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Molecular basis for the unusual ring reconstruction in fungal meroterpenoid biogenesis

Nature Chemical Biology volume 13, pages 1066–1073 (2017)Cite this article

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Abstract

Trt14 from Aspergillus terreus is involved in unusual skeletal reconstruction during the biosynthesis of the fungal meroterpenoid terretonin. Detailed in vitro characterization revealed that this novel multifunctional enzyme catalyzes not only the D-ring expansion via intramolecular methoxy rearrangement, but also the hydrolysis of the expanded D-ring. The X-ray crystal structures of Trt14, in complex with substrate or product, and two Trt14 homologs, AusH and PrhC from Aspergillus nidulans and Penicillium brasilianum, respectively, indicated similar overall structures to those of the NTF2-like superfamily of enzymes, despite lacking sequence and functional similarities. Moreover, we gained structural insight into the mechanism of the Trt14-catalyzed ring reconstruction from the in-crystal enzyme reaction and site-directed mutagenesis to show that this reaction involves sequential ester bond cleavage and formation. Structural comparison of Trt14 and its homologs suggests that the enzymes in this new superfamily employ similar acid–base chemistry to diversify the molecular architecture of fungal meroterpenoids.

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Figure 1: Biosynthesis of terretonin.
Figure 2: In vitro Trt14 enzyme reactions.
Figure 3: Comparison of the overall structures and the active sites of Trt14, AusH, and PrhC.
Figure 4: Comparison of the active site structures of Trt14 with different ligands.
Figure 5: In vitro enzyme reactions of the wild-type and mutants of Trt14.
Figure 6: Proposed mechanism for the Trt14-catalyzed D-ring expansion.

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Acknowledgements

We thank H. Nakamura (The University of Tokyo) for critical reading of the manuscript. 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 Number JP15H01836 and JP16H06443 to I.A.). The synchrotron radiation experiments were performed at the BL41XU of SPring-8, BL-17A, NE3A, and NW12A of the Photon Factory with proposal No. 2015B2031 and 2015G530, respectively.

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

    Takahiro Mori, Taiki Iwabuchi, Shotaro Hoshino, Hang Wang, Yudai Matsuda & Ikuro Abe

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  1. Takahiro Mori
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Contributions

T.M. and I.A. designed the experiments. T.M. expressed, purified, crystallized, and solved the structure of the protein, and performed in vitro experiments. T.I. designed constructs and cloned, expressed, and purified protein, and performed in vivo experiments. S.H. determined the structure of compounds. H.W. expressed, and purified, and crystallized protein. T.M., Y.M., 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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The authors declare no competing financial interests.

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Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–2 and Supplementary Figures 1–12. (PDF 10866 kb)

Supplementary Note 1

Characterization of Chemical Compounds. (PDF 10989 kb)

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Mori, T., Iwabuchi, T., Hoshino, S. et al. Molecular basis for the unusual ring reconstruction in fungal meroterpenoid biogenesis. Nat Chem Biol 13, 1066–1073 (2017). https://doi.org/10.1038/nchembio.2443

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