Synthesis of defined mono-de-<i>N</i>-acetylated β-(1→6)-<i>N</i>-acetyl-<scp>d</scp>-glucosamine oligosaccharides to characterize PgaB hydrolase activity

Forman, Adam; Pfoh, Roland; Eddenden, Alexander; Howell, P. Lynne; Nitz, Mark

doi:10.1039/c9ob02079a

Cited by 13 publications

(9 citation statements)

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“…The selective interaction with locations of high PNAG density at the cells’ surface may aid in the function of DspB to disperse biofilms as it would limit competitive binding by PNAG oligosaccharides and promote biofilm localization. PNAG oligosaccharides would be generated in high quantities during DspB-based hydrolysis of a biofilm, or they may be generated as a product of PNAG production. , As a probe, the low affinity of GFP-DiPP allows specific binding to high local concentrations of PNAG allowing direct interrogation of the high density PNAG locations at the cells’ surface during the formation of a biofilm.…”

Section: Resultsmentioning

confidence: 99%

“…PNAG oligosaccharides would be generated in high quantities during DspB-based hydrolysis of a biofilm, or they may be generated as a product of PNAG production. 32,39 As a probe, the low affinity of GFP-DiPP allows specific binding to high local concentrations of PNAG allowing direct interrogation of the high density PNAG locations at the cells' surface during the formation of a biofilm.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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An Inactive Dispersin B Probe for Monitoring PNAG Production in Biofilm Formation

et al. 2020

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The bacterial exopolysaccharide poly-β-1,6-N-acetylglucosamine is a major extracellular matrix component in biofilms of both Gram-positive and Gram-negative organisms. We have leveraged the specificity of the biofilm-dispersing glycoside hydrolase Dispersin B (DspB) to generate a probe (Dispersin B PNAG probe, DiPP) for monitoring PNAG production and localization during biofilm formation. Mutation of the active site of Dispersin B gave DiPP, which was an effective probe despite its low affinity for PNAG oligosaccharides (K D ∼ 1−10 mM). Imaging of PNAG-dependent and -independent biofilms stained with a fluorescent-protein fusion of DiPP (GFP-DiPP) demonstrated the specificity of the probe for the structure of PNAG on both single-cell and biofilm levels, indicating a high local concentration of PNAG at the bacterial cell surface. Through quantitative bacterial cell binding assays and confocal microscopy analysis using GFP-DiPP, discrete areas of local high concentrations of PNAG were detected on the surface of early log phase cells. These distinct areas were seen to grow, slough from cells, and accumulate in interbacterial regions over the course of several cell divisions, showing the development of a PNAG-dependent biofilm. A potential helical distribution of staining was also noted, suggesting some degree of organization of PNAG production at the cell surface prior to cell aggregation. Together, these experiments shed light on the early stages of PNAG-dependent biofilm formation and demonstrate the value of a low-affinity-highspecificity probe for monitoring the production of bacterial exopolysaccharides Special Issue: Chemical Microbiology

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Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

An Inactive Dispersin B Probe for Monitoring PNAG Production in Biofilm Formation

et al. 2020

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“…Given the importance of PNAG for biofilm integrity and its widespread distribution amongst Gram-positive and Gram-negative bacteria, enzymes that catalyze the hydrolysis of PNAG are potentially useful for treating diverse biofilm infections (11,12). Two PNAG glycosidases, DspB and the C-terminal domain of PgaB, have been described to date, but both suffer from relatively low catalytic activity (29,30,35). For example, the activity of DspB measured with a variety of substrate analogs has resulted in observed rates in the order of 2-60 M -1 s -1 (30, 32) nearly 4-6 orders of magnitude slower than comparable rates observed with other GH20 enzymes (40,53).…”

Section: Discussionmentioning

confidence: 99%

“…PgaB is the bifunctional carbohydrate esterase/glycosyl hydrolase enzyme required for PNAG biosynthesis in Gram-negative bacteria (24,34). The PgaB GH domain catalyzes endoglycosidic cleavage of partially de-Nacetylated PNAG substrates containing a glucosamine (GlcN) in the -3 binding site (29,35). Despite interest in these enzymes as biofilm dispersal agents and as treatments for biofilmdependent infections, there is still relatively little known about the specific binding interactions required for recognition of their respective PNAG substrates.…”

Section: Introductionmentioning

confidence: 99%

Anionic amino acids support hydrolysis of poly-β-(1,6)-N-acetylglucosamine exopolysaccharides by the biofilm dispersing glycosidase Dispersin B

Breslawec

Wang

et al. 2021

Journal of Biological Chemistry

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The exopolysaccharide poly- β -(1→6)- 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 on de- N -acetylated PNAG oligosaccharides, but the molecular basis for this increased activity is not known. 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.

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“…The first, Dispersin B (DspB), is an exported CAZy glycosyl hydrolase family 20 enzyme produced by Aggregatibacter actinomycetemcomitan that has been shown to hydrolyze PNAG utilizing both exo ‐ and endo ‐glycosidic cleavage activity . The second, PgaB, is the first member of the novel CAZy glycosyl hydrolase family 153 and specifically catalyzes endoglycosidic cleavage of PNAG substrates containing d ‐glucosamine (GlcN) in the −3 binding site . These enzymes share little sequence similarity and use different mechanisms for PNAG hydrolysis.…”

Section: Figurementioning

confidence: 99%

A Colorimetric Assay to Enable High‐Throughput Identification of Biofilm Exopolysaccharide‐Hydrolyzing Enzymes

Wang

Breslawec

et al. 2020

Chemistry A European J

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Glycosidase enzymes that hydrolyze the biofilm exopolysaccharide poly‐β‐(1→6)‐N‐acetylglucosamine (PNAG) are critical tools to study biofilm and potential therapeutic biofilm dispersal agents. Function‐driven metagenomic screening is a powerful approach for the discovery of new glycosidase but requires sensitive assays capable of distinguishing between the desired enzyme and functionally related enzymes. Herein, we report the synthesis of a colorimetric PNAG disaccharide analogue whose hydrolysis by PNAG glycosidases results in production of para‐nitroaniline that can be continuously monitored at 410 nm. The assay is specific for enzymes capable of hydrolyzing PNAG and not related β‐hexosaminidase enzymes with alternative glycosidic linkage specificities. This analogue enabled development of a continuous colorimetric assay for detection of PNAG hydrolyzing enzyme activity in crude E. coli cell lysates and suggests that this disaccharide probe will be critical for establishing the functional screening of metagenomic DNA libraries.

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Synthesis of defined mono-de-N-acetylated β-(1→6)-N-acetyl-d-glucosamine oligosaccharides to characterize PgaB hydrolase activity

Abstract: Mono-de-N-acetylated β-(1→6)-N-acetyl-d-glucosamine penta- and hepta-saccharides were obtained using a convergent synthesis. The site of de-N-acetylation drives the selectivity of hydrolysis by PgaB.

Cited by 13 publications

References 70 publications

An Inactive Dispersin B Probe for Monitoring PNAG Production in Biofilm Formation

An Inactive Dispersin B Probe for Monitoring PNAG Production in Biofilm Formation

Anionic amino acids support hydrolysis of poly-β-(1,6)-N-acetylglucosamine exopolysaccharides by the biofilm dispersing glycosidase Dispersin B

A Colorimetric Assay to Enable High‐Throughput Identification of Biofilm Exopolysaccharide‐Hydrolyzing Enzymes

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