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Molecular basis for the P450-catalyzed C–N bond formation in indolactam biosynthesis

Nature Chemical Biology volume 15, pages 1206–1213 (2019)Cite this article

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Abstract

The catalytic versatility of cytochrome P450 monooxygenases is remarkable. Here, we present mechanistic and structural characterizations of TleB from Streptomyces blastmyceticus and its homolog HinD from Streptoalloteichus hindustanus, which catalyze unusual intramolecular C–N bond formation to generate indolactam V from the dipeptide N-methylvalyl-tryptophanol. In vitro analyses demonstrated that both P450s exhibit promiscuous substrate specificity, and modification of the N13-methyl group resulted in the formation of indole-fused 6/5/6 tricyclic products. Furthermore, X-ray crystal structures in complex with substrates and structure-based mutagenesis revealed the intimate structural details of the enzyme reactions. We propose that the generation of a diradical species is critical for the indolactam formation, and that the intramolecular C(sp2)–H amination is initiated by the abstraction of the N1 indole hydrogen. After indole radical repositioning and subsequent removal of the N13 hydrogen, the coupling of the properly-folded diradical leads to the formation of the C4–N13 bond of indolactam.

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Fig. 1: Biosynthetic pathway of indolactams.
Fig. 2: Enzyme reactions of TleB and HinD.
Fig. 3: Enzyme reactions of TleB and HinD with various substrate analogs.
Fig. 4: The binding modes of substrates in the active sites of TleB and HinD.
Fig. 5: Enzyme reactions of TleB and HinD mutants.
Fig. 6: Proposed mechanism for the TleB and HinD enzymatic reaction.

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Data availability

The coordinates and structure factor amplitudes for the apo structures of TleB and HinD and those complexed with ligands were deposited to the Protein Data Bank (PDB) under accession codes 6J82, 6J83, 6J84, 6J85, 6J86, 6J87 and 6J88.

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Acknowledgements

We thank A. Arisawa (MicroBiopharm Japan) for giving us the plasmids of the P450 redox partner CamA and CamB. We thank T. Eguchi and F. Kudo (Tokyo Institute of Technology) for assistance with the stopped-flow experiments. The synchrotron radiation experiments were performed at the BL-1A, and NW12A of the Photon Factory and at beamline TPS-05A and BL15A1 at NSRRC. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI grant nos. JP16H06443, JP17H04763 and JP18K19139), Japan Science and Technology Agency (JST SICORP grant no. JPMJSC1701), Kobayashi International Scholarship Foundation and the Takeda Science Foundation.

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Author notes

  1. Miroslava Alblova

    Present address: Department of Structural Biology of Signaling Proteins, Division Biotechnology and Biomedicine Center of the Academy of Sciences and Charles University in Vestec, Institute of Physiology, The Czech Academy of Sciences, Prague, Czech Republic

  2. These authors contributed equally: Fei He, Takahiro Mori, Iori Morita.

Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

    Fei He, Takahiro Mori, Iori Morita, Hitomi Nakamura, Miroslava Alblova, Shotaro Hoshino, Takayoshi Awakawa & Ikuro Abe

  2. Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo, Japan

    Takahiro Mori, Takayoshi Awakawa & Ikuro Abe

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Contributions

T.M., H.N. and I.A. designed the experiments. F.H., T.M. and M.A. performed in vitro analysis and crystallization experiments. I.M. measured pre-steady-state kinetic values. I.M. and H.N. synthesized substrate analogs. S.H. performed structure determination of enzyme reaction products. F.H., T.M., H.N., T.A. and I.A. analyzed the data. T.M. and I.A. wrote the paper.

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Correspondence to Ikuro Abe.

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Supplementary Tables 1–6 and Supplementary Figures 1–10.

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He, F., Mori, T., Morita, I. et al. Molecular basis for the P450-catalyzed C–N bond formation in indolactam biosynthesis. Nat Chem Biol 15, 1206–1213 (2019). https://doi.org/10.1038/s41589-019-0380-9

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