title: Biochemical and Spectroscopic Characterization of the Non-Heme Fe(II)- and 2-Oxoglutarate-Dependent Ethylene-Forming Enzyme from Pseudomonas syringae pv. phaseolicola PK2.
abstract: 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 and three CO2molecules 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 activity greater than that 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 cosubstrate (forming glutaric acid) during the hydroxylation of l-Arg, with this reaction unlinked from ethylene formation. Kinetic constants were determined for both 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. On the basis of our results and what is generally known about EFE and Fe(II)- and 2OG-dependent oxygenases, an updated model for the reaction mechanism is presented.
authors.affiliation: Department of Microbiology and Molecular Genetics and ‡Department of Biochemistry and Molecular Biology, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Salette
authors.initials: S
authors.lastname: Martinez
authors.affiliation: Department of Microbiology and Molecular Genetics and ‡Department of Biochemistry and Molecular Biology, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Hausinger
mesh_terms: D005296:Ferrous Compounds / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism*; D007700:Kinetics; D008190:Lyases / Q000737:chemistry / Q000378:metabolism*; D044224:Pseudomonas syringae / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet
chemical_list: D005296:Ferrous Compounds; D007656:Ketoglutaric Acids; D008190:Lyases; C034693:ethylene forming enzyme