Bacteria in a biofilm are enmeshed in a self-synthesized extracellular polysaccharide matrix (PGA) which is a linear polymer of N-acetylglucosamine residues in β(1,6)-linkage. Dispersin B (DspB), a soluble glycoside hydrolase produced by Actinobacillus actinomycetemcomitans (Aa) degrades PGA. DspB, is an (β/α) TIM-barrel protein and belongs to family 20 glycosyl hydrolases in which a conserved amino acid pair, aspartate-glutamate, is present (Asp183-Glu184, DspB numbering). In addition, the active site of DspB contains another acidic residue Glu332 at about 5Å away from Glu184. Objective: To understand the role of each of these acidic residues in the hydrolytic mechanism. Methods: Using site-directed mutagenesis, biological and biochemical characterization, we investigated the role of Asp183, Glu184 and Glu332. Results: We found that Glu184 and Glu332 residues are essential for DspB activity. Mutation of each of these causes a significant reduction in the enzymatic activity. The variant Glu184Gln requires a 10-fold increase in enzyme concentration (>1000 nM) for measurable activity in kinetic as well as biofilm assay whereas Glu332Gln is inactive even at 1000 nM. In contrast, both DspB and Asp183Ala exhibited similar kinetics at 100 nM concentration; however, Asp183Ala showed a 12-fold loss in activity compared to DspB. Similar results were obtained in a 96-well biofilm detachment assay as well. Conclusion: The loss of activity in the Glu184 and Glu332 variants suggests that DspB might hydrolyze PGA through a mechanism other than substrate-assisted mechanism. In family 20 hydrolases, the Asp183 equivalent residue plays a significant role in the substrate-assisted mechanism and participates in holding the substrate in a specific orientation. However, in DspB, Asp183 might play only a subtle role by participating in substrate binding. This work was supported by USPHS Grant DE16291.

Back to the Microbiology / Immunology and Infection Control Program
Back to the IADR/AADR/CADR 85th General Session and Exhibition (March 21-24, 2007)

Top Level Search