Control Of Sugar Transport & Metabolism In Oral Bacteria

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Previous studies in this laboratory pertaining to the mechanisms of transport and metabolism of sugars by microorganisms, led to the discovery of a large, but previously unrecognized family of glycosyl hydrolases (GH). These novel enzymes catalyze the cleavage of a wide variety of phosphorylated disaccharides including maltose-6?P, cellobiose-6?P and, most remarkably, the five phosphorylated isomers of sucrose. However, the characteristics that distinguish these hydrolases (designated Family GH4) from all others in the > 90 families comprising the Glycosyl Hydrolase superfamily, are their obligate requirements for NAD+, divalent metal ion and reducing conditions for activity. Whether these unique cofactors functioned in a catalytic or structural capacity was, until recently, unknown. However, our collaborations with international investigators in the past year, have provided the crystal structure of phospho - alpha - glucosidase (GlvA) from Bacillus subtilis in complex with its ligands to 2.05 Angstrom resolution. Analyses of the active site architecture, in conjunction with mechanistic studies and solvent isotope exchange, suggest a novel mechanism of glycoside hydrolysis requiring participation of both NAD(H) and Mn(2+) ion. The proposed four -step reaction involves hydride extraction at C3, and NAD+ mediated oxidation of the 3-OH group to a ketone. This oxidation step causes acidification of the C2 proton, and facilitates deprotonation by an enzymatic base. Thereafter, an acid -catalyzed reaction causes elimination of the glycosidic oxygen, and attendant formation of a 1,2 -unsaturated intermediate. This Michael-like acceptor undergoes base-catalyzed attack by water to generate the 3-keto form of glucose 6-phosphate (G6P). Finally, this keto - intermediate is reduced by the ?on-board? NADH to yield G6P, thereby completing the cycle, and returning the glycosyl hydrolase to its initial NAD/Mn(2+)-liganded active state. Related studies with researchers at Argonne National Lab, Northwestern University and York University have resulted in the first crystallization, and determination of structure of a phospho-beta-glucosidase (BglT) from Family 4. The native structure of the protein was determined by single-wavelength anomalous dispersion (SAD) methods at 2.85 Angstrom resolution. Complexes of the enzyme with NAD+/Mn2+ and Glc6P were determined at 2.55 Angstrom resolution. Comparison of the active-centre structure of BglT with GlvA, reveals a striking degree of architectural similarity, that in light of kinetic isotope effects, allows postulation of a common reaction mechanism for both alpha- and beta-glycosidases. These structural comparisons suggest that simple steric factors, including subtle modifications to protein fold, are sufficient to modulate specificity on a common catalytic framework. Sucrose is the precursor for glycan synthesis that facilitates attachment of oral pathogens e.g., Streptococcus mutans to the tooth surface. Subsequent fermentation of this and other disaccharides (to lactic acid), initiates dental caries by promoting demineralization of tooth enamel. The belief that microorganisms are unable to metabolize the five isomers of sucrose, suggests the potential of these ?sweet? non-cariogenic compounds as substitutes for dietary sucrose in order to combat the etiology of dental caries. However, innovative studies conducted in the Microbial Biochemistry and Genetics Section have revealed rapid dissimilation of these isomers, namely : trehalulose, turanose, maltulose, leucrose and palatinose) by several bacterial species including Fusobacteria, Klebsiella, Bacillus and Clostridia. Unique transport proteins and the NAD+/Mn(2+)-dependent phospho-alpha-glycosylhydrolases participate in the bacterial metabolism of sucrose isomers. The relevant genes have been cloned, sequenced, and proteins expressed for biochemical characterizatioon. The absence of these genes in oral streptococci including S. mutans, explains the failure of these species to ferment the isomeric compounds. Current studies with Dr. Berhard Erni (University of Berne) have facilitated the transfer of the relevant transport and phospho-hydrolysis genes to Escherichia coli. In the presence of an active component of the glucose:phosphotransferase system (EIIAglc), this organism also aquires the capacity to grow at the expense of sucrose isomers and related alpha-linked glucosides.