Complexities of the Reaction Mechanisms of CC Double Bond Reduction in Mammalian Fatty Acid Synthase Studied with Quantum Mechanics/Molecular Mechanics Calculations

Abstract

Mammalian fatty acid synthase is a mega-enzyme responsible for de novo fatty acid biosynthesis. NADPH-dependent enoyl reductase (ER) is one of its seven different catalytic domains. The “classical” reduction mechanism of CC bonds catalysed by ER is postulated to take place through a hydride plus proton transfer to the substrate double bond. This mechanism was recently challenged because of the very unexpected experimental detection of two NADPH-substrate covalent adducts (A2 and A4) in enzymes similar to mammalian ER (mER). The fact that 16% of known enzymes used NADPH as a cofactor, mostly as a hydride donor, makes the discovery of previously unknown cofactor-substrate covalent intermediates very interesting. We studied the mechanism of reaction of ER using quantum mechanics/molecular mechanics (QM/MM) calculations, using three layers, two of them described by QM [the very accurate DLPNO-CCSD­(T)/CBS and B3LYP/6-311+G­(2d,2p)]. The rate-limiting step of the classical pathway was the formation of an enolate intermediate upon hydride transfer (ΔG ‡ of 14.7 kcal·mol–1, which is in very good agreement with the experimental value). Two alternative pathways, considering the recently detected A2 and A4 intermediates, were subsequently studied. The barriers for forming A2-wild type and A4-mutant (17.0 and 19.3 kcal·mol–1) also agreed very well with the experimental values. However, these species were incapable of forming the final product because of the very high Gibbs energy barriers to do so (>70 and >50 kcal·mol–1). They proved to be dead-end branches on the Gibbs energy surface, in chemical equilibrium with the intermediate enolate of the classical pathway. In summary, the classical reaction mechanism seems to hold in mER, but the discovery of unprecedented mechanistic hypotheses challenges the solidity and thoroughness with which we define and explore enzyme reaction mechanisms.

How the Destabilization of a Reaction Intermediate Affects Enzymatic Efficiency: The Case of Human Transketolase

“…The presence of a single imaginary vibrational frequency confirmed the nature of the transition state. Only vibrational temperatures larger than 100 K have been accounted during the calculation of entropy contribution, according with a validated procedure , and successfully proposed in other works. ,, The final free energy profiles were obtained adding the Zero Point Energy (ZPE), the empirical D3-dispersion correction, and rigid rotor/harmonic oscillator entropy contributions, as reported in Tables S2 and S3. Accurate energetic profiles were obtained through a relaxed scan along the dihedral angle involved in the distortion observed in the INT1 (C5–S–C2–C2x).…”

Animal Fatty Acid Synthase: A Chemical Nanofactory

“…This hypothesis was also anticipated in the past. 349 This result emphasizes that the experimental observation of a reaction intermediate does not necessarily mean that the intermediate belongs to the chemical pathway of the reaction. It can be a dead-end in the reaction landscape, as are the two adducts experimentally detected in the crotonyl-CoA carboxylase enzyme.…”

“…342,344 It was in this complex context that QM/MM computer simulations were carried out to try to shed some light in the controversy surrounding the true chemical mechanism of animal ER. 349 The detailed mechanistic picture that emerged is described below.…”

“…Both the "classical mechanism" and the "covalent adduct mechanism" start with the transfer of a hydride from the NADPH C 4 to the substrate C β . 349 QM/MM computer simulations have shown that the hydride transfer occurs with a Gibbs energy barrier of 14.7 kcal•mol −1 , leading to an enolate intermediate (Figure 14B).…”

Reengineering the programming of a functional domain of an iterative highly reducing polyketide synthase

“…This correlates with recent QM/ MM studies of the related vFAS ER by Fernandes and coworkers which showed typical b-carbon distances to the reactive hydride of ca 3 A, although the s-cis vs. s-trans conformation of the bound substrate was not addressed in this study. 23 In specic cases in silico modelling was veried in vitrofor example prediction that mutation F1941A would specically convert 4S,6S-tetraketide 11p from an inhibitor in the case of the WT protein to a substrate of the mutant protein was veried. Likewise, mutation F2157A increases the volume of the active site pocket and signicantly improves the substrate specicity of longer substrates.…”