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PreprintCited by 8Microbial Natural Products and BiosynthesisAntimicrobial Peptides and ActivitiesBiochemical and Structural Characterization

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Mining the biosynthetic landscape of lactic acid bacteria unearths a new family of RiPPs assembled by a novel type of ThiF-like adenylyltransferases

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Wang, Mengjiao, Wu, Mengyue, Han, Meng, Niu, Xiaogang, Fan, Aili, Zhu, Shaozhou, Tong, Yigang·2024·ACS Omega
DOI 10.1101/2024.05.13.593846PMC PMC11256085

Preprint — published as 10.1021/acsomega.4c03760

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Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are chemically diverse natural products of ribosomal origin. These peptides, which frequently act as signals or antimicrobials, are biosynthesized by conserved enzymatic machineries, making genome mining a powerful strategy for unearthing previously uncharacterized members of their class. Herein, we investigate the untapped biosynthetic potential of Lactobacillales (i.e. lactic acid bacteria) – an order of Gram-positive bacteria closely associated with human life, including pathogenic species and industrially-relevent fermenters of dairy products. Through genome mining methods, we systematically explored the distribution and diversity of ThiF-like adenylyltransferases-utilizing RiPP systems in lactic acid bacteria and identified a number of unprecedented biosynthetic gene clusters. In one of these clusters, we found a previously undescribed group of macrocyclic imide biosynthetic pathways containing multiple transporters and may be involved in potential quorum sensing (QS) system. Through in vitro assays, we determined that one such adenylyltranferase specifically catalyzes the intracyclization of its precursor peptide through macrocyclic imide formation. Incubating the enzyme with various primary amines revealed it could effectively amidate the C-terminus of the precursor peptide. This new transformation adds to the growing list of Nature’s peptide macrocyclization strategies and expands the impressive catalytic repertoire of the adenylyltransferase family. The diverse RiPP systems identified herein represent a vast, unexploited landscape for discovery of novel class of natural products and potential QS systems. Abstract Figure: (For Table of Contents use only)

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Figure 1 (A) Representative structures of known cyclic peptides with isopeptide bonds. (B) Known RiPP biosynthetic gene clusters containing ThiF enzymes, including pantocin A, microcin C, and microcin C analogue.
Figure 1 (A) Representative structures of known cyclic peptides with isopeptide bonds. (B) Known RiPP biosynthetic gene clusters containing ThiF enzymes, including pantocin A, microcin C, and microcin C analogue.CC BY-NC-ND · PubMed Central
Figure 2 ThiF-RiPP Network in Lactobacillales. (A) The sequence similarity network of ThiF-RiPP gene clusters from 8 groups (edge % identity of 20) based on the sequence of the ThiF genes. Color coding indicates the distribution of each subfamily among Lactobacillales strains. The subfamilies are named based on conserved motifs within each precursor peptide. (B) The representative biosynthetic gene cluster for each of the subfamilies in panel (A). The genes are color-coded, as indicated in the figure. Precursor peptide logo plots for ThiF-RiPP subfamilies are shown on the right.
Figure 2 ThiF-RiPP Network in Lactobacillales. (A) The sequence similarity network of ThiF-RiPP gene clusters from 8 groups (edge % identity of 20) based on the sequence of the ThiF genes. Color coding indicates the distribution of each subfamily among Lactobacillales strains. The subfamilies are named based on conserved motifs within each precursor peptide. (B) The representative biosynthetic gene cluster for each of the subfamilies in panel (A). The genes are color-coded, as indicated in the figure. Precursor peptide logo plots for ThiF-RiPP subfamilies are shown on the right.CC BY-NC-ND · PubMed Central
Figure 3 (A) WWIII-type cluster was identified in E. faecalis FDAARGOS_397 (WWIVI). (B) SDS-PAGE of the purified EnfB. Lane 1 is the protein marker and lane 2 is the final purified EnfB after size-exclusion chromatography. (C) In vitro cyclization and amidation of EnfA precursor peptide by EnfB, as monitored by LC-MS. Extracted ion chromatograms showing the [M + 3H]3+ ion (m/z = 824.4707) of the precursor peptide, [M + 3H]3+ ion (m/z = 858.8237) of the amidated peptide, and [M + 3H]3+ ion (m/z = 818.4674) of the cyclized peptide. (D) Identified fragment ions are highlighted.
Figure 3 (A) WWIII-type cluster was identified in E. faecalis FDAARGOS_397 (WWIVI). (B) SDS-PAGE of the purified EnfB. Lane 1 is the protein marker and lane 2 is the final purified EnfB after size-exclusion chromatography. (C) In vitro cyclization and amidation of EnfA precursor peptide by EnfB, as monitored by LC-MS. Extracted ion chromatograms showing the [M + 3H]3+ ion (m/z = 824.4707) of the precursor peptide, [M + 3H]3+ ion (m/z = 858.8237) of the amidated peptide, and [M + 3H]3+ ion (m/z = 818.4674) of the cyclized peptide. (D) Identified fragment ions are highlighted.CC BY-NC-ND · PubMed Central
Figure 4 (A) Kinetic characterization of the cyclization catalyzed by EnfB. (B) proposed mechanism of EnfB-catalyzed cyclization and amidation of the C-terminal of a precursor peptide. After activation by ATP, the C-terminal acyl-AMP anhydride of EnfA is attacked either by the nitrogen from the peptide bond at Ile19 to form a macrocyclic imide or by the nitrogen in small organic molecules to amidate the C-terminus.
Figure 4 (A) Kinetic characterization of the cyclization catalyzed by EnfB. (B) proposed mechanism of EnfB-catalyzed cyclization and amidation of the C-terminal of a precursor peptide. After activation by ATP, the C-terminal acyl-AMP anhydride of EnfA is attacked either by the nitrogen from the peptide bond at Ile19 to form a macrocyclic imide or by the nitrogen in small organic molecules to amidate the C-terminus.CC BY-NC-ND · PubMed Central
Figure 5 (A) Results of substrate specificity assays with EnfB and EnfA analogues. (B) Results of substrate specificity assays with EnfB and the amino compounds. The cyclization ratio or the amidation ratio was shown alongside.
Figure 5 (A) Results of substrate specificity assays with EnfB and EnfA analogues. (B) Results of substrate specificity assays with EnfB and the amino compounds. The cyclization ratio or the amidation ratio was shown alongside.CC BY-NC-ND · PubMed Central
Figure 6 (A) Evolution of the precursor peptide for these diverse macrocyclase. The substrates could be classified into 4 clades based on the organization of their biosynthetic gene clusters. (B) Representative biosynthetic gene cluster for each of the subfamilies in panel (A).
Figure 6 (A) Evolution of the precursor peptide for these diverse macrocyclase. The substrates could be classified into 4 clades based on the organization of their biosynthetic gene clusters. (B) Representative biosynthetic gene cluster for each of the subfamilies in panel (A).CC BY-NC-ND · PubMed Central
Figure 7 (A) Proposed schematic of enterofaecin biosynthesis. Precursor peptide EnfA is first either cyclized or amidated. A portion of these peptides are then exported by diverse transporters while triggering the potential QS system.
Figure 7 (A) Proposed schematic of enterofaecin biosynthesis. Precursor peptide EnfA is first either cyclized or amidated. A portion of these peptides are then exported by diverse transporters while triggering the potential QS system.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Wang, M., Wu, M., Han, M., Niu, X., Fan, A., Zhu, S., & Tong, Y.
Affiliations
  • Beijing University of Chemical Technology (CN)
  • Peking University (CN)
  • Tsinghua University (CN)
  • National Institutes for Food and Drug Control (CN)
Corresponding author
  • Fan, Aili
  • Zhu, Shaozhou
  • Tong, Yigang
ORCID
Venue
ACS Omega
Publisher
openRxiv
Published
2024-05-13
Volume
9
Issue
28
Pages
30891-30903
ISSN
2470-1343 (ISSN-L)
DOI
10.1101/2024.05.13.593846
PMC
PMC11256085
OpenAlex
W4400297230
Cited by
8
References
1
Citations by year
2026: 3 · 2025: 4 · 2024: 1
FWCI
2.27
Type
journal article
Language
en
Open access
Licence
cc-by-nc-nd
Locations
  • ACS Omega (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Natural Science Foundation of China (21706005)
  • Peking University
  • Beijing Municipal Natural Science Foundation (7202107)
  • National Key Research and Development Program of China (2022YFC2804900)
Topics
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Microbial Natural Products and Biosynthesis
  • Life Sciences › Immunology and Microbiology › Microbiology › Antimicrobial Peptides and Activities
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
Keywords
ribosomally synthesized and post-translationally modified peptides, lactic acid bacteria, Lactobacillales, genome mining, biosynthetic gene clusters, quorum sensing, peptide transporters, C-terminal amidation, natural product discovery

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