AB5 toxins comprise an A subunit that corrupts essential eukaryotic cell functions, and pentameric B subunits that direct target cell uptake after binding surface glycans. Subtilase cytotoxin (SubAB) is an AB5 toxin secreted by Shiga toxigenic Escherichia coli (STEC)1, which causes serious gastrointestinal disease in humans2. SubAB causes haemolytic uraemic syndrome-like pathology in mice3 via SubA-mediated cleavage of BiP/GRP78, an essential endoplasmic reticulum chaperone4. Here we show that SubB has a strong preference for glycans terminating in the sialic acid N-glycolylneuraminic acid (Neu5Gc), a monosaccharide not synthesised in humans. Structures of SubB-Neu5Gc complexes revealed the basis for this specificity, and mutagenesis of key SubB residues abrogated in vitro glycan recognition, cell binding and cytotoxicity. SubAB specificity for Neu5Gc was confirmed using mouse tissues with a human-like deficiency of Neu5Gc and human cell lines fed with Neu5Gc. Despite human lack of Neu5Gc biosynthesis, assimilation of dietary Neu5Gc creates high-affinity receptors on human gut epithelia and kidney vasculature. This, together with the human lack of Neu5Gc-containing body fluid competitors, confers susceptibility to the gastrointestinal and systemic toxicities of SubAB. Ironically, foods rich in Neu5Gc are the most common source of STEC contamination. Thus a bacterial toxin’s receptor is generated by metabolic incorporation of an exogenous factor derived from food.

Legionella pneumophila is the predominant cause of Legionnaires disease, a severe and potentially fatal form of pneumonia. Recently, we identified an ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila, termed Lpg1905, which enhances intracellular replication of L. pneumophila in eukaryotic cells. Lpg1905 is the first prokaryotic member of the CD39/NTPDase1 family of enzymes, which are characterized by the presence of five apyrase conserved regions and the ability to hydrolyze nucleoside tri-and diphosphates. Here we examined the substrate specificity of Lpg1905 and showed that apart from ATP and ADP, the enzyme catalyzed the hydrolysis of GTP and GDP but had limited activity against CTP, CDP, UTP, and UDP. Based on amino acid residues conserved in the apyrase conserved regions of eukaryotic NTPDases, we generated five site-directed mutants, Lpg1905E159A, R122A, N168A, Q193A, and W384A. Although the mutations E159A, R122A, Q193A, and W384A abrogated activity completely, N168A resulted in decreased activity caused by reduced affinity for nucleotides. When introduced into the lpg1905 mutant strain of L. pneumophila, only N168A partially restored the ability of L. pneumophila to replicate in THP-1 macrophages. Following intratracheal inoculation of A/J mice, none of the Lpg1905 mutants was able to restore virulence to an lpg1905 mutant during lung infection, thereby demonstrating the importance of NTPDase activity to L. pneumophila infection. Overall, the kinetic studies undertaken here demonstrated important differences to mammalian NTPDases and different sensitivities to NTPDase inhibitors that may reflect underlying structural variations.Legionella pneumophilia is the major causative agent of Legionnaires disease, a severe systemic disease characterized by acute pneumonia. During infection of the human lung, L. pneumophila is internalized by alveolar macrophages where the bacteria replicate within an intracellular vacuole that evades fusion with the endocytic pathway. Instead the L. pneumophila containing vacuole intercepts early secretory vesicles and following maturation exhibits properties of the endoplasmic reticulum (1-3). Establishment of the unique Legionella vacuole and the ability to replicate within eukaryotic cells requires the Dot/Icm type IV secretion system, which translocates bacterial effector proteins into the cytoplasm of the host cell (4 -7).The ability of L. pneumophila to replicate within mammalian cells appears to be a consequence of its association in the natural environment with free living protozoa. The three sequenced L. pneumophila genomes encode an unusually high number of proteins with similarity to eukaryotic proteins (8 -10), which may interfere with host cell processes by functional mimicry (11). We recently showed that Lpg1905 from L. pneumophila is a secreted member of the CD39/ NTPDase1 family of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases; gene family ENTPD).5 NTPDases are extremely rare in bacteria, and Lpg1905 is the first prokaryotic...

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Many pathogenic bacteria have sophisticated mechanisms to interfere with the mammalian immune response. These include the disruption of host extracellular ATP levels that, in humans, is tightly regulated by the nucleoside triphosphate diphosphohydrolase family (NTPDases). NTPDases are found almost exclusively in eukaryotes, the notable exception being their presence in some pathogenic prokaryotes. To address the function of bacterial NTPDases, we describe the structures of an NTPDase from the pathogen Legionella pneumophila (Lpg1905/Lp1NTPDase) in its apo state and in complex with the ATP analog AMPPNP and the subtype-specific NTPDase inhibitor ARL 67156. Lp1NTPDase is structurally and catalytically related to eukaryotic NTPDases and the structure provides a basis for NTPDase-specific inhibition. Furthermore, we demonstrate that the activity of Lp1NTPDase correlates directly with intracellular replication of Legionella within macrophages. Collectively, these findings provide insight into the mechanism of this enzyme and highlight its role in host-pathogen interactions.

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Fatty acid catabolism by ␤-oxidation mainly occurs in mitochondria and to a lesser degree in peroxisomes. Polyunsaturated fatty acids are problematic for ␤-oxidation, because the enzymes directly involved are unable to process all the different double bond conformations and combinations that occur naturally. In mammals, three accessory proteins circumvent this problem by catalyzing specific isomerization and reduction reactions. Central to this process is the NADPH-dependent 2,4-dienoyl-CoA reductase. We present high resolution crystal structures of human mitochondrial 2,4-dienoylCoA reductase in binary complex with cofactor, and the ternary complex with NADP ؉ and substrate trans-2,trans-4-dienoyl-CoA at 2.1 and 1.75 Å resolution, respectively. The enzyme, a homotetramer, is a shortchain dehydrogenase/reductase with a distinctive catalytic center. Close structural similarity between the binary and ternary complexes suggests an absence of large conformational changes during binding and processing of substrate. The site of catalysis is relatively open and placed beside a flexible loop thereby allowing the enzyme to accommodate and process a wide range of fatty acids. Seven single mutants were constructed, by site-directed mutagenesis, to investigate the function of selected residues in the active site thought likely to either contribute to the architecture of the active site or to catalysis. The mutant proteins were overexpressed, purified to homogeneity, and then characterized. The structural and kinetic data are consistent and support a mechanism that derives one reducing equivalent from the cofactor, and one from solvent. Key to the acquisition of a solvent-derived proton is the orientation of substrate and stabilization of a dienolate intermediate by Tyr-199, Asn-148, and the oxidized nicotinamide.Essential fatty acids and derivatives that mammals acquire from exogenous sources or by endogenous biosynthesis fulfill critical functions in numerous metabolic pathways, in endocrine, and signaling processes (1, 2). Fatty acids also provide much of the cells energy following degradation in a sequence of four enzyme-catalyzed reactions termed the ␤-oxidation cycle (1, 3, 4), a highly exergonic metabolic process so named because oxidation occurs at C␤ of a fatty acyl-coenzyme A (CoA) 1 derivative preceding cleavage of the C␣-C␤ bond. The initial step leading into ␤-oxidation is fatty acid activation by formation of a thiolester bond with CoA in a reaction catalyzed by acyl-CoA synthetase. The oxidation of the C␣-C␤ bond produces an olefin, then hydration and oxidation produce a carbonyl group at C␤. The fourth step is cleavage of the ␤-keto ester in a reverse Claisen condensation resulting in a new acyl-CoA derivative truncated by two C atoms, which are used to produce acetyl-CoA. These reactions proceed until eventually only acetyl-CoA is produced. Extensive studies have afforded a very clear picture of structure, mechanism, and specificity of the four enzymes (acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-L-hydrox...