Catalytic Mechanisms of Fe(II)- and 2-Oxoglutarate-dependent Oxygenases

Abstract

Mononuclear non-heme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenases comprise a large family of enzymes that utilize an Fe(IV)-oxo intermediate to initiate diverse oxidative transformations with important biological roles. Here, four of the major types of Fe(II)/2OG-dependent reactions are detailed: hydroxylation, halogenation, ring formation, and desaturation. In addition, an atypical epimerization reaction is described. Studies identifying several key intermediates in catalysis are concisely summarized, and the proposed mechanisms are explained. In addition, a variety of other transformations catalyzed by selected family members are briefly described to further highlight the chemical versatility of these enzymes.

Keywords: 2-oxoglutarate; desaturation; dioxygenase; enzyme mechanism; halogenase; hydroxylase; iron; metalloenzyme; ring-formation.

FIGURE 1.

Examples of the diverse reactions catalyzed by Fe(II)/2OG oxygenases. The four major types of oxidative reactions are highlighted using representative enzymes as emphasized in the text, with several other types of Fe(II)/2OG-dependent chemistry only briefly discussed. A, the reaction of TauD (green highlight), a representative hydroxylase; the initial product of taurine hydroxylation is unstable and spontaneously decomposes as shown. B, the reaction catalyzed by SyrB2 (gray highlight), a model halogenase. The primary substrate of this enzyme is l-Thr in thioester linkage to a phosphopantetheine group bound to the SyrB1 protein. C, the trifunctional enzyme CAS; in addition to its oxidative ring-forming reaction (highlighted in blue), this enzyme also catalyzes hydroxylation and desaturation reactions. D, reactions of CarC, illustrating both desaturation (highlighted in pink) and epimerization (highlighted in purple). E, ring expansion reaction catalyzed by DAOCS. R = δ-(l-α-aminopropyl). F, sequential hydroxylation and epoxidation reactions of H6H. G, formation of an endoperoxide by FtmOx1. See text for definition of the abbreviations.

FIGURE 2.

Proposed mechanism of hydroxylation by Fe(II)/2OG oxygenases. Top, the structure of the archetype hydroxylase within this enzyme family, TauD, is depicted as a graphic (red, α helices; yellow, β strands, and green, loops) with 2OG and the coordinating side chains in stick mode and Fe as a sphere to illustrate the position of the metallocenter relative to the double-stranded β-helix fold (Protein Data Bank (PDB) access code 1GY9) (40). Bottom, an expanded view of the Fe(II)/2OG metallocenter surrounded by species that are proposed to be formed during the enzymatic mechanism. The green cycle illustrates the consensus view of the reaction, whereas the yellow arc depicts an alternative proposal for the final steps in the reaction. See text for details.

FIGURE 3.

Proposed mechanisms for halogenation, ring-cyclization, and desaturation reactions by Fe(II)/2OG oxygenases. A, halogenases within this family possess a 2-His-1-Cl facial triad that binds 2OG in the same manner as the hydroxylases (PDB access code 2FCT) (58). Chlorination occurs via haloferryl abstraction of a substrate hydrogen atom followed by recombination with the chloride atom (gray highlight). B, three potential mechanisms for oxidative cyclization are illustrated with CAS (blue highlight). In path a (red arrows), the ferryl intermediate abstracts a hydrogen atom from C4′ of substrate, the resulting Fe(III)-OH abstracts a hydrogen atom from the C3 hydroxyl group, and radical coupling closes the ring. In path b (cyan arrows), the ferryl intermediate again abstracts the C4′ hydrogen atom, electron transfer to the metallocenter creates a substrate carbocation, and hydroxyl anion attack completes the reaction. In path c (purple arrows), the ferryl intermediate abstracts a hydrogen atom from the C3 hydroxyl group, radical cleavage of the substrate yields aminopropanal and a carbon-centered radical that rearranges as shown, and cycloaddition provides the product. C, three possible desaturation mechanisms are illustrated for CarC (pink highlight). Path a (cyan arrows) depicts sequential hydrogen atom abstractions by ferryl and Fe(III)-OH intermediates with subsequent radical coupling. Path b (red arrows) shows hydroxyl radical rebound as found in the hydroxylases with subsequent elimination of hydroxide and rearrangement to form product. Path c (purple arrows) includes electron transfer from the substrate radical to the Fe(III)-OH, forming a carbocation that rearranges to the product. D, the epimerization mechanism proposed for CarC (purple highlight).