Dehydratase (DH) is a catalytic domain of the mammalian fatty acid synthase (mFAS), a multidomain enzyme with seven different active sites that work in tandem to carry out the biosynthesis of palmitic acid for de novo lipogenesis. DH catalyzes the dehydration of the β-hydroxyacyl to an α,β-unsaturated acyl intermediate. We have conducted hybrid QM/MM calculations to clarify the catalytic mechanism for the DH domain at the ONIOM­(DFT/Amber) level of theory. The results have shown that the dehydration step occurs in two stages: (i) the His878-imidazole acts as a base deprotonating the Cα of the β-hydroxyacyl (HAC) substrate and (ii) the β-elimination of the β-hydroxyl of HAC proceeds with late protonation of the leaving hydroxide by the Asp1033-carboxylic group, forming a water molecule as a byproduct. The α-deprotonation depends on an oxyanion hole mechanism where the HAC’s α-carbonyl is anchored by two strong hydrogen bonds from the neighboring Gly888 and the intramolecular β-hydroxyl, positioning the Cα of HAC for deprotonation by His878. A positively charged His1037 improves the acidic character of Asp1033 and completes the catalytic triad in DH, because when His1037 is neutral the positively charged His878 behaves as the acid in the β-elimination step. We observe that the positively charged His1037 renders the β-elimination step more thermodynamically favorable (Δr G of −15.9 kcal·mol–1). The β-elimination step exhibits a Gibbs energy barrier of 14.1 kcal·mol–1 and it is the rate-limiting step of the reaction (in agreement with the experimental barrier of ∼17 kcal·mol–1. Nevertheless, the rate-limiting step does not seem to be dependent on the protonation of His1037. Through evaluation of the electrostatic effect per residue on the rate-limiting step, we concluded also that the electrostatic contribution of the enzyme’s body does not seem significant, even though there are many positively and negatively charged residues close to the leaving β-hydroxyl group of HAC.

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.