Hybrid radical-polar pathway for excision of ethylene from 2-oxoglutarate by an iron oxygenase

Copeland, Rachelle A.; Zhou, Shengbin; Schaperdoth, Irene; Shoda, Tokufu Kent C.; Bollinger, J. Martin; Krebs, Carsten

doi:10.1126/science.abj4290

Cited by 16 publications

(38 citation statements)

References 30 publications

(83 reference statements)

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“…Bollinger and Krebs first experimentally established the key Fe(IV)O oxidant in 2003 . The nonheme Fe(IV)O in this supergroup of enzymes is a powerful oxidant for which whose action results in a wide range of oxidative outcomes such as hydroxylation, desaturation, epimerization, epoxidation, ring closure, ring expansion, and C–C or C–N bond-coupling reactions. ,,,, Because of their versatility, these enzymes have several important roles in biological processes, including the post-translational modification of collagen, fatty acid metabolism, oxygen sensing, DNA and RNA repair, ethylene production, and the biosynthesis of many antibiotics. ,,,− …”

Section: Oxygenation Reactions With α-Ketoglutarate As a Cosubstratementioning

confidence: 99%

Charge Maintenance during Catalysis in Nonheme Iron Oxygenases

Traore

Liu

2022

ACS Catal.

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Here, the choice of the first coordination shell of the metal center is analyzed from the perspective of charge maintenance in a binary enzyme–substrate complex and an O2-bound ternary complex in the nonheme iron oxygenases. Comparing homogentisate 1,2-dioxygenase and gentisate dioxygenase highlights the significance of charge maintenance after substrate binding as an important factor that drives the reaction coordinate. We then extend the charge analysis to several common types of nonheme iron oxygenases containing either a 2-His-1-carboxylate facial triad or a 3-His or 4-His ligand motif, including extradiol and intradiol ring-cleavage dioxygenases, thiol dioxygenases, α-ketoglutarate-dependent oxygenases, and carotenoid cleavage oxygenases. After forming the productive enzyme–substrate complex, the overall charge of the iron complex at the 0, +1, or +2 state is maintained in the remaining catalytic steps. Hence, maintaining a constant charge is crucial to promote the reaction of the iron center beginning from the formation of the Michaelis or ternary complex. The charge compensation to the iron ion is tuned not only by protein-derived carboxylate ligands but also by substrates. Overall, these analyses indicate that charge maintenance at the iron center is significant when all the necessary components form a productive complex. This charge maintenance concept may apply to most oxygen-activating metalloenzymes systems that do not draw electrons and protons step-by-step from a separate reactant, such as NADH, via a reductase. The charge maintenance perception may also be useful in proposing catalytic pathways or designing prototypical reactions using artificial or engineered enzymes for biotechnological applications.

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Section: Oxygenation Reactions With α-Ketoglutarate As a Cosubstratementioning

confidence: 99%

Charge Maintenance during Catalysis in Nonheme Iron Oxygenases

Traore

Liu

2022

ACS Catal.

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“…[21][22][23][24][25][26] Upon dioxygen activation in the presence of substrate and co-substrate 2OG, [27][28][29][30][31][32][33][34][35] the wellcharacterized ferryl-oxo active species can be generated to initiate the reaction via either hydrogen atom abstraction from the substrate or oxygen atom transfer to the substrate. [15,[36][37][38][39][40][41][42][43][44][45][46][47][48] As the first Fe/2OG-dependent endoperoxidase,t he mechanism of FtmOx1 remains elusive at current stage.L i and co-authors performed isotopic experiments and demonstrated that both oxygen atoms inserted into the structure of verruculogen arise from as ingle O 2 molecule. [9] They also found that ascorbate was essential for the enzymatic endoperoxide conversion by FtmOx1.…”

Section: Introductionmentioning

confidence: 99%

“…This family of enzymes employ a conserved 2‐His‐1‐carboxylate facial triad in the active pocket to coordinate a ferrous iron cofactor [21–26] . Upon dioxygen activation in the presence of substrate and co‐substrate 2OG, [27–35] the well‐characterized ferryl–oxo active species can be generated to initiate the reaction via either hydrogen atom abstraction from the substrate or oxygen atom transfer to the substrate [15, 36–48] …”

Section: Introductionmentioning

confidence: 99%

Structural Insight into the Catalytic Mechanism of the Endoperoxide Synthase FtmOx1

Zhou

Wang

et al. 2022

Angew Chem Int Ed

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The 2-oxoglutarate (2OG)-dependent non-heme enzyme FtmOx1 catalyzes the endoperoxide biosynthesis of verruculogen. Although several mechanistic studies have been carried out, the catalytic mechanism of FtmOx1 is not well determined owingtothe lackofareliable complex structure of FtmOx1 with fumitremorgin B. Herein we providet he X-ray crystal structure of the ternary complex FtmOx1•2OG•fumitremorgin Bataresolution of 1.22 .Our structures showthat the binding of fumitremorgin Bi nduces significant compression of the active pocket and that Y68 is in close proximityt o C26 of the substrate.F urther MD simulation and QM/MM calculations support aCarC-like mechanism, in which Y68 acts as the Hatom donor for quenching the C26-centered substrate radical. Our results are consistent with all available experimental data and highlight the importance of accurate complex structures in the mechanistic study of enzymatic catalysis.

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“…EFE generally catalyzes two reactions: (a) the oxidative decarboxylation of αKG to form a succinate concomitant with C 5 -hydroxylation of l -arginine, where the hydroxyarginine intermediate is decomposed to guanidine, and (b) the conversion of αKG into ethylene and three molecules of CO 2 . − ,,, Kinetic studies with l -Arg versus deuterated l -Arg proposed that the l -Arg hydroxylation pathway proceeds via an iron(IV)-oxo intermediate (Figure , right), while the kinetics of ethylene formation are not affected by deuteration of l -Arg. , They concluded, therefore, that the ethylene formation does not pass an iron(IV)-oxo species and diverges from the l -arginine hydroxylation pathway at an earlier stage and possibly is formed from the conversion of the iron(III)-superoxo species into a peroxic anhydride complex (bottom-right structure in Figure ). Nevertheless, l -arginine plays a critical role in the ethylene formation reaction catalyzed by EFE .…”

Section: Introductionmentioning

confidence: 99%

Cluster Model Study into the Catalytic Mechanism of α-Ketoglutarate Biodegradation by the Ethylene-Forming Enzyme Reveals Structural Differences with Nonheme Iron Hydroxylases

et al. 2022

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Ethylene is an important signaling molecule in plants that triggers the growth of leaves, flowers, and fruits. One of the enzymes involved in the biosynthesis of ethylene is the ethylene-forming enzyme (EFE), which is an usual nonheme iron enzyme that biodegrades α-ketoglutarate into three CO 2 molecules and ethylene. As the detailed mechanism of EFE in the biosynthesis of ethylene remains controversial and particularly the function of the co-substrate L-arginine, we decided to pursue a density functional theory study on the possible pathways of the enzyme leading to ethylene biosynthesis and test many possible pathways and mechanisms. A large active site cluster model of 322 atoms was created, which contains all the features of the firstand second-coordination sphere of the active site and substrate (αketoglutarate) binding pockets. The calculations identify a persuccinate intermediate that triggers a bifurcation pathway in the enzyme and either react with a molecule of CO 2 to form a carbonate or forms a high-valent iron(IV)-oxo species through heterolytic dioxygen bond cleavage. Our studies show that both the bifurcation pathways converge to the same intermediate again and can lead to ethylene products, although the two pathways have different kinetics. Interestingly, our studies also show that the iron(IV)-oxo itself can form a carbonate and ethylene but through much higher barriers. As a matter of fact, these barriers are higher in energy than the typical aliphatic hydroxylation barriers and may not be competitive with arginine hydroxylation. Inclusion of the L-arginine co-substrate into the model leads to minor changes in the structure and fold, but its charge and dipole moment does not seem to affect the first stage of the catalytic cycle. Moreover, the key activation barriers seem less affected by the inclusion of L-arginine into the model. We, therefore, believe that the role of L-arginine is to lock α-ketoglutarate and its products into a tight binding pocket to enable its degradation and to prevent early release of CO 2 . Our studies show that due to the distinct differences in α-ketoglutarate positioning between different arginine activating nonheme iron dioxygenases in the co-substrate binding pocket and its tighter binding in EFE, we predict that the release of CO 2 is prevented in the first stage of the oxygen activation mechanism. This enables attack of CO 2 on a persuccinate complex to form carbonate products, leading to ethylene formation. This work gives suggestions on the engineering of EFE into a hydroxylase or improving the ethylene biosynthesis.

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Hybrid radical-polar pathway for excision of ethylene from 2-oxoglutarate by an iron oxygenase

Cited by 16 publications

References 30 publications

Charge Maintenance during Catalysis in Nonheme Iron Oxygenases

Charge Maintenance during Catalysis in Nonheme Iron Oxygenases

Structural Insight into the Catalytic Mechanism of the Endoperoxide Synthase FtmOx1

Cluster Model Study into the Catalytic Mechanism of α-Ketoglutarate Biodegradation by the Ethylene-Forming Enzyme Reveals Structural Differences with Nonheme Iron Hydroxylases

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