Background:The bacterial second messenger cyclic di-GMP (c-di-GMP) is degraded by EAL phosphodiesterases. Results:The isolated EAL domain is active only as a homodimer. Substrate binding is coupled with EAL dimerization. Conclusion: Activity of many full-length EAL phosphodiesterases may be regulated by catalytic domain dimerization. Significance: A generic mechanism for the regulation of a central node of c-di-GMP signaling is provided.

ADP-ribosylation is one of the favored modes of cell intoxication employed by several bacteria. Clostridium difficile is recognized to be an important nosocomial pathogen associated with considerable morbidity and attributable mortality. Along with its two well known toxins, Toxin A and Toxin B, it produces an ADP-ribosylating toxin that targets monomeric actin of the target cell. Like other Clostridial actin ADP-ribosylating toxins, this binary toxin, known as C. difficile toxin (CDT), is composed of two subunits, CDTa and CDTb. In this study, we present high resolution crystal structures of CDTa in its native form (at pH 4.0, 8.5, and 9.0) and in complex with ADP-ribose donors, NAD and NADPH (at pH 9.0). The crystal structures of the native protein show "pronounced conformational flexibility" confined to the active site region of the protein and "enhanced" disorder at low pH, whereas the complex structures highlight significant differences in "ligand specificity" compared with the enzymatic subunit of a close homologue, Clostridium perfringens iota toxin. Specifically in CDTa, two of the suggested catalytically important residues (Glu-385 and Glu-387) seem to play no role or a less important role in ligand binding. These structural data provide the first detailed information on protein-donor substrate complex stabilization in CDTa, which may have implications in understanding CDT recognition.Clostridium difficile infection is a major problem as a healthcare-associated infection. The bacterium causes nosocomial, antibiotic-associated diarrhea and pseudomembranous colitis in patients treated with broad spectrum antibiotics (1-3). Elderly patients are most at risk from these potentially life-threatening diseases, and incidents of hospital infection have increased dramatically over the last 10 years.Strains of C. difficile produce a variety of virulence factors, notable among which are several protein toxins: Toxin A, Toxin B (4 -6), and, in some strains, the binary toxin CDT, 3 which is similar to Clostridium perfringens iota toxin and Clostridium botulinum C2 toxin (7-9). Toxins A and B are large protein cytotoxins that play a key role in the pathology of infection and most probably are involved in the gut colonization process. Outbreaks of C. difficile infection have been reported with Toxin A-negative/Toxin B-positive strains, and a recent report (10) suggests that Toxin B plays a major role in the disease pathology. Little is presently known about the contribution of the binary toxin to C. difficile infection.CDT binary toxin belongs to the family of actin-specific ADP-ribosylating toxin (ADPRT) (for a recent review see Ref.11), composed of two independently produced components: a transport component of 99 kDa (CDTb) that facilitates translocation of the enzymatic component of 49 kDa (CDTa) into the target cell that is capable of transferring ADP-ribose group of NAD/NADPH to monomeric actin molecules in target cells (9,12,13). This irreversible modification of G-actin at 14) blocks its polymerization and thus...

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Histo-blood group antigens (HBGAs) are a source of antigenic variation between individuals that modulates resistance and susceptibility to pathogens and is a barrier to the spread of enveloped viruses. HBGAs are also produced by a few prokaryotes where they are synthesized by glycosyltransferases (GTs) related to human HBGA synthases. Here we report the first structure of a bacterial GT of this family, from an intestinal resident, Bacteroides ovatus. Unlike its mammalian homologues and other GTs with similar folds, this protein lacks a metal-binding Asp-X-Asp motif and is fully active in the absence of divalent metal ions, yet is strikingly similar in structure and in its interactions with substrates to structurally characterized mammalian metal-dependent mammalian homologues. This shows how an apparently major divergence in catalytic properties can be accommodated by minor structural adjustments and illustrates the structural underpinnings of horizontal transfer of a functional gene from prokaryotes to vertebrates.