SUMMARY
Dental caries is a polymicrobial disease that affects much of the human population worldwide, where the
equilibrium of a cooperative eco-organization among commensal microbes shifts towards a dysbiotic framework
with an overrepresentation of pathogenic microorganisms, especially by Streptococcus mutans (SM). The
progression of this disease begins with bacterial attachment and a complex cascade of events that include the
production of soluble (a-1,6-linked) and insoluble (a-1,3-linked) glucans by glucosyltransferases (Gtfs) from the
GH70 hydrolase family. This class of enzymes utilizes dietary sucrose to produce various isomeric glucan
polymers through two important steps: (i) first is the sucrase (invertase) activity, where cleavage of sucrose
results in glucose and fructose; (ii) subsequently, the polymerization of glucose molecules forms extended a-
glucans. SM’s three different Gtfs, GtfB, GtfC, and GtfD, have been biologically well characterized, and their
synthesis of glucans is crucial for biofilm formation, bacterial colonization, and virulence. However, despite more
than 30 years of research and numerous studies, there remains a fundamental knowledge gap on their enzymatic
mechanism; specifically, how do they produce the soluble and insoluble glucans via distinct functional domains?
The goal of this application is to determine the specific molecular mechanism(s) that drive SM’s Gtfs to produce
various glucans, particularly how they synthesize soluble and insoluble glucans. Our structural studies show that
the sucrase activity pocket is very tight and cannot accommodate an a-retaining double displacement reaction
like the GH13 enzymes. Therefore, polymerization must take place at another distinct site in the nearby vicinity.
We hypothesize that ‘The GH70 glucosyltransferases of SM adopt a novel enzymatic mechanism to produce the
soluble a-1,6-linked and insoluble a-1,3-linked glucans.’ We will address our hypothesis through three specific
aims (SAs) by (a) determining the structures of Glucosyltransferases (GtfB & GtfD) of SM (SA1), (b) elucidating
the catalytic mechanism of SM’s glucosyltransferases (SA2), and (c) characterizing the role of (i) the sucrase
site, (ii) the polymerization site, (iii) the glucan binding domains and (iv) their inhibitors on biofilms, colonization,
and virulence potential through in vitro and in vivo models of dental caries (SA3).
The results from this study will (1) determine the novel structures adopted by these Gtf enzymes, (2) assign
specific roles to domains/regions, (3) identify key residues involved in the dual-step enzymatic action, (4) specify
how each GtfB and GtfD polymerize glucans, (5) reveal the importance of the sucrase site, the polymerization
site, and the glucan binding domains on disease outcomes, and (6) develop inhibitors that selectively target each
Gtf’s enzymatic activity. These investigations would provide a mechanistic foundation for the 800+ GH70
hydrolases, as multiple biotechnology interests exist to engineer these glucansucrases to dispense varied sizes
of dextran for chromatography, food preservation, and pharmaceuticals. The long-term objective of this study is
to explore innovative strategies for specifically addressing the cariogenic dysbiosis mediated by SM’s Gtfs.
