Dehaloperoxidase (DHP) from the marine polychaete Amphitrite ornata is a multifunctional enzyme that possesses peroxidase, peroxygenase, oxidase and oxygenase activities. Previous studies have shown that DHP catalyzes the oxidation of a diverse panel of substrates, including halo/nitrophenols, haloindoles, pyrroles, guaiacols and cresols by these activities. Herein, we investigated the reactivity of DHP isoenzyme B with Bisphenol A (BPA) and its derivatives (Bisphenol E, Bisphenol F, tetrachlorobisphenol A, 2,2’-biphenol, 3,3’-biphenol, 4,4’-biphenol, and 3,3’-Br2-4,4’-biphenol). As a previously unknown class of substrate for DHP B, BPA is a representative endocrine disrupting compound and widely used in polycarbonate and epoxy resins, resulting in human exposure. Reactivity studies with these substrates were investigated using high performance liquid chromatography (HPLC), with BPA having the highest reactivity (86.0% conversion) and 3,3’-Br2-4,4’-biphenol the lowest (21.8% conversion) of the derivatives studied. The oxidation products for each substrate were determined by mass spectrometry (GC-MS/ LC-MS). 18O-labeling studies confirmed that the O atom incorporated into the products was derived exclusively from water, consistent with substrate oxidation via a peroxidase-based mechanism. The substrate-enzyme binding affinities were determined by UV-visible spectroscopy with 2,2’-biphenol exhibiting a relatively strong binding affinity (Kd = 68 µM) for DHP B when compared to previously studied substrates. The X-ray crystal structures of DHP bound with BPA (2.2 Å), Bisphenol E (3.5 Å), Bisphenol F (3.4 Å), 2,2’-biphenol (2.2 Å) and 3,3’-biphenol (3.7 Å) showed substrate binding sites are in the distal pocket of the heme cofactor. Stopped-flow UV-visible spectroscopy was utilized to investigate the mechanistic details and enzyme oxidation states during the turnover, and a preliminary reaction mechanism is proposed. The data presented here strongly suggest that DHP B could be used in bioremediation efforts of these pollutants.

Oxidation of bisphenol A (BPA) and its derivatives by the multifunctional catalytic globin dehaloperoxidase

ACS Spring 2022

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