Dissecting the Mechanism of the Nonheme Iron Endoperoxidase FtmOx1 Using Substrate Analogues

Abstract

FtmOx1 is a nonheme iron (NHFe) endoperoxidase, catalyzing three disparate reactions, endoperoxidation, alcohol dehydrogenation, and dealkylation, under in vitro conditions; the diversity complicates its mechanistic studies. In this study, we use two substrate analogues to simplify the FtmOx1-catalyzed reaction to either a dealkylation or an alcohol dehydrogenation reaction for structure–function relationship analysis to address two key FtmOx1 mechanistic questions: (1) Y224 flipping in the proposed COX-like model vs α-ketoglutarate (αKG) rotation proposed in the CarC-like mechanistic model and (2) the involvement of a Y224 radical (COX-like model) or a Y68 radical (CarC-like model) in FtmOx1-catalysis. When 13-oxo-fumitremorgin B ( 7 ) is used as the substrate, FtmOx1-catalysis changes from the endoperoxidation to a hydroxylation reaction and leads to dealkylation. In addition, consistent with the dealkylation side-reaction in the COX-like model prediction, the X-ray structure of the FtmOx1•Co II •αKG• 7 ternary complex reveals a flip of Y224 to an alternative conformation relative to the FtmOx1•Fe II •αKG binary complex. Verruculogen ( 2 ) was used as a second substrate analogue to study the alcohol dehydrogenation reaction to examine the involvement of the Y224 radical or Y68 radical in FtmOx1-catalysis, and again, the results from the verruculogen reaction are more consistent with the COX-like model.

Unusual Dioxygen-Dependent Reactions Catalyzed by Nonheme Iron Enzymes in Natural Product Biosynthesis

“… Accordingly, NvfI introduces three oxygen atoms in one catalytic cycle of the nonheme iron enzyme. This feature of the NvfI reaction is different from the reaction of the previously characterized Fe­(II)/αKG-dependent fumitremorgin B endoperoxidase FtmOx1, which only installs the endoperoxide group without forming an additional hydroxyl group. − Furthermore, assays using 18 O 2 and H 2 18 O suggested that the two oxygen atoms in the endoperoxide are derived from molecular oxygen while the oxygen atom in the hydroxyl group is derived from water, indicating that solvent exchange processes occur during the NvfI reaction.…”

“…Alternatively, the hydroxylation could occur via the hydrolysis of 60 , a hypothetical carbocation intermediate. In the related reaction of FtmOx1, the active site tyrosine residues Tyr224 and Tyr68 have been proposed to play key roles in H atom abstraction from the substrate or H atom donation to the radical intermediate. , However, the substitution of the active side tyrosine residue in NvfI did not abolish the endoperoxide activity of this enzyme. Thus, NvfI likely uses a strategy different from that of FtmOx1 to perform the iron-dependent endoperoxidation reaction …”

“…148 Another complex rearrangement reaction has been identified in the biosynthetic pathway of funiculolides, which are also fungal meroterpenoids discovered through a genome mining approach (Scheme 12B). 149 Investigation of the funiculolide (f nc) biosynthetic gene cluster revealed that an Fe(II)/αKGdependent enzyme FncG catalyzes the conversion of funiculolide B (111) to funiculolide D (115), which involves hydroxylation and C−C bond cleavage and formation steps (Scheme 12B). 149 FncG first catalyzes C6 hydroxylation to give a detectable alcohol intermediate.…”

Study and design of amino acid-based radical enzymes using unnatural amino acids

“…76 Liu and coworkers further argued that the COX-like mechanism is more feasible. 77 FtmOx1 is a tri-functional enzymes with endoperoxidation, alcohol dehydrogenation and dealkylation activities, which complicates mechanism investigation. By designing and synthesis of a substrate analogue, 13-oxofumitremorgin, and use it for crystallization and reaction, they can decouple the activities of FtmOx1 and showed structures and reactions that support rotation of Y224 during the reaction, favouring the original COX-like mechanism.…”

Chemoenzymatic Synthesis of 13-Oxoverruculogen

“…Buffer exchange with HEPES solvent or removal of L-ascorbate both resulted in no reaction. While Liu and co-workers have reported that the absence of L-ascorbate and the use of stoichiometric enzyme results in direct oxidation of fumitremorgin B to 13-oxoverruculogen, 24 13-epi-fumitremorgin B did not react in the absence of L-ascorbate with aerobically purified protein. Contrary to our work, their preparation of FtmOx1 was under strictly anaerobic conditions, and their reaction used EDTA treated protein without a hexahistidine tag.…”