Since their discovery in the 1960s, the family of Fe(II)/2-oxoglutarate-dependent oxygenases has undergone a tremendous expansion to include enzymes catalyzing a vast diversity of biologically important reactions. Recent examples highlight roles in controlling chromatin modification, transcription, mRNA demethylation, and mRNA splicing. Others generate modifications in tRNA, translation factors, ribosomes, and other proteins. Thus, oxygenases affect all components of molecular biology's central dogma, in which information flows from DNA to RNA to proteins. These enzymes also function in biosynthesis and catabolism of cellular metabolites, including antibiotics and signaling molecules. Due to their critical importance, ongoing efforts have targeted family members for the development of specific therapeutics. This review provides a general overview of recently characterized oxygenase reactions and their key biological roles.

The ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2 is a member of the mononuclear non-heme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenase superfamily. EFE converts 2OG into ethylene plus three CO2 molecules while also catalyzing the C5 hydroxylation of L-arginine (L-Arg) driven by the oxidative decarboxylation of 2OG to form succinate and CO2. Here we report eleven X-ray crystal structures of EFE that provide insight into the mechanisms of these two reactions. Binding of 2OG in the absence of L-Arg resulted in predominantly monodentate metal coordination, distinct from the typical bidentate metal-binding species observed in other family members. Subsequent addition of L-Arg resulted in compression of the active site, a conformational change of the carboxylate side chain metal ligand to allow for hydrogen bonding with the substrate, and creation of a twisted peptide bond involving this carboxylate and the following tyrosine residue. A reconfiguration of 2OG achieves bidentate metal coordination. The dioxygen binding site is located on the metal face opposite to that facing L-Arg, thus requiring reorientation of the generated ferryl species to catalyze L-Arg hydroxylation. Notably, a phenylalanyl side chain pointing towards the metal may hinder such a ferryl flip and promote ethylene formation. Extensive site-directed mutagenesis studies supported the importance of this phenylalanine and confirmed the essential residues used for substrate binding and catalysis. The structural and functional characterization described here suggests that conversion of 2OG to ethylene, atypical among Fe(II)/2OG oxygenases, is facilitated by the binding of L-Arg which leads to an altered positioning of the carboxylate metal ligand, a resulting twisted peptide bond, and the off-line geometry for dioxygen coordination.

The Escherichia coli gene initially named ygaT is located adjacent to lhgO, encoding L-2hydroxyglutarate oxidase/dehydrogenase, and the gabDTP gene cluster, utilized for γaminobutyric acid (GABA) metabolism. Because this gene is transcribed specifically during periods of carbon starvation, it was renamed csiD for carbon starvation induced. The CsiD protein was structurally characterized and shown to possess a double-stranded β-helix fold, characteristic of a large family of non-heme Fe(II)-and 2-oxoglutarate (2OG)-dependent oxygenases. Consistent with a role in producing the substrate for LhgO, CsiD was shown to be a glutarate L-2hydroxylase. We review the kinetic and structural properties of glutarate L-2-hydroxylase from E. coli and other species, and we propose a catalytic mechanism for this archetype 2OG-dependent hydroxylase. Glutarate can be derived from L-lysine within the cell, with the gabDT genes exhibiting expanded reactivities beyond those known for GABA metabolism. The complete CsiDcontaining pathway provides a means for the cell to obtain energy from the metabolism of L-lysine during periods of carbon starvation. To reflect the role of this protein in the the cell, a renaming of csiD to glaH has been proposed.

The ethylene‐forming enzyme (EFE) is a ferrous ion‐ and l ‐arginine ( l ‐Arg)‐dependent oxygenase that cleaves three carbon–carbon bonds of 2‐oxoglutarate (2OG) as it forms ethylene plus three molecules of carbon dioxide/bicarbonate. The enzyme also catalyzes the oxidative decarboxylation of 2OG (producing carbon dioxide and succinate) while hydroxylating the C5 position of l ‐Arg, with this intermediate species decomposing spontaneously to guanidine and L‐Δ‐1‐pyrroline‐5‐carboxylate. This secondary reaction is typical of the hydroxylation reactions catalyzed by a broad range of Fe(II)/2OG‐dependent oxygenases, whereas the ethylene‐generating reaction is unprecedented. Genes encoding homologs of EFE are present in specific fungi and bacteria, and the protein is proposed to be a virulence factor in some plant pathogens. The EFE sequence and structure are clearly related to those of 1‐aminocyclopropane‐1‐carboxylate oxygenase, an Fe(II)‐ and dioxygen‐dependent ethylene‐forming enzyme that does not require or utilize 2OG. By contrast, the EFE sequence is not significantly related to three Fe(II)/2OG‐dependent oxygenases (VioC, NapI, and OrfP) that catalyze other reactions with l ‐Arg (hydroxylation at C3, C4‐C5 desaturation, and C3,C4 dihydroxylation, respectively). The best characterized EFE is that from Pseudomonas syringae pv. phaseolicola strain PK2. A visible absorption spectrum associated with the Fe(II)/2OG metal‐to‐ligand charge‐transfer transition of EFE·Fe(II)·2OG is less intense than for other members of this enzyme family; this result is explained by the predominantly monodentate metal coordination of 2OG as seen in the corresponding EFE·Mn(II)·2OG crystal structure. Binding of l ‐Arg leads to an intensification of the Fe(II)/2OG visible absorption spectrum and a shift to bidentate metal coordination. In EFE·Mn(II)·2OG, the metal is bound by one oxygen atom of the Asp191 carboxylate, but in EFE·Mn(II)·2OG,· l ‐Arg this atom shifts to interact with l ‐Arg and the other carboxylate oxygen atom binds the metal. A reorientation of Tyr192 accompanies l ‐Arg binding, forming a hydrogen bond between the phenol hydroxyl group and the amino acid. The carboxylate shift and the aromatic side chain reorientation lead to a twisted Asp191‐Tyr192 peptide bond. The environment of 2OG in EFE is more hydrophobic compared to other family members. In combination, the altered position of the metal‐binding carboxylate, the twisted peptide bond, and the hydrophobic environment surrounding 2OG may all contribute to the ability of EFE to catalyze ethylene formation.