Biological formation of ethylene

Abstract

This review summarizes the structures, biochemical properties, and mechanisms of two major biological sources of ethylene, the ethylene-forming enzyme (EFE) and 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). EFE is found in selected bacteria and fungi where it catalyzes two reactions: (1) the oxygen-dependent conversion of 2-oxoglutarate (2OG) to ethylene plus three molecules of CO₂/bicarbonate and (2) the oxidative decarboxylation of 2OG while transforming l-arginine to guanidine and l-Δ¹-pyrroline-5-carboxylic acid. ACCO is present in plants where it makes the plant hormone by transforming ACC, O₂, and an external reductant to ethylene, HCN, CO₂, and water. Despite catalyzing distinct chemical reactions, EFE and ACCO are related in sequence and structure, and both enzymes require Fe(ii) for their activity. Advances in our understanding of EFE, derived from both experimental and computational approaches, have clarified how this enzyme catalyzes its dual reactions. Drawing on the published mechanistic studies of ACCO and noting the parallels between this enzyme and EFE, we propose a novel reaction mechanism for ACCO.

Fig. 1. Four biological processes for ethylene production. (A) ACCO conversion of ACC to ethylene, hydrogen cyanide, and carbon dioxide. (B) EFE oxidative transformation of 2OG to ethylene and three molecules of carbon dioxide in the presence of l-Arg, and its C5 hydroxylation of l-Arg as 2OG undergoes oxidative decarboxylation to form succinate. (C) NADH:Fe(iii) oxidoreductase promoted decomposition of KMBA to form ethylene, methanethiol, and two molecules of carbon dioxide. (D) MarBHDK reduction of MTE to ethylene, methanethiol, and water.

Fig. 2. Sequence comparison of Petunia x hybrida ACCO (top) and Pseudomonas savastanoi pv. phaseolicola strain PK2 EFE (bottom). Identical residues are highlighted in cyan. The metal-binding ligands are indicated by asterisks.

Fig. 3. Phylogenetic and structural dendrograms comparing selected sequences and structures of EFE, ACCO, and related Fe(ii)/2OG-dependent oxygenases. (A) A maximum likelihood phylogenetic tree constructed using the Poisson correction model and the MUSCLE alignment plugin in MEGA11. Gaps and missing data were eliminated using the complete deletion option. UniprotKB or JGI numbers identify sequences for EFE (red) from strain PK2 and Penicillium digitatum (P32021 and A0A7T7BQH3, respectively), ACCO (blue) including type 1 from Arabidopsis thaliana, Solanum lycoperscim, and Zea mays (Q06588, A0A3Q7F7I3, and Q6JN54, respectively), types 3 and 2 from A. thaliana (Q0WPW4 and Q9ZUN4 respectively), and type 4 from Dictyostelium mucoroides (A6BM06) and Volvierella volvacea (JGI 116615, JGI 111142, JGI 111930, and JGI118606), and other Fe(ii)/2OG oxygenases (green), including hyoscyamine 6-β-hydroxylase from Atropa belladonna (Q9XJ43), deacetoxycephalosporin C synthase from Streptomyces clavuligerus (P18548), verruculogen synthase from Aspergillus fumigatus (Q4WAW9), clavaminate synthase 1 from Streptomyces clavuligerus (Q05581), (5R)-carbapenem-3-carboxylate synthase from Pectobacterium carotovorum (Q9XB59), taurine/2-oxoglutarate dioxygenase from Escherichia coli (P37610), and L-threonyl-(threonyl carrier protein) 4-chlorinase from Pseudomonas syringae pv. syringae (Q9RBY6). (B) Hierarchical clustering of structures denoted by PDB access codes. Structures related to strain PK2 EFE (red, 5V2Z) were identified using DALI with a Z-score up to 12 and clustered using MEGA11. The two reported ACCO structures (5GJA and 5TCV) are shown in blue. Other Fe(ii)/2OG oxygenases (green) include anthocyanidin synthase from A. thaliana (1GP6),), feruloyl-CoA 6-hydroxylase from A. thaliana (4XAE), hyoscyamine 6-hydroxylase from Datura metel (6TTO), thebaine-6-O-demethylase from Papaver somniferum (5O7Y), gibberellin 2β-hydroxylase and gibberellin 3β-hydroxylase from Oryza sativa (6KU3 and 7EKD, respectively), deoxypodophyllotoxin synthase from Sinopodophyllum hexandrum (7E38), dioxygenase for auxin oxidation from O. sativa and A. thaliana (6KUN and 6KWA, respectively), SnoN epimerase from Streptomyces nogalater (5ERL), SnoK carbocyclase from S. nogalater (5EPA), the TlxIJ enzyme in meroterpenoid biosynthesis in Talaromyces purpureogenus (7VBQ), FtmOx1 from Aspergillus fumigatus (7ETL), TropC involved in tropolone biosynthesis in Talaromyces stipitatus (6XJJ), a halogenase from Actinomadura sp. ATCC 39365 (7W5S), thymine 7-hydroxylase from Neurospora crassa (5C3Q), isopenicllin N synthase from Emericella nidulans (7P3L), deacetoxycephalosporin C synthase from S. clavuligerus (1UNB), prolyl hydroxylase from Dictyostelium discoideum (6T8M), and other enzymes from various sources with still undefined functions (6LSV, 7V3N, 4XAA, 5ZM3, 3OOX, 3ON7, and 6JYV). The bootstrap method was used for each panel with 1000 replicates.

Fig. 4. Comparison of EFE and ACCO structures. Cartoon depiction of (A) strain PK2 EFE (cyan, PDB: 5V2Y) and (B) ACCO (sienna, PDB: 5TCV). (C) Active site of EFE with 2OG (yellow), l-Arg (dark blue), and the metal-binding ligands shown as sticks, and with inactive Mn (substituting for Fe) as a purple sphere. (D) Active site of ACCO with ACC (magenta) and the metal binding ligands shown as sticks, and with inactive Ni (also substituting for Fe) as a green sphere.

Fig. 5. Enthalpy changes associated with the binding of Fe(ii), 2OG, and l-Arg to strain PK2 EFE.

Fig. 6. UV-visible difference spectra for EFE·Fe(ii)·2OG (red) and EFE·Fe(ii)·2OG·l-Arg, with the spectrum of EFE·Fe(ii) subtracted. The concentrations were 226 μM protein, 1 mM Fe(ii), 2.4 mM 2OG, and (when present) 2.7 mM l-Arg.

Fig. 7. Structural changes at the active site induced by the binding of l-Arg. (A) EFE·Mn(ii)·2OG (PDB: 5V2X). (B) EFE·Mn(ii)·2OG·l-Arg (PDB: 5V2Y). l-Arg binding leads to a shift from monodentate to bidentate binding of 2OG, a switch in the oxygen atom of D191 that coordinates the metal, and a flip of Y192 (depicted with red carbon atoms) that creates a twist in the D191-Y192 peptide bond. (C) Comparison of off-line (as in B) and in-line binding modes of 2OG.

Fig. 8. Computed tunnels for oxygen access to the EFE active site. Tunnel-1 is represented in blue, and tunnel-2 is shown in yellow. The red sticks depict l-Arg, 2OG, and the three metal-binding side chains.

Fig. 9. Proposed reaction mechanisms of EFE. (A) EFE·Fe(ii)·2OG with an open coordination site opposite of H189. (B–F) States in the pathway of l-Arg hydroxylation as 2OG undergoes oxidative decarboxylation. (G–K) States in the pathway of ethylene biosynthesis. (L) Very minor side reaction that converts 2OG to 3-hydroxypropionate. H-R is the substrate l-Arg.

Fig. 10. Oxygen activation and l-Arg hydroxylation mechanism of EFE. (A) The first l-Arg conformation and an off-line Fe(iii)-superoxo reaction center (labeled AO-RC1) converts to an off-line ferryl state (AO-IM3) via the intermediates (IM) and transition states (TS) along the potential energy surface shown. (B) After undergoing a ferryl flip to a second ferryl state (AO-IM4), HAT and hydroxyl radical rebound occur along the second potential energy surface. Hydrogen atoms and CO₂ molecules are hidden for clarity. Bond lengths are labeled in angstroms. Energies are shown in kcal mol⁻¹.

Fig. 11. Ethylene formation mechanism of EFE. The second l-Arg conformation and an off-line Fe(iii)-superoxo reaction center (labeled BO-RC1) inserts the oxygen atoms into the 2OG C1-C2 bond (BO-IM1) forming an O-carboxy-3-hydroxypropionate (BO-IM2), that then decomposes to generate ethylene, two carbon dioxide, and ferrous ion coordinated bicarbonate along the potential energy surface shown. Bond lengths are labeled in angstroms. Energies are shown in kcal mol⁻¹.

Fig. 12. Reaction selectivities and energy barriers with respect to the strength of applied external electric field (EEF) along the Fe–O bond for different 2OG and l-Arg conformations of EFE. Reproduced from Chaturvedi et al. (2023).

Fig. 13. One version of a catalytic mechanism for ACCO derived from biochemical and computation studies. Binding of ACC and dioxygen accompanied by reduction and protonation yields a ferryl/N-radical intermediate (state E). Radical rearrangement and hydrogen atom abstraction provides an Fe(iii)-hydroxide/dual radical species (state G) that decomposes to form ethylene and cyanoformate (state H), with the latter molecule decomposing to cyanide and CO₂. Reduction and product release restores the enzyme to the starting state. Ascorbic acid is used as reductant in the laboratory and may function as well in the cell. Bicarbonate (not shown) is proposed to facilitate proton transfer steps.

Fig. 14. Newly proposed hypothetical enzymatic mechanism for ACCO. States A through D are equivalent to steps in Fig. 13. Newly proposed are the steps involving ferryl formation, HAT, and those highlighted in green in which a carbon centered radical is joined to the Fe(iii)-carbonate, facilitating ethylene elimination and formation of cyanoformate. The latter intermediate decomposes to hydrogen cyanide and CO₂. Also shown (highlighted in red) is a plausible side product of the reaction.