Identification and structural basis of a Chloroflexus protein with homology to Bacillus quorum sensing-related prenyltransferase

Quorum sensing in Gram-positive bacteria typically involves posttranslationally modified peptide pheromones. One such mechanism involves the prenyltransferase ComQ from Bacillus subtilis, which catalyzes tryptophan prenylation of the quorum-sensing peptide ComX. However, the structural basis for this unique modification has not been well understood.

In this study, researchers identified a related enzyme, StheQ from Sphaerobacter thermophilus, along with its cognate peptide substrate, StheX. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), they confirmed that StheQ catalyzes prenylation of a specific tryptophan residue in StheX. X-ray crystallography revealed that StheQ adopts the helical fold typical of the trans-isoprenyl diphosphate synthase (IPPS) superfamily, but with an active site adapted specifically for peptide-based indole prenylation.

The structures also showed a single magnesium-binding site near the first aspartic acid-rich region, with no evidence of metal coordination at the second aspartic acid-rich motif. Through site-directed mutagenesis, complex formation assays, and docking analyses, the team identified a peptide-binding pocket near the active site, suggesting that residue N215 plays a crucial role in positioning the tryptophan acceptor.

These findings clarify the structural basis of peptide prenylation by a ComQ-family enzyme, shedding light on the evolution of peptide-based indole prenylation within the IPPS superfamily, and highlighting that ComQ-family enzymes form a distinct functional branch specialized for this peptide modification.