Abstract
Many peroxy-containing secondary metabolites1,2 have been isolated and shown to provide beneficial effects to human health3,4,5. Yet, the mechanisms of most endoperoxide biosyntheses are not well understood. Although endoperoxides have been suggested as key reaction intermediates in several cases6,7,8, the only well-characterized endoperoxide biosynthetic enzyme is prostaglandin H synthase, a haem-containing enzyme9. Fumitremorgin B endoperoxidase (FtmOx1) from Aspergillus fumigatus is the first reported α-ketoglutarate-dependent mononuclear non-haem iron enzyme that can catalyse an endoperoxide formation reaction10,11,12. To elucidate the mechanistic details for this unique chemical transformation, we report the X-ray crystal structures of FtmOx1 and the binary complexes it forms with either the co-substrate (α-ketoglutarate) or the substrate (fumitremorgin B). Uniquely, after α-ketoglutarate has bound to the mononuclear iron centre in a bidentate fashion, the remaining open site for oxygen binding and activation is shielded from the substrate or the solvent by a tyrosine residue (Y224). Upon replacing Y224 with alanine or phenylalanine, the FtmOx1 catalysis diverts from endoperoxide formation to the more commonly observed hydroxylation. Subsequent characterizations by a combination of stopped-flow optical absorption spectroscopy and freeze-quench electron paramagnetic resonance spectroscopy support the presence of transient radical species in FtmOx1 catalysis. Our results help to unravel the novel mechanism for this endoperoxide formation reaction.
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Change history
19 May 2021
A Correction to this paper has been published: https://doi.org/10.1038/s41586-021-03527-x
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Acknowledgements
We thank H.-w. Liu, S. Elliott and A. Liu for comments on the manuscript. We also thank R. Fan and J. Lee for assistance with the pre-steady state kinetics studies, J. Caradonna for use of stopped-flow instruments, and A. Monzingo for assistance with crystallography software. This work is supported in part by grants from the National Institutes of Health (R01 GM093903 to P.L.; P41 GM104603 to C.E.C.; R01 GM104896 to Y.J.Z.; and R01 GM077387 to M.P.H.), the National Science Foundation (CHE-1309148 to P.L.; CHE-1126268 for the EPR spectrometer), the Welch Foundation (F-1778 to Y.J.Z.), the 973 program (2013CB734000 to L.Z), and Y.G. acknowledges financial support from Carnegie Mellon University. Crystallographic data collection was conducted at advanced light sources (Beamline 5.0.3) and advanced photon sources (BL23-ID-B), Department of Energy (DOE) National User Facility. L.Z. is an awardee of the National Distinguished Young Scholar Program in China (31125002).
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P.L., Y.J.Z. and L.Z. designed the study. W.Y. conducted the crystallization experiments and structure determination. H.S., F.S., A.S.H., S.W. and N. N. conducted the biochemical studies. C.-H.W., Y.P. and C.E.C. performed the MS–MS analyses. H.S., Y.G., M.P.H. and A.W. conducted the pre-steady state kinetics and EPR characterization. The manuscript was written by P.L., Y.J.Z. and L.Z. with input from all contributing authors.
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Extended data figures and tables
Extended Data Figure 1 Characterization of FtmOx1–α-KG complex.
a, Wild-type FtmOx1 and α-KG binding curve. The increase in absorbance at 520 nm as a function of α-KG concentration when it was added to a solution of wild-type FtmOx1 (0.9 mM) and Feii (0.72 mM) is plotted. On the basis of the equations described in the Methods (determining the α-KG dissociation constant), the Kd for wild-type FtmOx1 and α-KG is ∼185 ± 35 μM. b, Y224F-substituted FtmOx1 and α-KG binding curve. The increase of absorbance at 520 nm as a function of α-KG concentration when it was added to a solution of Y224F-substituted FtmOx1 (0.9 mM) and Feii (0.7 mM) is plotted. Kd for Y224F-substituted FtmOx1 and α-KG is ∼198 ± 58 μM. c, Y224A-substituted FtmOx1 and α-KG binding curve. The increase of absorbance at 520 nm as a function of α-KG concentration when it was added to a solution of Y224A-substituted FtmOx1 (0.7 mM) and Feii (0.51 mM) is plotted. Kd for Y224A-substituted FtmOx1 and α-KG is 204 ± 43 μM. In a–c, the Kd was calculated based on the concentration of iron-loaded FtmOx1. The experiments were replicated three times and error bars represent s.e.m.
Extended Data Figure 2 Suppression of DOPA formation by the presence of substrate fumitremorgin B.
There is no immediate evidence for the formation of DOPA upon the exposure of the FtmOx1–α-KG complex to O2 when the substrate fumitremorgin B is present. Spectra were recorded after FtmOx1 was used as the control to blank the UV–visible absorption reading.
Extended Data Figure 3 HPLC chromatograms of the FtmOx1 reaction enzyme-concentration dependence.
Chromatograms of FtmOx1 reactions with increasing amounts of FtmOx1 relative to the amount of substrate. The reaction mixture contained 100 mM Tris-HCl, (pH 7.5), 180 μM fumitremorgin B, 2 mM α-ketoglutarate, and variable amounts of FmOx1. Identities of the peaks were assigned based on subsequent NMR and MS characterizations of the isolated compounds. This experiment indicates that FtmOx1 is capable of catalysing endoperoxides formation in the absence of any other reductants.
Extended Data Figure 4 Stoichiometry determination for α-KG and O2 in FtmOx1 reaction.
a, b, Equivalents of endoperoxide products (2 and 3) produced as a function of the ratio of α-KG to iron-loaded FtmOx1 (a) and oxygen to iron-loaded FtmOx1 (b). The quantification was conducted based on the fumitremorgin B (1), compound 2, and compound 3 internal standards. All calculations were based on the concentration of iron-loaded FtmOx1. The experiments were replicated three times and error bars represent s.d.
Extended Data Figure 5 Structural comparison of the active site topologies between FtmOx1 and TauD.
a, Examination of the alternative configuration of α-KG in the FtmOx1–α-KG binary complex using the configuration of α-KG in the TauD–α-KG binary complex. We modelled α-KG in this alternative binding mode and calculated the difference map. In the Fo − Fc map, strong positive density (green) and negative density (red) are shown even when contoured to high level (3.3σ), indicating that this configuration is not correct for the FtmOx1–α-KG complex. b, The Fo − Fc map at the active site of the FtmOx1–fumitremorgin-B complex. A model of the substrate fumitremorgin B is superimposed onto the difference map, which is contoured at 2.8σ. c, Side-by-side comparison of FtmOx1 and TauD active-site topologies. In the left panel, the superimposition of the binary structures of FtmOx1–α-KG and FtmOx1–fumitremorgin-B (1) show that the remaining site for oxygen binding and activation is blocked from the substrate by Y224. d, In contrast, in the structure of the TauD–taurine–α-KG tertiary complex, the remaining site for O2 binding and activation directly faces the substrate (taurine).
Extended Data Figure 6 Characterization of FtmOx1 Y224F variant.
a, Self-hydroxylation reaction in Y224F-substituted FtmOx1. Formation of DOPA upon exposure of the Y224F-substituted FtmOx1–α-KG complex to O2. b–e, MS/MS analyses of Y224F-substituted FtmOx1. b, MS/MS spectrum of the triply charged parent ion at m/z 768.4109 of a tryptic digested peptide (residue 219–237) from wild-type FtmOx1. c, MS/MS spectrum of the triply charged parent ion at m/z 763.0793 of a tryptic digested peptide (residue 219–237) from Y224F-substituted FtmOx1. d, MS/MS spectrum of the triply charged parent ion at m/z 768.4109 of a tryptic digested peptide (residue 219–237) after exposure Y224F(FtmOx1)–α-KG tertiary complex to O2. e, MS/MS spectrum of the triply charged parent ion at m/z 773.7426 of a tryptic digested peptide (residue 219–237) for DOPA formed upon exposure of FtmOx(Y224F)–α-KG complex to O2 in the absence substrate fumitremorgin B.
Extended Data Figure 8 Pre-steady-state analyses of FtmOx1 reactions.
a, HPLC chromatograms for FtmOx1 reactions chemically quenched at the indicated times. The reaction mixture in 100 mM Tris-HCl (pH 7.5) buffer contained FtmOx1 (0.65 mM), Feii (0.58 mM), α-KG (12 mM), substrate (0.58 mM), and 20% glycerol. The mixture was mixed with O2-saturated buffer to initiate the reaction. There is an extra signal next to compound 3, which might be due to other chemicals released during the quench process. Results from the chemical quench experiment indicate that FtmOx1 catalysis is on the timescale of a few seconds per cycle. b, Time-dependent 420 nm absorption change (black solid curve) determined by stopped-flow optical absorption spectroscopy and the concentrations of the high-spin Fe3+ species (blue squares) and the g = 2 species (red dots) determined in the rapid-freeze-quench EPR experiments. The black solid curve is associated with the left y axis and is from the average of two stopped-flow trials. The blue squares and red dots are associated with the right y axis and are from the average of two rapid-freeze-quench EPR experiments. The experiments were repeated twice, and error bars reflect the uncertainty of the packing factor of rapid-freeze-quench EPR samples, which is around ±10%.
Extended Data Figure 9 EPR spectroscopic analyses of FtmOx1 reactions.
a, X-band EPR spectra measured at 19 K in reaction samples prepared at the indicated times. The black line shows the sample containing the FtmOx1–Feii–α-KG complex in the absence of O2. (There is a very small signal at g ≈ 4.3 region, only accounted for by <5 μM iron in the sample, which might be due to a very small amount of Fe3+ from inactive enzyme.) Bottom, the reaction sample freeze-quenched at ~0.2 s after mixing the FtmOx1–Feii–α-KG complex with O2. It has two signals: an Fe3+ (g = 4.54, 4.26, and 3.93) and a radical signal at the g = 2 region. b, X-band EPR spectra measured at 19 K for samples freeze-quenched at the indicated times showing the formation of high-spin ferric species on the time scale within 1 s. The reaction was initiated by mixing the FtmOx1–Feii–α-KG complex with O2. g-values are indicated in the figure. c, X-band EPR spectra measured at 19 K for samples freeze-quenched at 0.05 s and the spectral simulation for an S = 5/2 high-spin ferric species. The simulation parameters are: D = 0.3 cm−1, E/D = 0.266, σ(E/D) = 0.03, and g = 4.54, 4.26, 3.93. Measurement conditions in a–c: microwave frequency, 9.64 GHz; microwave power, 0.2 mW; modulation amplitude, 1 mT; and modulation frequency, 100 kHz.
Supplementary information
Supplementary Information
This file contains Supplementary Figures and Data. (PDF 1613 kb)
FtmOx1 binding to α-KG and substrate
When α-KG is bound to iron center, its configuration in coordinating iron leaves only one open site for water/O2 binding. But this site is blocked from substrate access by Y224. (MOV 16990 kb)
TauD binding to α-KG and substrate.
Unlike FtmOx1, α-KG associates with TauD in a different coordination. The open site for water molecule binding faces directly to the substrate binding pocket. (MOV 17087 kb)
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Yan, W., Song, H., Song, F. et al. RETRACTED ARTICLE: Endoperoxide formation by an α-ketoglutarate-dependent mononuclear non-haem iron enzyme. Nature 527, 539–543 (2015). https://doi.org/10.1038/nature15519
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DOI: https://doi.org/10.1038/nature15519
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