Probing the mechanism of flavin action in the oxidative decarboxylation catalyzed by salicylate hydroxylase

Abstract

Salicylate hydroxylase (NahG) is a FAD-dependent monooxygenase in which the reduced flavin activates O₂ coupled to the oxidative decarboxylation of salicylate to catechol or uncoupled from substrate oxidation to afford H₂O₂. This chapter presents different methodologies in equilibrium studies, steady-state kinetics, and identification of reaction products, which were important to understand the S_EAr mechanism of catalysis in NahG, the role of the different FAD parts for ligand binding, the extent of uncoupled reaction, and the catalysis of salicylate's oxidative decarboxylation. These features are likely familiar to many other FAD-dependent monooxygenases and offer a potential asset for developing new tools and strategies in catalysis.

Keywords: Activation; Biocatalysis; Flavoenzyme; Mechanism; One-component; Oxidoreductases.