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

Martinez, Salette; Hausinger, Robert P.

doi:10.1021/acs.biochem.6b00890

Cited by 50 publications

(94 citation statements)

References 40 publications

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“…A recombinant form of the ethylene-forming enzyme from PsEFE was produced in Escherichia coli and purified to near homogeneity. Using NMR and GC/MS-based assays, we found that, as reported (14,21), PsEFE-catalyzed ethylene production is 2OG-dependent, stimulated by the addition of Fe(II) [some Fe(II) likely copurifies with PsEFE], and is increased by the addition of ascorbate or DTT ( Fig. 1 C and D and SI Appendix, Fig.…”

Section: Resultssupporting

confidence: 61%

“…2). Formation of the bidentate 2OG intermediate complex I is supported by spectroscopic work (21). Turnover of 2OG to succinate also occurs (Fig.…”

Section: Significancementioning

confidence: 61%

“…Similar conclusions have recently been made by Martinez and Hausinger in work on the PsEFE from P. syringae pv. phaseolicola PK2 (21). These results imply a mechanism for ethylene production in which binding of arginine plays a key role.…”

Section: Significancementioning

confidence: 74%

“…S8). The implied typical mode of Fe-chelation by 2OG is supported by spectroscopic studies (21). A water occupies the site trans to His189, likely where dioxygen binds.…”

Section: Significancementioning

confidence: 76%

“…1). Variations are possible [e.g., fragmentation of the cyclic peroxide intermediate II to give ethylene (21)], but are less likely, given the prevalence of intermediates of type III in 2OG oxygenase catalysis. NOG (and by implication 2OG) and L-Arg were observed in two conformations correlating with different Asp191 binding modes (Fig.…”

Section: Significancementioning

confidence: 99%

See 4 more Smart Citations

Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate

Zhang

Smart

Choi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Ethylene is important in industry and biological signaling. In plants, ethylene is produced by oxidation of 1-aminocyclopropane-1-carboxylic acid, as catalyzed by 1-aminocyclopropane-1-carboxylic acid oxidase. Bacteria catalyze ethylene production, but via the fourelectron oxidation of 2-oxoglutarate to give ethylene in an argininedependent reaction. Crystallographic and biochemical studies on the Pseudomonas syringae ethylene-forming enzyme reveal a branched mechanism. In one branch, an apparently typical 2-oxoglutarate oxygenase reaction to give succinate, carbon dioxide, and sometimes pyrroline-5-carboxylate occurs. Alternatively, Grob-type oxidative fragmentation of a 2-oxoglutarate-derived intermediate occurs to give ethylene and carbon dioxide. Crystallographic and quantum chemical studies reveal that fragmentation to give ethylene is promoted by binding of L-arginine in a nonoxidized conformation and of 2-oxoglutarate in an unprecedented high-energy conformation that favors ethylene, relative to succinate formation.ethylene-forming enzyme | 2-oxoglutarate-dependent oxygenases | hydroxylase | plant development | oxidoreductase E thylene is of industrial importance and is a vital signaling molecule in plants, where it has roles in germination, senescence, and stress responses (1). Commercial manipulation of the natural ethylene response is agriculturally important in controlling fruit ripening (2). In higher plants, ethylene is produced from methionine, via oxidation of 1-aminocyclopropane-1-carboxylic acid (ACC) in an unusual reaction catalyzed by the Fe(II)-dependent ACC oxidase (ACCO) (3, 4), which is part of the 2-oxoglutarate (2OG)-dependent oxygenase superfamily, although it does not use a 2OG cosubstrate (Fig. 1A) (5-7). Ethylene is also produced in some microorganisms by oxidation of 2-oxo-4-methylthiobutyric acid in a reaction not directly enzyme catalyzed (8,9).In work aimed at producing industrial ethylene by biocatalysis, Pseudomonas strains, including plant pathogens, were shown to produce large amounts of ethylene (10)(11)(12)(13)(14). Bacteria engineered to produce ethylene using the Pseudomonas syringae pv. phaseolicola ethylene-forming enzyme (PsEFE) have been developed to ripen fruit as an alternative to the use of synthetic ethylene (15, 16). Ethylene-forming enzymes are being explored for biocatalysis in cyanobacteria (17-19). PsEFE-catalyzed ethylene production is 2OG-dependent and is stimulated by the addition of L-arginine (L-Arg), which is also converted by PsEFE into pyrroline-5-carboxylate (P5C; Fig. 1B) (13, 20). In contrast to the consensus 2OG oxygenase mechanism, which involves sequential binding of 2OG, substrate, and then oxygen, an unprecedented "dual circuit" mechanism is proposed for PsEFE (13).We describe biochemical, structural, and modeling studies supporting a branched mechanistic pathway for PsEFE that can lead either to ethylene via oxidative fragmentation of 2OG or to succinate via a more typical 2OG oxygenase reaction, which sometimes results in P5C formation (Fig....

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Section: Resultssupporting

confidence: 61%

“…2). Formation of the bidentate 2OG intermediate complex I is supported by spectroscopic work (21). Turnover of 2OG to succinate also occurs (Fig.…”

Section: Significancementioning

confidence: 61%

Section: Significancementioning

confidence: 74%

“…S8). The implied typical mode of Fe-chelation by 2OG is supported by spectroscopic studies (21). A water occupies the site trans to His189, likely where dioxygen binds.…”

Section: Significancementioning

confidence: 76%

Section: Significancementioning

confidence: 99%

See 3 more Smart Citations

Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate

Zhang

Smart

Choi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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NonhemeFe(II)‐ and 2‐Oxoglutarate‐Dependent Ethylene‐Forming Enzyme

Hausinger

Herr

2019

Encyclopedia of Inorganic and Bioinorganic Chemistry

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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.

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Second‐Coordination Sphere Effects on Selectivity and Specificity of Heme and Nonheme Iron Enzymes

Visser

2020

Chemistry A European J

117

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Mononuclear iron‐containing enzymes are highly versatile oxidants that often react stereospecifically and/or regioselectively with substrates. Combined experimental and computational studies on heme monooxygenases, nonheme iron dioxygenases and halogenases have revealed the intricate details of the second‐coordination sphere, which determine this specificity and selectivity. These second‐coordination sphere effects originate from the positioning of the substrate and oxidant, which involve the binding of the co‐factors and substrate into the active site of the protein. In addition, some enzymes affect the selectivity and reactivity through charge‐stabilization from nearby bound cations/anions, an induced electric field or through the positioning of salt bridges and hydrogen‐bonding interactions to first‐coordination sphere iron ligands and/or the substrate. Examples of all of these second‐coordination sphere effects in iron‐containing enzymes and how these influence structure and reactivity are given.

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

Cited by 50 publications

References 40 publications

Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate

Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate

NonhemeFe(II)‐ and 2‐Oxoglutarate‐Dependent Ethylene‐Forming Enzyme

Second‐Coordination Sphere Effects on Selectivity and Specificity of Heme and Nonheme Iron Enzymes

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