearing a positive charge at the appropriate position leads to the recovery of 2.5–2.7 kcal/mol (about 60%) of the 4.4 kcal/mol by chemical rescue. Exogenous addition of ethylamine to the K42A variant leads to a neglible recovery of activity (0.180 kcal/mol, roughly 7% rescue), whereas addition of propylamine caused an additional modest loss in catalytic power (0.056 kcal/mol, or −2% loss). This is consistent with the view that (a) the lysine-42 residue is required in a specific conformation to stabilize the transition state and (b) the correct conformation is achieved for a lysine-mimetic sidechain at site 42 in the course of loop closure, as expected for transition-state stabilization by the side chain ammonio-function. That the positive charge is the main effector of transition state stabilization is shown by the construction of a lysine-isosteric residue capable of exerting steric effects and hydrogen bonding but not electrostatic effects, leading to a modest increase of catalytic power (0.267 – 0.505 kcal/mol of catalytic free energy, or roughly 6 – 11% rescue).

Mechanism-based site-directed mutagenesis to shift the optimum pH of the phenylalanine ammonia-lyase from Rhodotorula glutinis JN-1

“…6 ). The optimum pH of an enzyme depends on the ionizable amino acids at active site which are involved in catalysis [10] , [11] or stabilization of transition state by electrostatic interaction [17] , [18] and enzyme substrate binding [6] . The Glu137 might be involved in the stabilization of transition state by electrostatic interaction.…”

“…The improvements of Rg PAL-Q137E at pH 7 may be due to the negative charge of Glu137 which facilitates stabilization of carbocation intermediate to reduce the energy barrier through electrostatic interaction. The k cat value provides an assessment of the specificity and is directly related to the free energy of activation of the transition state [8] , [17] , [18] . Therefore, the calculation of mutational effects using k cat provides insights on the contribution of electrostatics.…”

“…The “mutational effect” was determined by dividing the k cat value of the mutant enzyme by that of the wild type, and the free energy (ΔΔG ‡ ) was calculated from the following equation: ΔΔG ‡ = −RTln (mutational effect) as described by Olucha (2011, 2012) [17] , [18] .…”

Chorismate- and isochorismate converting enzymes: versatile catalysts acting on an important metabolic node

“…4A and B). [62][63][64]66,67 In SPR enzymes the chemical structure of the product formed is controlled by an intricate network of mainly positively and negatively charged amino acid residues in the active site. These residues form an electrostatic gradient over the active site that promotes product formation.…”

“…In a concerted, asynchronous reaction, the C-O bond between C3 of the cyclohexadiene ring and the enol ether is broken during pyruvate formation and aromatisation yielding salicylate (Scheme 1D). 8,21,59,66 In chorismate mutases the C4-hydroxyl group of the cyclohexadiene ring is additionally stabilised by a highly conserved glutamate residue, while isochorismate-pyruvate lyases lack this residue. The resulting lower density of the electrostatic network in isochorismate-pyruvate lyases is assumed to be the reason for the promiscuity of this subfamily in contrast to chorismate mutases (Fig.…”