The exopolysaccharide poly-β-(16)-N-acetylglucosamine (PNAG) is a major structural determinant of bacterial biofilms responsible for persistent and nosocomial infections. The enzymatic dispersal of biofilms by PNAG-hydrolyzing glycosidase enzymes, such as Dispersin B (DspB), is a possible approach to treat biofilm dependent bacterial infections. The cationic charge resulting from partial de-N-acetylation of native PNAG is critical for PNAG-dependent biofilm formation. We recently demonstrated that DspB has increased catalytic activity with de-N-acetylated PNAG oligosaccharides; however, there is still little known about the molecular interaction required for DspB to bind native de-N-acetylated PNAG polysaccharides. Here, we analyze the role of anionic amino acids surrounding the catalytic pocket of DspB in PNAG substrate recognition and hydrolysis using a combination of site directed mutagenesis, activity measurements using synthetic PNAG oligosaccharide analogs, and in vitro biofilm dispersal assays. The results of these studies support a model in which bound PNAG is weakly associated with a shallow anionic groove on the DspB protein surface with recognition driven by interactions with the 1 GlcNAc residue in the catalytic pocket. An increased rate of hydrolysis for cationic PNAG was driven, in part, by interaction with D147 on the anionic surface. Moreover, we identified that a DspB mutant with improved hydrolysis of fully acetylated PNAG oligosaccharides correlates with improved in vitro dispersal of PNAG dependent Staphylococcus epidermidis biofilms. These results provide insight into the mechanism of substrate recognition by DspB and suggest a method to improve DspB biofilm dispersal activity by mutation of the amino acids within the anionic binding surface.

中文翻译：

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阴离子氨基酸在生物膜分散糖苷酶Dispersin B水解聚-β-（1,6）-N-乙酰氨基葡糖胞外多糖中的作用

胞外多糖聚-β-（16）-N-乙酰基葡糖胺（PNAG）是负责持续性和医院感染的细菌生物膜的主要结构决定因素。通过PNAG水解糖苷酶（如Dispersin B（DspB））对生物膜的酶促分散是治疗依赖生物膜的细菌感染的一种可能方法。天然PNAG部分脱N-乙酰化产生的阳离子电荷对于PNAG依赖的生物膜形成至关重要。我们最近证明，DspB与去N-乙酰化的PNAG寡糖具有增强的催化活性。然而，仍有知之甚少为DSPB需要结合天然脱的分子相互作用Ñ-乙酰化的PNAG多糖。在这里，我们分析了DspB催化口袋周围的阴离子氨基酸在定点诱变，使用合成PNAG寡糖类似物的活性测量以及体外生物膜分散测定的组合中在PNAG底物识别和水解中的作用。这些研究的结果支持了一个模型，其中结合的PNAG与DspB蛋白表面上的浅阴离子凹槽弱相关，并且通过与催化口袋中1 GlcNAc残基的相互作用驱动识​​别。阳离子PNAG水解速率的提高部分是由于与阴离子表面上的D147相互作用所致。此外，我们发现，DspB突变体具有完全乙酰化的PNAG寡糖水解作用的改善，与体外改善的作用有关PNAG依赖的表皮葡萄球菌生物膜的分散。这些结果提供了对DspB识别底物的机理的见解，并提出了一种通过阴离子结合表面内氨基酸的突变来改善DspB生物膜分散活性的方法。