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Heterochiral coupling in non-ribosomal peptide macrolactamization

Abstract

Heterochiral coupling is favoured in abiotic peptide bond formation, whereas biotic peptide bond formation is dominated by homochiral coupling. Here, we report that heterochiral coupling is a rather general paradigm in the head-to-tail macrolactamization of non-ribosomal peptide biosynthesis. The canonical cis-acting offloading cyclases, such as type I thioesterase (TE) and terminal condensation-like domains, catalyse head-to-tail macrolactamization between N- and C-terminal residues with d- and l-configurations, respectively. In contrast, the penicillin-binding protein-type TEs, a recently identified family of trans-acting cyclases, couple heterochiral residues with complementary stereoselectivity to the canonical one. Thus, a suite of cis- and trans-TE non-ribosomal peptide synthetases could overcome the stereochemical constraints present in heterochiral head-to-tail macrolactam formation in bacterial non-ribosomal peptide biosynthesis. Furthermore, we provide the structural rationale for the C-terminal stereoselectivity of non-canonical offloading cyclases. Penicillin-binding protein-type TEs with broad substrate specificity are potentially applicable as biocatalysts and genetic tools for synthetic biology.

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Fig. 1: PBP-type TEs for non-ribosomal macrolactam biosynthesis.
Fig. 2: Crystal structure of SurE.
Fig. 3: Engineering surNRPS revealed the plasticity of SurE.

Data availability

The crystallographic data that support the findings of this study are available from the Protein Data Bank (http://www.rcsb.org). The coordinates and the structure factor amplitudes for the structures of SurE apo and SurE complexed with 4 have been deposited under accession codes 6KSU and 6KSV, respectively. All other data supporting the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank H. Ikeda (Kitazato University) for providing vectors and the E. coli strain used for genetic manipulation of S. albidoflavus NBRC12854, and A. Katsuyama (Hokkaido University) for technical assistance in the conformational energy calculation. The synchrotron radiation experiments were performed at beamline BL-1A of the Photon Factory. We also thank the beamline staff of the Photon Factory for their help in collecting X-ray diffraction data. This work was partly supported by the Takeda Science Foundation, the Asahi Glass Foundation, the Naito Foundation, the Uehara Memorial Foundation, the Japan Agency for Medical Research and Development (AMED grant no. JP19ae0101045) and Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (JSPS KAKENHI grant nos. JP16703511, JP16H06443, JP18056499, JP19178402 and JP20H00490).

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Contributions

K.M., T.M., I.A. and T.W. designed the experiments. R.Z. and T.M. performed the crystallization experiment. K.M., R.Z., T.M. and M.K. performed in vitro analysis. K.M. performed kinetic analysis. M.K. and A.S. synthesized substrate analogues. K.M., M.K. and T.W. performed structure determination of enzyme reaction products. M.K. performed genetic manipulation of Streptomyces. K.M., R.Z., T.M., M.K., A.S., I.A. and T.W. analysed the data. K.M., T.M., I.A. and T.W. wrote the paper.

Corresponding authors

Correspondence to Ikuro Abe or Toshiyuki Wakimoto.

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Supplementary information

Supplementary Information

Supplementary Methods, Tables 1 and 2 and Figs. 1–41.

Reporting Summary

Supplementary Data Dataset 1

Model of N-formyl-d-Leu tethered on Ser63 of SurE apo structure.

Supplementary Data Dataset 2

Model of N-formyl-l-Leu tethered on Ser63 of SurE apo structure.

Supplementary Data Dataset 3

Model of I1P tethered on Ser63 of SurE holo structure.

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Matsuda, K., Zhai, R., Mori, T. et al. Heterochiral coupling in non-ribosomal peptide macrolactamization. Nat Catal 3, 507–515 (2020). https://doi.org/10.1038/s41929-020-0456-7

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