A QM/MM study of the reaction mechanism of human β-ketoacyl reductase

Abstract

Human fatty acid synthase (hFAS) is a multifunctional enzyme involved in a wide diversity of biological functions. For instance, it is a precursor of phospholipids and other complex processes such as the de novo synthesis of long chain fatty acid. Human FAS is also a component of biological membranes and it is implicated in the overexpression of several types of cancers. In this work, we describe the catalytic mechanism of β-ketoreductase (KR), which is a catalytic domain of the hFAS enzyme that catalyzes the reduction of β-ketoacyl to β-hydroxyacyl with the concomitant oxidation of the NADPH cofactor. The catalysis by KR is an intermediate step in the cycle of reactions that elongate the substrate's carbon chain until the final product is obtained. We study and propose the catalytic mechanism of the KR domain determined using the hybrid QM/MM methodology, at the ONIOM(B3LYP/6-311+G(2d,2p):AMBER) level of theory. The results indicate that the reaction mechanism occurs in two stages: (i) nucleophilic attack by a NADPH hydride to the β-carbon of the substrate, together with an asynchronous deprotonation of the Tyr2034 by the oxygen of the β-alkoxide to hold the final alcohol product; and (ii) an asynchronous deprotonation of the hydroxyl in the NADP's ribose by Tyr2034, and of the Lys1995 by the resulting alkoxide in the former ribose to restore the protonation state of Tyr2034. The reduction step occurs with a Gibbs energy barrier of 11.7 kcal mol and a Gibbs reaction energy of -10.6 kcal mol. These results have provided an understanding of the catalytic mechanism of the KR hFAS domain, a piece of the heavy hFAS biosynthetic machinery.

The Path to Actinorhodin: Regio- and Stereoselective Ketone Reduction by a Type II Polyketide Ketoreductase Revealed in Atomistic Detail

“…Subsequently, Tyr157 moves to coordinate to a NADP+ ribose hydroxyl (Figure 6d), ready for reprotonation through a proton shuttle likely involving the ribose and Lys161. [26][27] Notably, our simulations show energetically feasible reactions whilst the cyclized octaketide is bound to actACP, confirming that the ACP-PPant tether does not need to be broken prior to ketoreduction by actKR (in contrast to what is expected for hedamycin KR). 9 The barriers to reaction are not significantly different between the three isomer-conformers, suggesting that actKR can facilitate ketoreduction to a similar extent in all three, via axial hydride attack at C9 (Scheme 1, Figure 6b-d).…”

QM Cluster or QM/MM in Computational Enzymology: The Test Case of LigW-Decarboxylase

“…A rectangular box was used to solvate the system up to 12.0 Å of the metal center. During the optimizations, all the water molecules and protein atoms in the 18 Å from the active site were kept frozen, as proposed by a recent work (Medina et al, 2017 ). The final model contains 11,895 atoms with 118 atoms of QM region.…”

Path to Actinorhodin: Regio- and Stereoselective Ketone Reduction by a Type II Polyketide Ketoreductase Revealed in Atomistic Detail

“…The main (second) transformation in act KR—its ketoreduction of act ACP- 2 —is both regio- and stereoselective, 5 producing an alcohol group on C9 ( Scheme 1 ). The first and rate-determining 26 step in this reaction involves hydride transfer from the act KR-bound NADPH to C9 7 , 27 and (asynchronous concerted) proton abstraction by O9 from act KR/Tyr157. Stabilization is provided throughout the reaction by a hydrogen bond between O9 and act KR/Ser144 ( Scheme 1 ).…”