Characterising a cyclodipeptide synthase from Porphyromonas gingivalis

Abstract

Oral diseases affect around half of the world’s population, incurring significant treatment costs. Natural peptide products represent a class of medicinally significant compounds and of these, a diverse family of small, highly stable, cyclic dipeptides exhibit a wide range of pharmacological and biological activities. Given that these molecules are known to function in bacterial communication and exhibit potent antimicrobial and antivirulence effects, they may represent a novel means of treating oral dysbiosis. Therefore, the primary aim of the present study was to identify and characterise additional cyclodipeptide synthases - the enzymes responsible for tRNA-dependent cyclic dipeptide biosynthesis - in a number of human pathogens and, given the association between oral and systemic disease, attention was focused on bacteria associated with periodontitis.

Iterative homology searching identified putative cyclodipeptide synthase genes in three periodontopathogens - Porphyromonas gingivalis (PgCDPS), Aminiphilus circumscriptus (AcCDPS) and Selenomonas sputigena (SsCDPS) - and through a combination of sequence alignment and phylogenetic analysis, all were determined to be XYP CDPS subfamily members. Of these, PgCDPS was successfully purified and crystallised, with its structure solved using molecular replacement which confirmed its XYP subfamily status. By global metabolic profiling of spent reaction matrix via LC-ESI-MS, utilising a combination of cyclic dipeptide and adduct transition lists, as well as the authentic chemical standard (cLQ), PgCDPS was determined to synthesise cyclo(Leu-Gln). Site directed mutagenesis of the active site serine (S8A), resulted in complete loss of the m/z 242.15 [cIQ/cLQ] feature, thus confirming catalytic requirement for peak detection. Finally, following intact mass analysis, a mass corresponding to cIQ/cLQ was bound to wild type PgCDPS on a number of occasions, with this mass absent from the mutant (S8A) protein. This suggests a possible method of allosteric regulation and, if correct, is the first case known to be reported for a cyclodipeptide synthase.

Date of Award	2 Jul 2025
Original language	English
Awarding Institution	University of St Andrews
Supervisor	Clarissa Melo Czekster (Supervisor)