Catalytic mechanism of acetolactate decarboxylase from Brevibacillus brevis towards both enantiomers of α-acetolactate

Acetolactate decarboxylase catalyzes both enantiomers of α-acetolactate to give a single product, (R)-acetoin, however, the reaction details are still ambiguous. In this paper, the catalytic mechanism of ALDC using both enantiomers of α-acetolactate as substrates has been investigated by means of the combined quantum mechanical/molecular mechanical (QM/MM) approach based on the recently obtained crystal structures of ALDC in complex with the designed transition state mimics. The conversion of (S)-α-acetolactate only contains two elementary steps: the direct decarboxylation of the substrate to form an enolate intermediate, and the protonation of the intermediate to generate the final product. The decarboxylation corresponds to an energy barrier of 13.5 kcal mol−1. In the protonation process, E253 is suggested to be the more likely proton donor, and the overall energy barrier of the catalytic reaction is 23.1 kcal mol−1. The direct conversion of the non-natural substrate (R)-α-acetolactate is calculated to be difficult. It should be firstly rearranged to the natural substrate (S)-α-acetolactate by a carboxylate migration, then the converted substrate undergoes a rotation to enter the decarboxylation manifold of (S)-α-acetolactate. Since the energy barrier of carboxylate migration of (R)-AL is calculated to be only 11.2 kcal mol−1, considering the fact that the conversion of (R)-α-acetolactate to (R)-acetoin by ALDC is at a lower rate, the weak binding of (R)-α-acetolactate in the active site is thus suggested to be the main factor to lower its conversion rate.