Transition Path Sampling Study of the Feruloyl Esterase Mechanism

Abstract

Serine hydrolases cleave peptide and ester bonds and are ubiquitous in nature, with applications in biotechnology, in materials, and as drug targets. The serine hydrolase two-step mechanism employs a serine–histidine–aspartate/glutamate catalytic triad, where the histidine residue acts as a base to activate poor nucleophiles (a serine residue or a water molecule) and as an acid to allow the dissociation of poor leaving groups. This mechanism has been the subject of debate regarding how histidine shuttles the proton from the nucleophile to the leaving group. To elucidate the reaction mechanism of serine hydrolases, we employ quantum mechanics/molecular mechanics-based transition path sampling to obtain the reaction coordinate using the Aspergillus niger feruloyl esterase A (AnFaeA) as a model enzyme. The optimal reaction coordinates include terms involving nucleophilic attack on the carbonyl carbon and proton transfer to, and dissociation of, the leaving group. During the reaction, the histidine residue undergoes a reorientation on the time scale of hundreds of femtoseconds that supports the “moving histidine” mechanism, thus calling into question the “ring flip” mechanism. We find a concerted mechanism, where the transition state coincides with the tetrahedral intermediate with the histidine residue pointed between the nucleophile and the leaving group. Moreover, motions of the catalytic aspartate toward the histidine occur concertedly with proton abstraction by the catalytic histidine and help stabilize the transition state, thus partially explaining how serine hydrolases enable poor nucleophiles to attack the substrate carbonyl carbon. Rate calculations indicate that the second step (deacylation) is rate-determining, with a calculated rate constant of 66 s–1. Overall, these results reveal the pivotal role of active-site dynamics in the catalytic mechanism of AnFaeA, which is likely similar in other serine hydrolases.

Reaction Mechanism of MHETase, a PET Degrading Enzyme

“…In addition to the last energy barrier, we further detected the formation of a tetrahedral intermediate TI1 (Figure ), analogous to those reported in the literature for serine hydrolases both computationally and experimentally, − corresponding to a local minimum of 1.97 ± 0.17 kcal·mol –1 relative to the subsequent transition state (TS2) in the free energy curve. The energy barrier for the resolution of TI1 is above RT and, according to the k cat value estimated from transition state theory, would have a lifetime half-life of about 3 ps.…”

“…Although that the species is short-lived and may even be an artifact of the B3LYP density functional, the fact that the tetrahedral intermediate is found toward the product state is an important indicator that the oxyanion hole provided by high p K a (backbone) amide N–H groups can be used to design enzymes with improved rates toward polymer degradation. This finding matched the results observed with enzymes that catalyze the same type of reaction, − albeit tetrahedral intermediates for MHETase have yet been detected with experimental techniques and will not be easy given their short lifetime.…”

Crystal structure of the feruloyl esterase from Lentilactobacillus buchneri reveals a novel homodimeric state

“…A histidine-ring flip mechanism has been proposed based on NMR studies for the protonation of the leaving group ( Ash et al, 2000 ). Recently, results from quantum mechanics/molecular mechanics (QM/MM)-based transition-path sampling (TPS) on A. niger FaeA has provided strong support for an alternative mechanism whereby the histidine shuttles a proton from the nucleophile to the leaving group as a concerted event without ring flip ( Silveira et al, 2021 ). In our proposed interaction network, His233 would indeed be suitably oriented to shuttle a proton from Ser114 to the leaving group in a concerted mechanism without the need for conformational changes.…”

Perspective: Path Sampling Methods Applied to Enzymatic Catalysis

“…The past 3 years has seen a significant expansion in the application of TPS to understanding enzymatic chemistry. We count 9 separate systems from 6 separate groups ,− being studied in this fashion. The technique is clearly gaining in importance, but there remain basic hurdles to more general application.…”