Global and local molecular dynamics of a bacterial carboxylesterase provide insight into its catalytic mechanism

Abstract

Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of xenobiotics. In humans, substrates for these enzymes are far-ranging, and include the street drug heroin and the anticancer agent irinotecan. Hence, their ability to bind and metabolize substrates is of broad interest to biomedical science. In this study, we focused our attention on dynamic motions of a CE from B. subtilis (pnbCE), with emphasis on the question of what individual domains of the enzyme might contribute to its catalytic activity. We used a 10 ns all-atom molecular dynamics simulation, normal mode calculations, and enzyme kinetics to understand catalytic consequences of structural changes within this enzyme. Our results shed light on how molecular motions are coupled with catalysis. During molecular dynamics, we observed a distinct C-C bond rotation between two conformations of Glu310. Such a bond rotation would alternately facilitate and impede protonation of the active site His399 and act as a mechanism by which the enzyme alternates between its active and inactive conformation. Our normal mode results demonstrate that the distinct low-frequency motions of two loops in pnbCE, coil_5 and coil_21, are important in substrate conversion and seal the active site. Mutant CEs lacking these external loops show significantly reduced rates of substrate conversion, suggesting this sealing motion prevents escape of substrate. Overall, the results of our studies give new insight into the structure-function relationship of CEs and have implications for the entire family of α/β fold family of hydrolases, of which this CE is a member.

Computational Studies of Glycoside, Carboxylic Ester, and Thioester Hydrolase Mechanisms: A Review

“…183 A C−C bond rotation of Glu310 can facilitate or impede protonation of the active-site His399 residue, causing the enzyme to alternate between active and inactive conformations. Low-frequency motions of two loops are important in substrate conversion by sealing the active site and preventing substrate escape.…”

“…MD simulation, normal mode calculations, and enzyme kinetics were employed by Yu et al to understand how structural changes in Bacillus subtilis CE affected its catalysis. 183 A C−C bond rotation of Glu310 can facilitate or impede protonation of the active-site His399 residue, causing the enzyme to alternate between active and inactive conformations. Low-frequency motions of two loops are important in substrate conversion by sealing the active site and preventing substrate escape.…”

Molecular Dynamics Investigation of the Substrate Binding Mechanism in Carboxylesterase

“…14 All-atom MD simulation of pnb-CE by Wadkins et al has shown that the dynamic motions of coil_5 (residues 61−82) and coil_21 (residues 408−422) located on the surface of pnbCE are critical to the substrate binding. 15 Here, we performed all-atom MD simulations of the wild type and mutant rPPE enzymes, both apo and in complex with (S)-AcO-CPA, to investigate the binding mechanisms of the substrate in different enzymes. Our studies provide insights into why the catalytic activity of the W187H mutant has increased from the perspective of the substrate binding.…”

“…From the analysis of the crystal structure of a Bacillus subtilis CE (pnb-CE), the “side-door” residues were found to be important for the metabolism of a water-soluble anticancer prodrug CPT-11 (7-ethyl-10-[4-(1-piperidino)-1-piperidino]­carbonyloxycamptothecin) . All-atom MD simulation of pnb-CE by Wadkins et al has shown that the dynamic motions of coil_5 (residues 61–82) and coil_21 (residues 408–422) located on the surface of pnbCE are critical to the substrate binding …”