QM/MM Study of Tungsten-Dependent Benzoyl-Coenzyme A Reductase: Rationalization of Regioselectivity and Predication of W vs Mo Selectivity

Abstract

The class II benzoyl-coenzyme A reductase (BCR) is a tungsten-dependent enzyme that catalyzes the Birch reduction of benzoyl-CoA to a cyclic diene. The reaction mechanism and regioselectivity of benzoyl-CoA were explored through QM/MM calculations using two different QM regions (124 atoms and 223 atoms) on the solvated enzyme. The reduction reaction involves two major chemical steps that both proceed in the triplet state or in the broken-symmetry singlet state. First, the tungsten-bound water molecule delivers a proton to the C4 of the benzoyl-CoA substrate, coupled with an electron transfer from the W center to the substrate. This leads to the formation of a W-radical intermediate, with a barrier of 23.2 kcal/mol in the broken-symmetry singlet state at the B3LYP-D3/def2-TZVPP:Charmm level. Subsequently, the protonated His260 residue delivers a second proton to C3 of the benzoyl-CoA substrate, concomitantly with the shift of the second electron from the pyranopterin cofactor rather than the W ion to the substrate, which has a barrier of 19.1 kcal/mol at the B3LYP-D3/def2-TZVPP:Charmm level and produces the cyclohexa-1,5-diene-1-carboxyl-CoA product. The reduction of the aromatic ring at other positions has also been considered; however, the barriers are much higher, which shows that the cyclohexa-1,5-diene-1-carboxyl-CoA product is exclusively formed during the benzoyl-CoA reduction. Moreover, molybdenum, tungsten's lighter congener, has also been considered to replace the tungsten ion in the benzoyl-CoA reductase. The molybdenum substituted enzyme (Mo-BCR) was found to have a quite higher barrier for the reduction reaction, but a feasible barrier for the reverse oxidation reaction.

Computational Understanding of the Selectivities in Metalloenzymes

“…The mechanism and selectivity of three different tungstoenzymes have been subjected to QM and QM/MM calculations, namely, acetylene hydratase (Liao et al, 2010; Liao and Himo, 2011), formaldehyde oxidoreductase (Liao et al, 2011b; Liao, 2013) and benzoyl CoA reductase (Culka et al, 2017; Qian and Liao, 2018).…”

“…Very recently, Qian and Liao performed QM/MM calculations (B3LYP-D3/def2-TZVPP:Charmm) to rationalize the regioselectivity of the benzoyl CoA reductase (Qian and Liao, 2018). They found a similar mechanism (Figure 15) as suggested by Culka et al (2017) on the basis of QM/MM calculations.…”

Revealing the Mechanism of Isethionate Sulfite-Lyase by QM/MM Calculations

“…The single-conformation QM/MM method − uses an X-ray structure corresponding to the time and space average of conformations taken over the protein to construct a computational model. The results obtained based on this method in QM/MM calculations are generally consistent with the experimentally observed kinetics. , In this study, the crystal structure of isethionate-bound IseG (PDB code 5YMR) was used to build up the model.…”

“…The single-conformation QM/MM method − uses an X-ray structure corresponding to the time and space average of conformations taken over the protein to construct a computational model. The results obtained based on this method in QM/MM calculations are generally consistent with the experimentally observed kinetics. , In this study, the crystal structure of isethionate-bound IseG (PDB code 5YMR) was used to build up the model. The crystal structure contains four monomers per asymmetric unit, and the active sites located at all four monomers are almost identical (see S1.1 and Figure S1).…”

“…In the past several decades, quantum mechanics/molecular mechanics (QM/MM) methods − have been used to elucidate the reaction mechanisms of many enzymes, including GREs such as 4-hydroxyl­phenyl­acetate decarboxylase and the B 12 -independent glycerol dehydratase . With the goal of advancing the mechanistic understanding of enzymatic isethionate degradation, here, we utilized the single-conformation QM/MM method − (see section ) to investigate the reaction mechanism of IseG, which can be compared with related GREs and B 12 -dependent enzymes. The previously suggested direct elimination mechanism is found unfavorable according to our calculations, and a new mechanism involving a 1,2-SO 3 – radical migration is suggested instead.…”