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An NmrA-like enzyme-catalysed redox-mediated Diels–Alder cycloaddition with anti-selectivity


The Diels–Alder cycloaddition is one of the most powerful approaches in organic synthesis and is often used in the synthesis of important pharmaceuticals. Yet, strictly controlling the stereoselectivity of the Diels–Alder reactions is challenging, and great efforts are needed to construct complex molecules with desired chirality via organocatalysis or transition-metal strategies. Nature has evolved different types of enzymes to exquisitely control cyclization stereochemistry; however, most of the reported Diels–Alderases have been shown to only facilitate the energetically favourable diastereoselective cycloadditions. Here we report the discovery and characterization of CtdP, a member of a new class of bifunctional oxidoreductase/Diels–Alderase, which was previously annotated as an NmrA-like transcriptional regulator. We demonstrate that CtdP catalyses the inherently disfavoured cycloaddition to form the bicyclo[2.2.2]diazaoctane scaffold with a strict α-anti-selectivity. Guided by computational studies, we reveal a NADP+/NADPH-dependent redox mechanism for the CtdP-catalysed inverse electron demand Diels–Alder cycloaddition, which serves as the first example of a bifunctional Diels–Alderase that utilizes this mechanism.

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Fig. 1: Fungal bicyclo[2.2.2]diazaoctane alkaloid biosynthesis and functional characterization of CtdP.
Fig. 2: Crystal structure analysis of CtdP complex.
Fig. 3: CtdP active site view and mutagenesis study.
Fig. 4: Proposed mechanism of CtdP-catalysed α-anti-selective IMDA reaction.

Data availability

Data supporting the findings of this work are available within the Article, Extended Data, Source Data and the Supplementary Information files. Data supporting the current study are also available from the corresponding author upon request. The genome sequences of P. citrinum ATCC 9849 are accessible from the GeneBank database (BCKA00000000.1). Coordinates and associated structure factors of CtdP have been deposited in the Protein Data Bank (PDB) database (PDB code: 7UF8). The crystallographic data of small molecules have been deposited at the CCDC (2127333 for 5 and 2127332 for 10). Energies and molecular coordinates of calculated structures are provided in the Supplementary Information file. Source data are provided with this paper.


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We thank L. Kürti and Y.-D. Kwon for their assistance with the high-resolution mass spectrometry experiments, J. D. Hartgerink for sharing the instruments for electronic circular dichroism measurements, C. Ajo-Franklin and S. Li for sharing the anaerobic workstation, and J. Smith for providing generous resources for protein crystallization, APS beamline access, and software for structure determination; and finally the 23-IDB@GM/CA beamline staff. This work was supported by a National Institute of Health (NIH) grant (R35GM138207) and the Robert A. Welch Foundation (C-1952) to X.G., an NIH grant (R35GM11810) and the Hans W. Vahlteich Professorship to D.H.S., and an NIH grant (AI141481) to K.N.H. J.N.S. acknowledges the support of the National Institute of General Medical Sciences (NIGMS) of the NIH under an F32 individual postdoctoral fellowship (F32GM122218). S.R. acknowledges support from the Michigan Chemistry–Biology Interface Training Program, funded by the NIGMS, NIH T32 Training Grant (5T32GM132046). Computational resources for DFT computations were provided by the UCLA Institute for Digital Research and Education (IDRE) and by the San Diego Supercomputing Center (SDSC) through XSEDE (ACI-1548562). Microsecond molecular dynamics simulations were performed using Anton 2 computer time provided by the Pittsburgh Supercomputing Center (PSC) through grant R01GM116961 from the NIH. The Anton 2 machine at PSC was generously made available by D.E. Shaw Research.

Author information

Authors and Affiliations



Z.L. carried out compound purification, synthesis and structural determination experiments, and in vitro assays and mutagenesis. Z.L., F.Z., H.-W.S. and S.P. performed protein expression and purification. Z.L., F.Z., Q.N. and S.L. performed in vivo gene deletion experiments. Z.L. and S.L. did the CtdP refolding experiments. Z.L. and Q.N. performed (S)-[4-2H] NADPH assays. J.D.F. and G.N.P. assisted in the single-crystal X-ray diffraction analysis of small molecules. S.R. and S.A.N. carried out the protein crystallization and crystal structure determinations. Z.L., S.R., S.A.N., W.X. and M.D.M. analysed the protein crystal structure. J.N.S. performed the DFT computations and MD simulations. Z.L., S.R., S.A.N., J.N.S., K.N.H., D.H.S. and X.G. analysed all the data and prepared the manuscript. All authors revised the manuscript.

Corresponding authors

Correspondence to K. N. Houk, David H. Sherman or Xue Gao.

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The authors declare no competing interests.

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Nature Chemistry thanks Andrew Gulick, Shina Kamerlin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Representations of different classes of Diels–Alderases and the proposed biosynthetic pathway of 21R-citrinadin A.

a, Chemical structures of representative natural products synthesized by different types of Diels–Alderases. b, Chemical structures of natural PIAs with α-anti bicyclo[2.2.2]diazaoctane rings63,64. c, The proposed biosynthetic pathway of 21R-citrinadin A (1)24. PT: prenyltransferase, DAase: Diels–Alderase.

Extended Data Fig. 2 Comparison analysis of the biosynthetic gene clusters of PIAs and in vivo characterization of NmrA-like proteins CtdO, CtdR, and CtdP.

a, Annotation of the biosynthetic gene clusters of PIAs. 21R-citrinadin A (ctd in P. citrinum ATCC 9849), citrinadin A (cnd in P. citrinum DSM1997)24, paraherquamides (phq in P. fellutanum ATCC20841)65, (−)-notoamide A (not in A. protuberus MF297-2)66, (+)-notoamide A (not’ in A. versicolor NRRL35600)66 and malbrancheamide (mal in Malbranchea aurantiaca RRC1813)8. NRPS: non-ribosomal peptide synthetase; FPMO: flavoprotein monooxygenase; P450: cytochrome P450; SDR: short-chain dehydrogenase/reductase. SDRs MalC and PhqE, and NmrA-like oxidoreductase CtdP are characterized as Diels–Alderases. b, LC–MS analysis (extracted ion chromatogram, EIC) of P. citrinum wild-type (WT) and mutant ΔctdN. Four ΔctdN mutants all retained the production of compound 1. c, LC–MS analysis (EIC) of P. citrinum WT and mutants ΔctdO, ΔctdR, ΔctdN, and ΔctdP. The ΔctdR mutant major produces R1, which is identified as an α-anti product by NMR (Supplementary Table 10) and ECD (Supplementary Fig. 6b) analysis, indicating that CtdR might work on the biosynthetic steps after Diels–Alder cycloaddition. The ΔctdO mutant showed reduced production of citrinadin compounds, which possibly serves as a regulator. Both ΔctdR and ΔctdO produce trace amounts of 5 and 6, indicating the deletions of these genes have some influence and result in accumulations of the shunt products. Only the ΔctdP mutant could abolish the production of 1 and 2, as well as have a remarkable accumulation of 5 and 6.

Extended Data Fig. 3 In vitro assays of 3 in anaerobic and aerobic conditions.

a, EIC traces of in vitro assays of 3 with CtdP, CtdN, and MalC in an anaerobic chamber, respectively. b, Enzymatic and spontaneous transforms of 3. c, Ultraviolet (UV) spectrum of 12. d, X-ray structure of 5. e, Spontaneous reactions of 3. f, In vitro assays of 3 with CtdO and CtdR in aerobic conditions, respectively. The symbol * represents the compound identified by MS and UV spectra (Supplementary Figs. 4, 5).

Extended Data Fig. 4 Sequence alignment of CtdP and homologous NmrA-like proteins.

CtdP, HSCARG from Homo sapiens (PDB: 2exx), and NmrA-like family domain-containing protein 1 from H. sapiens (PDB: 2wm3) are aligned using the Clustal Omega server67. Secondary structural elements of CtdP are marked above the alignment. Residues are coloured based on their conservation using the ESPript 3.0 server68. The η symbol refers to a 310-helice.

Extended Data Fig. 5 Superposition of the different chains in CtdP.

Superposition of the different chains and calculated r.m.s.d. values. Chains A-D are coloured forest green, cyan, wheat, and warm pink, respectively. NADP+ (yellow) and penicimutamide E (10, salmon) are shown as sticks.

Extended Data Fig. 6 C-terminal tail and electrostatic surface structures of CtdP.

a, Interactions of C-terminal tail (orange) in CtdP chain A. NADP+ (yellow) and penicimutamide E (10, salmon) shown as sticks. Distances in angstroms shown in red. b, Electrostatic surface potential of CtdP-NADP+-10 complex (chain A, calculated by Adaptive Pisson-Boltzmann Sovler plugin,

Extended Data Fig. 7 Density functional theory calculations for tautomerization and redox mechanisms.

a, The redox-mediated pathway of CtdP catalysis in (S)-[4-2H] NADPH assay. b, Study of the inherent stereoselectivity of the IMDA reaction via the redox-mediated pathway by comparing the four diastereomeric transition states in the absence of the CtdP enzyme. c, DFT computations comparing the redox properties of CtdP substrate 3 and MalC substrate 9. The free energy change for the reaction above (∆G = +4.5 kcal mol–1) corresponds to CtdP substrate 3, with a 6-membered methylpiperidine ring, being over 1000 times harder to oxidize than the corresponding substrate 9-red with a 5-membered pyrrolidine ring.

Extended Data Fig. 8 MD simulations of substrate 3-ox in CtdP active site.

a, Tracking the distance between the carbon atom that gets oxidized to form 3-ox and the hydride transferred to form NADPH during three 1.2-microsecond MD simulations. b, The hydrogen bond between substrate 3-ox and NADPH in MD simulations. The amide N-H of substrate 3-ox is the H-bond donor and the ribose 2′-OH of NADPH is the H-bond acceptor. The plot illustrates the N-O distance as a function of time over three 1.2-microsecond MD simulations. c, MD snapshot of CtdP active site in which substrate 3-ox has an α-anti conformation and the shortest DA bond lengths.

Extended Data Fig. 9 Sequence similarity network analysis of NmrA-like protein CtdP.

Sequence similarity network of CtdP generated by the EFI-EST69 server at an E-value of 5 and alignment score of 35, 2000 sequences from UniProt (2022-02) database were used, filtered by 50% representative nodes, visualized in Cytoscape v3.9.170.

Supplementary Information

Supplementary Information

Supplementary Tables 1–14, Figs. 1–17, and energies and molecular coordinates of calculated structures.

Reporting Summary

Supplementary Data 1

Crystallographic data of the protein and small molecules.

Supplementary Data 2

Cif file with crystallographic data for compound 5.

Supplementary Data 3

Cif file with crystallographic data for compound 10.

Source data

Source Data Fig. 1

Source data of Fig. 3c and Supplementary Fig. 7a,d.

Source Data Fig. 2

Uncropped and unprocessed scan of SDS–PAGE images.

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Liu, Z., Rivera, S., Newmister, S.A. et al. An NmrA-like enzyme-catalysed redox-mediated Diels–Alder cycloaddition with anti-selectivity. Nat. Chem. (2023).

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