QM/MM Study of the Reaction Mechanism of the Dehydratase Domain from Mammalian Fatty Acid Synthase

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
(ΔrG 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.