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Abstract: Bacteria are constantly challenged by bacteriophage (phage) infection and have developed multitudinous and varied resistance mechanisms. Bacteriophage Exclusion (BREX) systems protect from phage infection by generating methylation patterns at non-palindromic 6 bp sites in host bacterial DNA, to distinguish and block replication of non-self DNA. Type 1 BREX systems are comprised of six conserved core genes. Here, we present the first reported structure of a BREX core protein, BrxA from the phage defence island of Escherichia fergusonii ATCC 35469 plasmid pEFER, solved to 2.09 Å. BrxA is a monomeric protein in solution, with an all α-helical globular fold. Conservation of surface charges and structural homology modelling against known phage defence systems highlighted that BrxA contains two helix-turn-helix motifs, juxtaposed by 180°, positioned to bind opposite sides of a DNA major groove. BrxA was subsequently shown to bind dsDNA. This new understanding of BrxA structure, and first indication of BrxA biological activity, suggests a conserved mode of DNA-recognition has become widespread and implemented by diverse phage defence systems.

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Abstract: Solar H2O2 produced by O2 reduction provides a green, efficient, and ecological alternative to the industrial anthraquinone process and H2/O2 direct-synthesis. We report efficient photocatalytic H2O2 production at a rate of 73.4 mM h–1 in the presence of a sacrificial donor on a structurally engineered catalyst, alkali metal-halide modulated poly(heptazine imide) (MX → PHI). The reported H2O2 production is nearly 150 and >4250 times higher than triazine structured pristine carbon nitride under UV–visible and visible light (≥400 nm) irradiation, respectively. Furthermore, the solar H2O2 production rate on MX → PHI is higher than most of the previously reported carbon nitride (triazine, tri-s-triazine), metal oxides, metal sulfides, and other metal–organic photocatalysts. A record high AQY of 96% at 365 nm and 21% at 450 nm was observed. We find that structural modulation by alkali metal-halides results in a highly photoactive MX → PHI catalyst which has a broader light absorption range, enhanced light absorption ability, tailored bandgap, and a tunable band edge position. Moreover, this material has a different polymeric structure, high O2 trapping ability, interlayer intercalation, as well as surface decoration of alkali metals. The specific C≡N groups and surface defects, generated by intercalated MX, were also considered as potential contributors to the separation of photoinduced electron–hole pairs, leading to enhanced photocatalytic activity. A synergy of all these factors contributes to a higher H2O2 production rate. Spectroscopic data help us to rationalize the exceptional photochemical performance and structural characteristics of MX → PHI.

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Abstract: Bacterial cell division is a complex process requiring the coordination of multiple components to allow the appropriate spatial and temporal control of septum formation and cell scission. Peptidoglycan (PG) is the major structural component of the septum, and our recent studies in the human pathogen Staphylococcus aureus have revealed a complex, multistage PG architecture that develops during septation. Penicillin-binding proteins (PBPs) are essential for the final steps of PG biosynthesis; their transpeptidase activity links the peptide side chains of nascent glycan strands. PBP1 is required for cell division in S. aureus, and here, we demonstrate that it has multiple essential functions associated with its enzymatic activity and as a regulator of division. Loss of PBP1, or just its C-terminal PASTA domains, results in cessation of division at the point of septal plate formation. The PASTA domains can bind PG and thereby potentially coordinate the cell division process. The transpeptidase activity of PBP1 is also essential, but its loss leads to a strikingly different phenotype of thickened and aberrant septa, which is phenocopied by the morphological effects of adding the PBP1-specific β-lactam, meropenem. Together, these results lead to a model for septal PG synthesis where PBP1 enzyme activity is required for the characteristic architecture of the septum and PBP1 protein molecules enable the formation of the septal plate.

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Abstract: Nitroreductases activate nitroaromatic antibiotics and cancer prodrugs to cytotoxic hydroxylamines and reduce quinones to quinols. Using steady-state and stopped-flow kinetics we show that the E. coli nitroreductase NfsA is 20-50 fold more active with NADPH than with NADH and that product release may be rate-limiting. The crystal structure of NfsA with NADP+ shows that a mobile loop forms a phosphate-binding pocket. The nicotinamide ring and nicotinamide ribose are mobile, as confirmed in molecular dynamics (MD) simulations. We present a model of NADPH bound to NfsA. Only one NADP+ is seen bound to the NfsA dimers, and MD simulations show that binding of a second NADP(H) cofactor is unfavourable, suggesting that NfsA and other members of this protein superfamily may have a half-of-sites mechanism.

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Abstract: The optimization of compounds with multiple targets is a difficult multidimensional problem in the drug discovery cycle. Here, we present a systematic, multidisciplinary approach to the development of selective antiparasitic compounds. Computational fragment-based design of novel pteridine derivatives along with iterations of crystallographic structure determination allowed for the derivation of a structure–activity relationship for multitarget inhibition. The approach yielded compounds showing apparent picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, and selective submicromolar inhibition of parasite dihydrofolate reductase (DHFR) versus human DHFR. Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei while retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds, and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.