Identification and structural basis of a Chloroflexus protein with homology to Bacillus quorum sensing-related prenyltransferase
- 1Kitasato University
Abstract
Quorum sensing in Gram-positive bacteria commonly relies on posttranslationally modified peptide pheromones. In Bacillus subtilis, the prenyltransferase ComQ catalyzes tryptophan prenylation of the quorum-sensing peptide ComX, but the structural basis of this unique peptide modification has remained unclear. Here we identified a previously uncharacterized ComQ homolog, StheQ, and its cognate peptide substrate, StheX, from Sphaerobacter thermophilus and investigated their structural and functional relationship. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis demonstrated that StheQ catalyzes prenylation of the tryptophan residue located second from the C-terminus of StheX. Crystal structures of apo StheQ and its complexes with a farnesyl pyrophosphate analog revealed that StheQ adopts the all--helical fold of the trans-isoprenyl diphosphate synthase (IPPS) superfamily while possessing an active-site architecture adapted for peptide-based indole prenylation. The structures identified a single Mg2+-binding site associated with the first aspartic acid-rich motif and showed no evidence for metal coordination at the pseudo-second aspartic acid-rich motif. Site-directed mutagenesis, complex formation assays, and docking analyses identified a peptide-binding pocket adjacent to the active site and suggested that N215 contributes to productive positioning of the acceptor tryptophan. These findings establish the structural basis for peptide prenylation by a ComQ-family enzyme, providing insight into the evolution of peptide-based indole prenylation within the IPPS superfamily, and support the view that ComQ-family enzymes constitute a distinct functional branch specialized for peptide modification.
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BibTeX
@misc{matsui2026identification,
title = {{Identification and structural basis of a Chloroflexus protein with homology to Bacillus quorum sensing-related prenyltransferase}},
author = {T. Matsui and S. Inoue and S. Yanagimoto and A. Kaneko and R. Tago and A. Suto and M. Odagi and Y. Kodera and H. Morita and I. Abe and M. Okada},
howpublished = {bioRxiv},
year = {2026},
month = aug,
doi = {10.64898/2026.08.29.745113},
url = {https://www.biorxiv.org/content/10.64898/2026.08.29.745113v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Identification and structural basis of a Chloroflexus protein with homology to Bacillus quorum sensing-related prenyltransferase
AU - T. Matsui
AU - S. Inoue
AU - S. Yanagimoto
AU - A. Kaneko
AU - R. Tago
AU - A. Suto
AU - M. Odagi
AU - Y. Kodera
AU - H. Morita
AU - I. Abe
AU - M. Okada
PY - 2026
DA - 2026/08/29
PB - bioRxiv
DO - 10.64898/2026.08.29.745113
UR - https://www.biorxiv.org/content/10.64898/2026.08.29.745113v1
AB - Quorum sensing in Gram-positive bacteria commonly relies on posttranslationally modified peptide pheromones. In Bacillus subtilis, the prenyltransferase ComQ catalyzes tryptophan prenylation of the quorum-sensing peptide ComX, but the structural basis of this unique peptide modification has remained unclear. Here we identified a previously uncharacterized ComQ homolog, StheQ, and its cognate peptide substrate, StheX, from Sphaerobacter thermophilus and investigated their structural and functional relationship. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis demonstrated that StheQ catalyzes prenylation of the tryptophan residue located second from the C-terminus of StheX. Crystal structures of apo StheQ and its complexes with a farnesyl pyrophosphate analog revealed that StheQ adopts the all--helical fold of the trans-isoprenyl diphosphate synthase (IPPS) superfamily while possessing an active-site architecture adapted for peptide-based indole prenylation. The structures identified a single Mg2+-binding site associated with the first aspartic acid-rich motif and showed no evidence for metal coordination at the pseudo-second aspartic acid-rich motif. Site-directed mutagenesis, complex formation assays, and docking analyses identified a peptide-binding pocket adjacent to the active site and suggested that N215 contributes to productive positioning of the acceptor tryptophan. These findings establish the structural basis for peptide prenylation by a ComQ-family enzyme, providing insight into the evolution of peptide-based indole prenylation within the IPPS superfamily, and support the view that ComQ-family enzymes constitute a distinct functional branch specialized for peptide modification.
ER -