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.

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 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. This enzyme is reported to simultaneously catalyze the conversion of 2OG into ethylene plus three CO2 and the Cδ hydroxylation of L-arginine (L-Arg) while oxidatively decarboxylating 2OG to form succinate and carbon dioxide. A new plasmid construct for expression in recombinant Escherichia coli cells allowed for the purification of large amounts of EFE with greater activity than previously recorded. A variety of assays were used to quantify and confirm the identity of the proposed products, including the first experimental demonstration of L-Δ1-pyrroline-5-carboxylate and guanidine derived from 5-hydroxyarginine. Selected L-Arg derivatives could induce ethylene formation without undergoing hydroxylation, demonstrating that ethylene production and L-Arg hydroxylation activities are not linked. Similarly, EFE utilizes the alternative α-keto acid 2-oxoadipate as a co-substrate (forming glutaric acid) during the hydroxylation of L-Arg, with this reaction unlinked from ethylene formation. Kinetic constants were determined for both the ethylene formation and L-Arg hydroxylation reactions. Anaerobic UV-visible difference spectra were used to monitor the binding of Fe(II) and substrates to the enzyme. Based on our results and what is generally known about EFE and Fe(II)/2OG-dependent oxygenases, an updated model for the reaction mechanism is presented.

Nitrile hydratase (NHase) catalyzes the hydration of nitriles to their corresponding commercially valuable amides at ambient temperatures and physiological pH. Several reaction mechanisms have been proposed for NHase enzymes however, the source of the nucleophile remains a mystery. Boronic acids have been shown to be potent inhibitors of numerous hydrolytic enzymes due to the open shell of boron, which allows it to expand from a trigonal planar (sp2) form to a tetrahedral form (sp3). Therefore, we examined the inhibition of the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) by boronic acids via kinetics and X-ray crystallography. Both 1-butaneboronic acid (BuBA) and phenylboronic acid (PBA) function as potent competitive inhibitors of PtNHase. X-ray crystal structures for BuBA and PBA complexed to PtNHase were solved and refined at 1.5, 1.6 and 1.2 Å resolution. The resulting PtNHase-boronic acid complexes represent a “snapshot” of reaction intermediates and implicate the cysteine-sulfenic acid ligand as the catalytic nucleophile, a heretofore unknown role for the αCys113-OH sulfenic acid ligand. Based on these data, a new mechanism of action for the hydration of nitriles by NHase is presented.