The exosporium layer of Bacillus anthracis spores is rich in L-rhamnose, a common bacterial cell-wall component, which often contributes to the virulence of pathogens by increasing their adherence and immune evasion. The biosynthetic pathway used to form the activated L-rhamnose donor dTDP-L-rhamnose consists of four enzymes (RfbA, RfbB, RfbC and RfbD) and is an attractive drug target because there are no homologs in mammals. It was found that co-purifying and screening RfbC (dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase) from B. anthracis in the presence of the other three B. anthracis enzymes of the biosynthetic pathway yielded crystals that were suitable for data collection. RfbC crystallized as a dimer and its structure was determined at 1.63 Å resolution. Two different ligands were bound in the protein structure: pyrophosphate in the active site of one monomer and dTDP in the other monomer. A structural comparison with RfbC homologs showed that the key active-site residues are conserved across kingdoms.

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Polyamines are simple yet critical molecules with diverse roles in numerous pathogenic and non-pathogenic organisms. Regulating polyamine concentrations affects the transcription and translation of genes and proteins important for cell growth, stress, and toxicity. One way polyamine concentrations are maintained within the cell is via spermidine/spermine N-acetyltransferases (SSATs) that acetylate intracellular polyamines so they can be exported. The bacterial SpeG enzyme is an SSAT that exhibits a unique dodecameric structure and allosteric site compared to other SSATs that have been previously characterized. While its overall 3D structure is conserved, its presence and role in different bacterial pathogens are inconsistent. For example, not all bacteria have speG encoded in their genomes; in some bacteria, the speG gene is present but has become silenced, and in other bacteria, it has been acquired on mobile genetic elements. The latter is the case for methicillin-resistant Staphylococcus aureus (MRSA) USA300, where it appears to aid pathogenesis. To gain a greater understanding of the structure/function relationship of SpeG from the MRSA USA300 strain (SaSpeG), we determined its X-ray crystal structure in the presence and absence of spermine. Additionally, we showed the oligomeric state of SaSpeG is dynamic, and its homogeneity is affected by polyamines and AcCoA. Enzyme kinetic assays showed that pre-incubation with polyamines significantly affected the positive cooperativity toward spermine and spermidine and the catalytic efficiency of the enzyme. Furthermore, we showed bacterial SpeG enzymes do not have equivalent capabilities to acetylate aminopropyl versus aminbutyl ends of spermidine. Overall, this study provides new insight that will assist in understanding the SpeG enzyme and its role in pathogenic and non-pathogenic bacteria at a molecular level.

Bacillus anthracis is the causative agent of the deadly disease Anthrax. Its use in bioterrorism and its ability to re-emerge have brought renewed interest in this organism. B. anthracis is a Gram-positive bacterium that adds L-rhamnose to its cell-wall polysaccharides using the activated donor dTDP-β-L-rhamnose. The enzymes involved in the biosynthesis of the activated donor are absent in humans, which make them ideal targets for therapeutic development to combat pathogens. Here, the 2.65 Å resolution crystal structure of the fourth enzyme in the dTDP-β-L-rhamnose-biosynthetic pathway from B. anthracis, dTDP-4-dehydro-β-L-rhamnose reductase (RfbD), is presented in complex with NADP. This enzyme catalyzes the reduction of dTDP-4-dehydro-β-L-rhamnose to dTDP-β-L-rhamnose. Although the protein was co-crystallized in the presence of Mg, the protein lacks the conserved residues that coordinate Mg.

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