Catalytic Cycle of Penicillin Acylase from Escherichia coli: QM/MM Modeling of Chemical Transformations in the Enzyme Active Site upon Penicillin G Hydrolysis

Abstract

Penicillin acylase from Escherichia coli is a unique enzyme that belongs to the recently discovered superfamily of N-terminal nucleophile hydrolases. It catalyzes selective hydrolysis of the side chain amide bond of penicillins and cephalosporins while leaving the labile amide bond in the βlactam ring intact. Despite wide applications of penicillin acylase in the industry of β-lactam antibiotics and production of chiral amino compounds, its catalytic mechanism at atomic resolution has not yet been characterized. The complete cycle of chemical transformations of the most specific substrate of the enzyme, penicillin G, leading to formation of 6-aminopenicillanic and phenylacetic acids was modeled following quantum mechanics−molecular mechanics (QM/MM) calculations of the minimum energy reaction profile. The active site residues and the substrate were included in the QM part, and the rest of the system was treated applying molecular mechanics and classical force field parameters. The 3D structures in the enzyme active site corresponding to the noncovalent enzyme−substrate complex, the covalent acylenzyme intermediate, the noncovalent enzyme−product complex, the tetrahedral intermediates, and the respective transition states have been identified. QM/MM studies have shown that the α-amino group of the N-terminal catalytic βSer1 plays a key role in the catalytic machinery and directly assists its hydroxyl group in a proton relay at major stages of penicillin acylase catalytic mechanism, formation and hydrolysis of the covalent acylenzyme intermediate, which are characterized by close energy barriers. The βSer1 residue together with the oxyanion hole residues βAla69 and βAsn241 as well as βArg263 and βGln23 constitute a buried active site interaction network responsible for stabilization of tetrahedral intermediates, transition states, orientation of substrate and catalytic residues. βArg263 and βGln23 maintain the integrity of the catalytic machinery: βArg263 participates in orientation of the substrate as well as the α-amino group of βSer1 and coordinates the oxyanion hole residue βAsn241 across the whole catalytic cycle, whereas the backbone of βGln23 is responsible for orientation of both the βSer1 and the substrate. These results deliver insight into the earlier unknown ability of Nterminal amino acid to activate its own nucleophilic group directly as well as into organization of the stabilizing interaction network in penicillin acylase's active site and will be used to design more effective enzyme variants for synthesis of new penicillins and cephalosporins.

Analysis of proton wires in the enzyme active site suggests a mechanism of c‐di‐GMP hydrolysis by the EAL domain phosphodiesterases

“…33,34 This method was successfully employed in numerous applications, including simulations of the c-GMP hydrolysis in water, 19 studies of proton transfer routes in the photocycle of the green fluorescent protein, 35 modeling chemical transformations upon penicillin G hydrolysis by penicillin acylase. 36 In every case a reaction mechanism was revealed by computing a set of stationary points on the potential energy surface corresponding to reaction intermediates and transition states. When moving along a selected reaction coordinate a current value of this coordinate was fixed and positions of all protein atoms except those on the exterior of a protein were optimized in QM/MM calculations.…”

Structural analysis of a penicillin V acylase from Pectobacterium atrosepticum confirms the importance of two Trp residues for activity and specificity

“…Penicillin acylases also undergo post-translational autocatalytic processing to expose the N-terminal residue, the a-amino group of this residue acts as the base in the catalytic reaction Grigorenko et al, 2014). However, the processing mechanism differs in different penicillin acylases.…”

Ring Contraction Catalyzed by the Metal-Dependent Radical SAM Enzyme: 7-Carboxy-7-deazaguanine Synthase from B. multivorans. Theoretical Insights into the Reaction Mechanism and the Influence of Metal Ions

“…To answer these questions, in this work, the combined quantum mechanics and molecular mechanics (QM/MM) method 33,34 has been employed to explore the catalytic mechanism of QueE from B. multivorans. The QM/MM method has been widely applied in elucidating the catalytic mechanism of extended systems, 35 29 provided good templates for our theoretical studies. Both structural and biochemical analysis indicate that QueE is a homodimer with two identical monomers, and two identical active sites locate at the two monomers, respectively.…”