Biochemical and Spectroscopic Characterization of the Non-Heme Fe(II) and 2-Oxoglutarate Dependent Ethylene-Forming Enzyme from Pseudomonas syringae pv. phaseolicola PK2

摘要

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.