Stacking interactions and hydrolytic mechanism of anti-biofilm agent Dispersin B

Dispersin B (DspB) from Actinobacillus actinomycetemcomitans (Aa) is an enzyme that degrades homopolymer of β-(1,6)linked glucosamine (PGA) residues. A member of family 20 glycosyl hydrolases, DspB possesses four structurally conserved aromatic residues in the active site. Among these, Trp237 and Tyr278 residues are highly exposed based on the previously determined crystal structure of DspB. Their juxtaposition suggests that the aromatic rings might be involved in stacking interaction with sugar rings of the substrate. Objective: To determine whether or not stacking interactions involving these aromatic residues contribute to the catalytic efficiency of DspB in the hydrolysis of PGA. Methods: Site-directed mutagenesis using PCR was carried out to generate DspB variants Trp237Ala and Tyr278Ala. These were expressed in an E. coli expression system as His.tagged proteins and purified using Ni-affinity chromatography. Results: The alanine substitution exhibited decreased biofilm detachment activity compared to wild-type DspB in a biofilm assay (ED₅₀ for DspB was 2.5 minutes at 0.1 μM compared to 7 minutes for Y237A at 2.8 μM and 3 minutes for Y278A at 0.6 μM). Kinetic experiments utilizing p-nitrophenyl-β-N-acetylglucosamine showed that the enzymatic activities of the mutants were negligible even at 1000 nM (a 10-fold higher concentration compared to DspB). Conclusion: Correlation with crystal structure of DspB suggests that the side chains of W237 and Y278, parallel to each other, may be part of a GlcNAc binding subsite in which the bound GlcNAc might have stacking interactions. Reduced activities of the mutants suggest that stacking interactions involving these two aromatic residues play important roles in hydrolysis of PGA. Additionally, -OH of Tyr278 might contribute in positioning the N-acetyl group of the bound GlcNAc residue. Finally, these results suggest that Trp237 and Tyr278 are essential for catalytic efficiency of DspB. This work was supported by USPHS Grant DE16951.