The healthy human vaginal microbiota is generally dominated by Lactobacilli, and the transition to a more diverse community of anaerobic microbes is associated with a number of health risks. While the mechanisms underlying the stability of Lactobacillus-dominated vaginal communities are not fully understood, competition for nutrients is a likely contributing factor. Glycogen secreted by epithelial cells is widely believed to support the growth of vaginal microbes. However, the mechanism by which bacteria access sugars from this complex polymer is unclear, with evidence to support a role for both microbial and human enzymes. To shed light on the potential contribution from microbial enzymes, here we biochemically characterize six glycogen-degrading enzymes predicted to be secreted by vaginal bacteria and confirm their ability to support the growth of an amylase-deficient strain of L. crispatus on glycogen. We reveal significant differences in the pH tolerance between enzymes from different organisms, suggesting the adaptation of Lactobacilli to an acidic vaginal environment. Using a simple assay specific for the microbial enzymes, we confirm their presence in cervicovaginal lavage samples. Finally, we demonstrate the selective inhibition of glycogen-degrading enzymes from two vaginal microbes associated with dysbiosis. This work provides biochemical evidence to support the role of vaginal bacterial amylase enzymes in the breakdown of glycogen, providing insight into factors that shape the vaginal microbiota and highlighting the possibility of manipulating community structure via non-antibiotic small molecules.

Enzyme‐catalyzed β‐lactone formation from β‐hydroxy acids is a crucial step in bacterial biosynthesis of β‐lactone natural products and membrane hydrocarbons. We developed a novel, continuous assay for β‐lactone synthetase activity using synthetic β‐hydroxy acid substrates with alkene or alkyne moieties. β‐Lactone formation is followed by rapid decarboxylation to form a conjugated triene chromophore for real‐time evaluation by UV/Vis spectroscopy. The assay was used to determine steady‐state kinetics of a long‐chain β‐lactone synthetase, OleC, from the plant pathogen Xanthomonas campestris. Site‐directed mutagenesis was used to test the involvement of conserved active site residues in Mg2+ and ATP binding. A previous report suggested OleC adenylated the substrate hydroxy group. Here we present several lines of evidence, including hydroxylamine trapping of the AMP intermediate, to demonstrate the substrate carboxyl group is adenylated prior to making the β‐lactone final product. A panel of nine substrate analogues were used to investigate the substrate specificity of X. campestris OleC by HPLC and GC‐MS. Stereoisomers of 2‐hexyl‐3hydroxyoctanoic acid were synthesized and OleC preferred the (2R,3S) diastereomer consistent with the stereo‐preference of upstream and downstream pathway enzymes. This biochemical knowledge was used to guide phylogenetic analysis of the β‐lactone synthetases to map their functional diversity within the acyl‐CoA synthetase, NRPS adenylation domain, and luciferase superfamily.