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ε-Poly-L-lysine dispersity is controlled by a highly unusual nonribosomal peptide synthetase

Nature Chemical Biology volume 4, pages 766–772 (2008)Cite this article

Abstract

ε-Poly-L-lysine (ε-PL) consists of 25–35 L-lysine residues in isopeptide linkages and is one of only two amino acid homopolymers known in nature. Elucidating the biosynthetic mechanism of ε-PL should open new avenues for creating novel classes of biopolymers. Here we report the purification of an ε-PL synthetase (Pls; 130 kDa) and the cloning of its gene from an ε-PL–producing strain of Streptomyces albulus. Pls was found to be a membrane protein with adenylation and thiolation domains characteristic of the nonribosomal peptide synthetases (NRPSs). It had no traditional condensation or thioesterase domain; instead, it had six transmembrane domains surrounding three tandem soluble domains. These tandem domains iteratively catalyzed L-lysine polymerization using free L-lysine polymer (or monomer in the initial reaction) as acceptor and Pls-bound L-lysine as donor, directly yielding chains of diverse length. Thus, Pls is a new single-module NRPS having an amino acid ligase–like catalytic activity for peptide bond formation.

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Figure 1: In vitro polymerization of L-lysine (1) with Pls purified from S. albulus NBRC14147.
Figure 2: Domain architecture of Pls.
Figure 3: Performic acid oxidation of the radiolabeled Pls formed in the Pls reaction with L-[U-14C]lysine.
Figure 4: Pls reactions with L-lysine esters.
Figure 5: Substrate specificities in the A domain and T domain of Pls.
Figure 6: In vitro production of heteropolymers consisting of L-lysine and the L-lysine analogs.
Figure 7: Pls polymerization mechanism and the proposed model of the Pls catalytic cavity with substrate specificity.

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Acknowledgements

We thank M. Kobayashi (The University of Tsukuba, Japan) for providing plasmid pHSA81. This work was supported in part by KAKENHI (18780061), a Grant-in-Aid for young scientists (B), to Y.H. from the Japan Society for the Promotion of Science (JSPS).

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Author notes

  1. Chitose Maruyama & Hiroshi Takagi

    Present address: Present addresses: Faculty of Medical Sciences, University of Fukui, 23-3 Matsuoka-shimoaizuki, Eiheiji-cho, Yoshida-gun, Fukui 910-1193, Japan (C.M.) and Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan (H.T.).,

  2. Kazuya Yamanaka and Yoshimitsu Hamano: These authors contributed equally to this work.

Authors and Affiliations

  1. Yokohama Research Center, Chisso Corporation, 5-1 Ookawa, Kanazawa-Ku, Yokohama, 236-8605, Japan

    Kazuya Yamanaka

  2. Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Yoshida-gun, 910-1195, Fukui, Japan

    Chitose Maruyama, Hiroshi Takagi & Yoshimitsu Hamano

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  1. Kazuya Yamanaka
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Contributions

Y.H., K.Y. and H.T. conceived and designed the experiments; Y.H., K.Y. and C.M. performed the experiments; and K.Y. and Y.H. wrote the paper.

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Correspondence to Yoshimitsu Hamano.

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Competing interests

Y.H. and K.Y. have submitted a patent application related to the practical application of polylysine synthetase; some claims in this application are supported by this work.

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Supplementary Figures 1–3, Supplementary Tables 1–6 and Supplementary Methods (PDF 2803 kb)

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Yamanaka, K., Maruyama, C., Takagi, H. et al. ε-Poly-L-lysine dispersity is controlled by a highly unusual nonribosomal peptide synthetase. Nat Chem Biol 4, 766–772 (2008). https://doi.org/10.1038/nchembio.125

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