Computational Analysis of the Nicotine Oxidoreductase Mechanism by the ONIOM Method.

Ibrahim Yildiz

doi:10.1021/acsomega.1c03357

Abstract

Nicotine oxidoreductase (NicA2) is a monoamine oxidase (MAO)-based flavoenzyme that catalyzes the oxidation of S-nicotine into N-methylmyosmine. Due to its nanomolar binding affinity toward nicotine, it is seen as an ideal candidate for the treatment of nicotine addiction. Based on the crystal structure of the substrate-bound enzyme, hydrophobic interactions mainly govern the binding of the substrate in the active site through Trp108, Trp364, Trp427, and Leu217 residues. In addition, Tyr308 forms H-bonding with the pyridyl nitrogen of the substrate. Experimental and computational studies support the hydride transfer mechanism for MAO-based enzymes. In this mechanism, a hydride ion transfers from the substrate to the flavin cofactor. In this study, computational models involving the ONIOM method were formulated to study the hydride transfer mechanism based on the crystal structure of the enzyme–substrate complex. The geometry and energetics of the hydride transfer mechanism were analyzed, and the roles of active site residues were highlighted.

Highlights

Nicotine oxidoreductase (NicA2) was identified as the primary enzyme in the S16 bacterium that degrades S-nicotine into fumaric acid in a number of steps.[1]
From collective experimental and computational studies for monoamine oxidase (MAO) enzymes,[4−8] in particular for L-6-hydroxynicotine oxidase (LHNO)[9−12] a flavoenzyme structurally related to NicA2, the direct hydride transfer mechanism from deprotonated S-nicotine to FAD is proposed for NicA2.2,3 Based on this mechanism, a hydride anion is transferred from the α-carbon of S-nicotine (1 in Figure 1) to the N5 nitrogen of the isoalloxazine ring at FAD, forming reduced FAD (FADH−) and an iminium intermediate, Nmethylmyosmine (2 Figure 1)
Computational studies for the hydride transfer mechanism for a number of enzymes provided useful insights into understanding the mechanism as well as the contribution of the active site residues.[12,26−31] ONIOM calculations involve a hybrid method which is composed of quantum mechanics (QM)- and molecular mechanics (MM)-generated invaluable mechanistic information for a variety of enzymatic systems.[32−37] With this method, the substrate and active site residues are treated with density functional theory (DFT) functionals, whereas a model region around the active site is considered with MM force fields

Summary

INTRODUCTION

Nicotine oxidoreductase (NicA2) was identified as the primary enzyme in the S16 bacterium that degrades S-nicotine into fumaric acid in a number of steps.[1]. Computational studies for the hydride transfer mechanism for a number of enzymes provided useful insights into understanding the mechanism as well as the contribution of the active site residues.[12,26−31] ONIOM calculations involve a hybrid method which is composed of quantum mechanics (QM)- and molecular mechanics (MM)-generated invaluable mechanistic information for a variety of enzymatic systems.[32−37] With this method, the substrate and active site residues are treated with density functional theory (DFT) functionals, whereas a model region around the active site is considered with MM force fields In this regard, the protein environment around the active site as well as chemical interactions with the surrounding residues is taken into consideration. The role of active site residues was highlighted, and the energetics of the hydride transfer was evaluated by calculating the activation

COMPUTATIONAL DETAILS AND METHODOLOGY

RESULTS AND DISCUSSION

CONCLUSIONS

■ REFERENCES