Group A carbohydrate (GAC) is a bacterial peptidoglycan-anchored surface rhamnose polysaccharide (RhaPS) that is essential for growth of Streptococcus pyogenes and contributes to its ability to infect the human host. In this study, using molecular and synthetic biology approaches, biochemistry, radiolabeling techniques, and NMR and MS analyses, we examined the role of GacB, encoded in the S. pyogenes GAC gene cluster, in the GAC biosynthesis pathway. We demonstrate that GacB is the first characterized α-d-GlcNAc-β-1,4-l-rhamnosyltransferase that synthesizes the committed step in the biosynthesis of the GAC virulence determinant. Importantly, the substitution of S. pyogenes gacB with the homologous gene from Streptococcus agalactiae (Group B Streptococcus), Streptococcus equi subsp. zooepidemicus (Group C Streptococcus), Streptococcus dysgalactiae subsp. equisimilis (Group G Streptococcus), or Streptococcus mutans complemented the GAC biosynthesis pathway. These results, combined with those from extensive in silico studies, reveal a common phylogenetic origin of the genes required for this priming step in >40 pathogenic species of the Streptococcus genus, including members from the Lancefield Groups B, C, D, E, G, and H. Importantly, this priming step appears to be unique to streptococcal ABC transporter–dependent RhaPS biosynthesis, whereas the Wzx/Wzy-dependent streptococcal capsular polysaccharide pathways instead require an α-d-Glc-β-1,4-l-rhamnosyltransferase. The insights into the RhaPS priming step obtained here open the door to targeting the early steps of the group carbohydrate biosynthesis pathways in species of the Streptococcus genus of high clinical and veterinary importance.

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A new chiral hydrogen-bonded organic framework (HOF) denoted as ZIOC-1 based on a l-prolineamide substituted diarylacetylene dicarboxylic acid (3,3′-ethyne-1,2-diylbis­[6-(l-prolylamino)­benzoic acid]) has been synthesized and structurally characterized. Crystal structures of two forms of ZIOC-1, i.e., anhydrous and dihydrate phases, were identified by powder X-ray analysis. It was found that the novel HOF material demonstrated a framework flexibility: its crystalline phases transform reversibly into each other by a change in either humidity or ambient temperature. This fact in conjunction with the retention of crystal identity to the temperature as high as 265 °C indicates a robustness of the framework formed by a hydrogen bonding system. The catalytic performance of the synthesized HOF was probed in aldol condensation and Michael reaction.

The opportunistic pathogen Pseudomonas aeruginosa produces two major cell surface lipopolysaccharides, characterized by distinct O antigens, called common polysaccharide antigen (CPA) and O-specific antigen (OSA). CPA contains a polymer of Drhamnose (D-Rha) in ␣1-2 and ␣1-3 linkages. Three putative glycosyltransferase genes, wbpX, wbpY, and wbpZ, are part of the CPA biosynthesis cluster. To characterize the enzymatic function of the wbpZ gene product, we chemically synthesized the donor substrate GDP-D-Rha and enzymatically synthesized GDP-D-[ 3 H]Rha. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that WbpZ transferred one D-Rha residue from GDP-D-Rha in ␣1-3 linkage to both GlcNAc-and GalNAcdiphosphate-lipid acceptor substrates. WbpZ is also capable of transferring D-mannose (D-Man) to these acceptors. Therefore, WbpZ has a relaxed specificity with respect to both acceptor and donor substrates. The diphosphate group of the acceptor, however, is required for activity. WbpZ does not require divalent metal ion for activity and exhibits an unusually high pH optimum of 9. WbpZ from PAO1 is therefore a GDP-D-Rha:GlcNAc/GalNAc-diphosphate-lipid ␣1,3-D-rhamnosyltransferase that has significant activity of GDP-D-Man:GlcNAc/GalNAc-diphosphate-lipid ␣1,3-D-mannosyltransferase. We used site-directed mutagenesis to replace the Asp residues of the two DXD motifs with Ala. Neither of the mutant constructs of wbpZ (D172A or D254A) could be used to rescue CPA biosynthesis in the ⌬wbpZ knockout mutant in a complementation assay. This suggested that D172 and D254 are essential for WbpZ function. This work is the first detailed characterization study of a D-Rha-transferase and a critical step in the development of CPA synthesis inhibitors. IMPORTANCEThis is the first characterization of a D-rhamnosyltransferase and shows that it is essential in Pseudomonas aeruginosa for the synthesis of the common polysaccharide antigen. P seudomonas aeruginosa is a Gram-negative bacterium that is ubiquitous in the environment and is an opportunistic pathogen that can cause life-threatening infections in humans whose defenses are compromised, e.g., those with immune deficiency, burn wounds, cancer, or cystic fibrosis (CF). Specific epidemic strains have been shown to cause local outbreaks and hospital-associated infections (1). In the airways of CF patients, P. aeruginosa thrives, adopting a biofilm lifestyle, and often becomes resistant to antibiotic treatment. Therefore, it is important to understand the mechanisms by which P. aeruginosa synthesizes its virulence factor, in order to develop new antibacterial strategies.Lipopolysaccharides (LPS) on the outer membrane of P. aeruginosa are required for its survival against host defense mechanisms; hence, LPS is one of the major virulence factors (2-4). These bacteria are unusual in that they simultaneously synthesize two distinct forms of LPS differing in the O-antigen structures (5). Each of these O antigens is synthesized by a different pathway.

Dolichyl-phosphate-mannose (Dol-P-Man) synthase catalyzes the reversible formation of a key intermediate that is involved as a mannosyl donor in at least three different pathways for the synthesis of glycoconjugates important for eukaryotic development and viability. The enzyme is found associated with membranes of the endoplasmic reticulum (ER), where it transfers mannose from the water soluble cytoplasmic donor, guanosine 5'-diphosphate (GDP)-Man, to the membrane-bound, extremely hydrophobic, and long-chain polyisoprenoid acceptor, dolichyl-phosphate (Dol-P). The enzyme from Saccharomyces cerevisiae has been utilized to investigate the structure and activity of the protein and interactions of the enzyme with Dol-P and synthetic Dol-P analogs containing fluorescent probes. These interactions have been explored utilizing fluorescence resonance energy transfer (FRET) to establish intramolecular distances within the protein molecule as well as intermolecular distances to determine the localization of the active site and the hydrophobic substrate on the enzyme's surface. A three-dimensional (3D) model of the enzyme was produced with bound substrates, Dol-P, GDP-Man, and divalent cations to delineate the binding sites for these substrates as well as the catalytic site. The FRET analysis was used to characterize the functional properties of the enzyme and to evaluate its modeled structure. The data allowed for proposing a molecular mechanism of catalysis as an inverting mechanism of mannosyl residue transfer.