Metabolic Usage and Glycan Destinations of GlcNAz in E. coli.

Bacteria use a diverse range of carbohydrates to generate a profusion of glycans, with amino sugars, such as N-acetylglucosamine (GlcNAc), being prevalent in the cell wall and in many exopolysaccharides. The primary substrate for GlcNAc-containing glycans, UDP-GlcNAc, is the product of the bacterial hexosamine pathway and a key target for bacterial metabolic glycan engineering. Using the strategy of expressing NahK, to circumvent the hexosamine pathway, it is possible to directly feed the analogue of GlcNAc, N-azidoacetylglucosamine (GlcNAz), for metabolic labeling in Escherichia coli. The cytosolic production of UDP-GlcNAz was confirmed by using fluorescence-assisted polyacrylamide gel electrophoresis. The key question of where GlcNAz is incorporated was interrogated by analyzing potential sites including peptidoglycan (PGN), the biofilm-related exopolysaccharide poly-ß-1,6-N-acetylglucosamine (PNAG), lipopolysaccharide (LPS), and the enterobacterial common antigen (ECA). The highest levels of incorporation were observed in PGN with lower levels in PNAG and no observable incorporation in LPS or ECA. The promiscuity of the PNAG synthase (PgaCD) toward UDP-GlcNAz in vitro and the lack of undecaprenyl-pyrophosphoryl-GlcNAz intermediates generated in vivo confirmed the incorporation preferences. The results of this work will guide the future development of carbohydrate-based probes and metabolic engineering strategies.

The Metabolic Usage and Glycan Destinations of GlcNAz in E. coli.

Bacteria use a diverse range of carbohydrates to generate a profusion of glycans, with amino sugars such as N-acetylglucosamine (GlcNAc) being prevalent in the cell wall and in many exopolysaccharides. The primary substrate for GlcNAc-containing glycans, UDP-GlcNAc, is the product of the bacterial hexosamine pathway, and a key target for bacterial metabolic glycan engineering. Using the strategy of expressing NahK, to circumvent the hexosamine pathway, it is possible to directly feed the analogue of GlcNAc, N-azidoacetylglucosamine (GlcNAz), for metabolic labelling in E. coli. The cytosolic production of UDP-GlcNAz was confirmed using fluorescence assisted polyacrylamide gel electrophoresis. The key question of where GlcNAz is incorporated, was interrogated by analyzing potential sites including: peptidoglycan (PGN), the biofilm-related exopolysaccharide poly-ß-1,6-N-acetylglucosamine (PNAG), lipopolysaccharide (LPS) and the enterobacterial common antigen (ECA). The highest levels of incorporation were observed in PGN with lower levels in PNAG and no observable incorporation in LPS or ECA. The promiscuity of the PNAG synthase (PgaCD) towards UDP-GlcNAz in vitro and lack of undecaprenyl-pyrophosphoryl-GlcNAz intermediates generated in vivo confirmed the incorporation preferences. The results of this work will guide the future development of carbohydrate-based probes and metabolic engineering strategies.

Small Molecule Inhibition of an Exopolysaccharide Modification Enzyme is a Viable Strategy To Block Pseudomonas aeruginosa Pel Biofilm Formation.

Biosynthesis of the Pel exopolysaccharide in Pseudomonas aeruginosa requires all seven genes of the pelABCDEFG operon. The periplasmic modification enzyme PelA contains a C-terminal deacetylase domain that is necessary for Pel-dependent biofilm formation. Herein, we show that extracellular Pel is not produced by a P. aeruginosa PelA deacetylase mutant. This positions PelA deacetylase activity as an attractive target to prevent Pel-dependent biofilm formation. Using a high-throughput screen (n = 69,360), we identified 56 compounds that potentially inhibit PelA esterase activity, the first enzymatic step in the deacetylase reaction. A secondary biofilm inhibition assay identified methyl 2-(2-pyridinylmethylene) hydrazinecarbodithioate (SK-017154-O) as a specific Pel-dependent biofilm inhibitor. Structure-activity relationship studies identified the thiocarbazate as a necessary functional group and that the pyridyl ring could be replaced with a phenyl substituent (compound 1). Both SK-017154-O and compound 1 inhibit Pel-dependent biofilm formation in Bacillus cereus ATCC 10987, which has a predicted extracellular PelA deacetylase in its pel operon. Michaelis-Menten kinetics determined SK-017154-O to be a noncompetitive inhibitor of PelA, while compound 1 did not directly inhibit PelA esterase activity. Cytotoxicity assays using human lung fibroblast cells showed that compound 1 is less cytotoxic than SK-017154-O. This work provides proof of concept that biofilm exopolysaccharide modification enzymes are important for biofilm formation and can serve as useful antibiofilm targets. IMPORTANCE Present in more than 500 diverse Gram-negative and 900 Gram-positive organisms, the Pel polysaccharide is one of the most phylogenetically widespread biofilm matrix determinants found to date. Partial de-N-acetylation of this α-1,4 linked N-acetylgalactosamine polymer by the carbohydrate modification enzyme PelA is required for Pel-dependent biofilm formation in Pseudomonas aeruginosa and Bacillus cereus. Given this and our observation that extracellular Pel is not produced by a P. aeruginosa PelA deactylase mutant, we developed an enzyme-based high-throughput screen and identified methyl 2-(2-pyridinylmethylene) hydrazinecarbodithioate (SK-017154-O) and its phenyl derivative as specific Pel-dependent biofilm inhibitors. Michaelis-Menten kinetics revealed SK-017154-O is a noncompetitive inhibitor and that its noncytotoxic, phenyl derivative does not directly inhibit P. aeruginosa PelA esterase activity. We provide proof of concept that exopolysaccharide modification enzymes can be targeted with small molecule inhibitors to block Pel-dependent biofilm development in both Gram-negative and Gram-positive bacteria.

Termination of Poly-N-acetylglucosamine (PNAG) Polymerization with N-Acetylglucosamine Analogues.

Bacteria require polysaccharides for structure, survival, and virulence. Despite their central role in microbiology, few tools are available to manipulate their production. In E. coli, the glycosyltransferase complex PgaCD produces poly-N-acetylglucosamine (PNAG), an extracellular matrix polysaccharide required for biofilm formation. We report that C6-substituted (H, F, N3, SH, NH2) UDP-GlcNAc substrate analogues are inhibitors of PgaCD. In vitro, the inhibitors cause PNAG chain termination, consistent with the mechanism of PNAG polymerization from the nonreducing terminus. In vivo, expression of the GlcNAc-1-kinase NahK in E. coli provided a non-native GlcNAc salvage pathway that produced the UDP-GlcNAc analogue inhibitors in situ. The 6-fluoro and 6-deoxy derivatives were potent inhibitors of biofilm formation in the transformed strain, providing a tool to manipulate this key exopolysaccharide. Characterization of the UDP-GlcNAc pool and quantification of PNAG generation support PNAG termination as the primary in vivo mechanism of biofilm inhibition by 6-fluoro UDP-GlcNAc.

Allosteric modulation of the adenosine A2A receptor by cholesterol.

Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid nanodiscs. This model system conveniently excludes possible effects arising from cholesterol-induced phase separation or receptor oligomerization and focuses on the question of allostery. GTP hydrolysis assays show that cholesterol weakly enhances the basal signaling of A2AR while decreasing the agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy shows that this enhancement arises from an increase in the receptor's active state population and a G-protein-bound precoupled state. 19F NMR of fluorinated cholesterol analogs reveals transient interactions with A2AR, indicating a lack of high-affinity binding or direct allosteric modulation. The combined results suggest that the observed allosteric effects are largely indirect and originate from cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.

Synthesis of C6-substituted UDP-GlcNAc derivatives.

UDP-sugar analogs are useful for the study of glycosyltransferases and the production of unnatural glycans. The preparation of five UDP-GlcNAc derivatives is reported with 6-deoxy, 6-azido, 6-amino, 6-mercapto, or 6-fluoro substitutions. A concise chemoenzymatic synthesis was developed using the kinase NahK (B. longum JCM1217) and the uridyl transferase GlmU (E. coli K12).

TBAF Effects 3,6-Anhydro Formation from 6-O-Tosyl Pyranosides.

3,6-Anhydro sugars are common structures in algal polysaccharides and occur in the furanodictine and sauropunol natural products. We have found that treatment of 6-O-tosylpyranosides with tetrabutylammonium fluoride provides a mild, high-yielding synthesis of 3,6-anhydro sugars. Using O-glycoside substrates, 3,6-anhydropyranosides are isolated and the use of N,O-dimethyl hydroxylamine glycosides yields 3,6-anhydrofuranosides. Applying this approach, concise synthetic routes to several 3,6-anhydro sugar natural products are reported, including furanodictine A and sauropunols A-D.

Altering the Y137-K164-K166 triad of mandelate racemase and its effect on the observed pKa of the Brønsted base catalysts.

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelate using a two-base mechanism with Lys 166 acting as the Brønsted base to abstract the α-proton from (S)-mandelate. The resulting intermediate is subsequently re-protonated by the conjugate acid of His 297 to yield (R)-mandelate. The roles of these amino acids are reversed when (R)-mandelate is the substrate. The side chains of Tyr 137, Lys 164, and Lys 166 form a H-bonding network and the proximity of the two ε-NH3+ groups is believed to lower the pKa of Lys 166. We used site-directed mutagenesis, kinetics, and pH-rate studies to explore the roles of Lys 164 (K164 C/M) and Tyr 137 (Y137  L/F/S/T) in catalysis. The efficiency (kcat/Km) was reduced ∼3.5 × 105-fold for K164C MR, relative to wild-type MR, indicating a major role for this residue in catalysis. The efficiency of Y137F MR, however, was reduced only 25-30-fold. pH-Rate profiles (log kcat vs. pH) revealed that substitution of Tyr 137 by Phe increased the kinetic pKa of Lys 166 from 5.88 ±â€¯0.02 to 7.3 ±â€¯0.2. Hence, Tyr 137 plays an important role in facilitating the reduction of the pKa of the Brønsted base Lys 166 by ∼1.4 units. Interestingly, the Phe substitution also increased the kinetic pKa of His 297 from 5.97 ±â€¯0.04 to 7.1 ±â€¯0.1. Thus, the Tyr 137-Lys 164-Lys 166 H-bonding network plays a broader role in modulating the pKa of catalytic residues by influencing the electrostatic character of the entire active site, not only by decreasing the observed pKa value of Lys 166, but also by decreasing the pKa of His 297 by 1.1 units.

Monosaccharide inhibitors targeting carbohydrate esterase family 4 de-N-acetylases.

The Carbohydrate Esterase family 4 contains virulence factors which modify peptidoglycan and biofilm-related exopolysaccharides. Despite the importance of this family of enzymes, a potent mechanism-based inhibition strategy has yet to emerge. Based on the postulated tridentate binding mode of the tetrahedral de-N-acetylation intermediate, GlcNAc derivatives bearing metal chelating groups at the 2 and 3 positions were synthesized. These scaffolds include 2-C phosphonate, 2-C sulfonamide, 2-thionoacetamide warheads as well as derivatives bearing thiol, amine and azide substitutions at the 3-position. The inhibitors were assayed against a representative peptidoglycan deacetylase, Pgda from Streptococcus pneumonia, and a representative biofilm-related exopolysaccharide deacetylase, PgaB from Escherichia coli. Of the inhibitors evaluated, the 3-thio derivatives showed weak to moderate inhibition of Pgda. The strongest inhibitor was benzyl 2,3-dideoxy-2-thionoacetamide-3-thio-ß-d-glucoside, whose inhibitory potency showed an unexpected dependence on metal concentration and was found to have a partial mixed inhibition mode (Ki = 2.9 ±â€¯0.6 µM).