Briefly, the reaction combination (20 l) consisted of 100 mM Tris-HCl (pH 8.0), 10 mM DTT, 0.5 mM diC16-phosphatidylserine (Avanti), 25 M PI-3,4,5-P3 (diC16; Echelon), and the indicated amount of CdtB, CdtABC, or PTEN (provided by G. in lymphocytes is related to this activity. The cytolethal distending toxins (Cdts)3 are a family of heat-labile protein cytotoxins produced by several different bacterial species including diarrheal disease-causing enteropathogens such as some isolates, species, (1C7). There is clear evidence that Cdts are encoded by three genes, designated gene by in vitro site-directed mutagenesis using oligonucleotide primer pairs made up of appropriate base changes (Table I). Site-directed mutagenesis was performed using the QuikChange II site-directed mutagenesis kit (Stratagene) according to the manufacturers directions. Amplification of the mutant plasmid was conducted using PfuUltra HF DNA polymerase (Stratagene) and pGEMCdtB as a template; construction and characterization of this plasmid was previously explained (27). All mutants were verified by DNA AM 2201 sequencing. Expression of the plasmids and purification of the mutant peptides is usually explained below. Table I CdtB mutant constructs gene (pGEMCdtB) was previously explained (27). In vitro expression of Cdt peptides and CdtB mutants was performed as previously explained using the Rapid Translation System (RTS 500 ProteoMaster; Roche Applied Science). Reactions were run according to the manufacturers specification (Roche Applied Science) using 10C15 g of template DNA. After 20 h at 30C, the reaction mix was removed and the expressed Cdt peptides were purified by nickel affinity chromatography as explained (27). Construction and expression of the plasmid made AM 2201 up of the genes for the holotoxin (pUCAacdtABChis) has previously been reported (28). The plasmid was constructed so that the genes were under control of the promotor and transformed into DH5. Cultures of transformed were produced in 1 L Luria-Bertani broth and induced with 0.1 mM IPTG for 2 h; bacterial cells were harvested, washed, and resuspended in 50 mM Tris (pH 8.0). The cells were frozen overnight, thawed, and sonicated. The histidine-tagged peptide holotoxin was isolated by nickel affinity chromatography as previously explained (9). Phosphatase assay Phosphatase activity was assessed by monitoring the dephosphorylation of PI-3,4,5-P3 as explained by Maehama et al. (29). Briefly, the reaction combination (20 l) consisted of 100 mM Tris-HCl (pH 8.0), 10 mM DTT, 0.5 mM diC16-phosphatidylserine (Avanti), 25 M PI-3,4,5-P3 (diC16; Echelon), and the indicated amount of CdtB, CdtABC, or PTEN (provided by G. Taylor, University or college of Nebraska Medical Center, Omaha, NE). Appropriate amounts of lipid solutions were deposited in 1.5-ml tubes, organic solvent was removed, the buffer was added, and a lipid suspension was formed by sonication. For experiments to determine substrate specificity, PI-3,4,5-P3 was replaced by the indicated phosphatidylinositol phosphate (Echelon). Phosphatase assays were conducted at 37C for 30 min; the reactions were terminated by the addition of 15 l of 100 mM with inositol polyphosphate 5-phosphatase and DNase I was Rabbit polyclonal to ZFYVE9 made using MUSTANG (www.cs.mu.oz.aw/~arun/mustang/) (30C33). Positional sequence conservation of CdtB was derived from combined alignments of CdtB and inositol polyphosphate 5-phosphatase homologs (34) and residue conservation was decided (35). Conservation indices were converted to colors (reddish, most conserved; green, intermediate; blue, least conserved) and mapped onto the CdtB structure using Bobscript (36). Results It has been proposed, based on sequence comparisons, that CdtB AM 2201 shares catalytic residues and comparable reaction mechanism with the large group of functionally diverse Mg2+-dependent phosphoesterases (23). DNase I was the first structurally characterized member of this diverse enzyme superfamily (32); subsequent structural characterization of inositol polyphosphate 5-phosphatases and CdtB confirmed the initial prediction (13, 30, 31). An alignment of these three structures shows striking conservation of catalytic and divalent ion-chelating residues, despite low overall sequence identity (Fig. 1). As a general rule, AM 2201 all enzymes in this superfamily hydrolyze phosphate esters and their exact function depends on what substrate(s) can be accommodated in the active site. CdtB was originally characterized as a DNase-like enzyme and the putative PI phosphatase activity was by no means formally tested despite its poor nuclease activity (18). Open in a separate window Physique 1 Structural alignment. Structural alignment of CdtB, inositol polyphosphate 5-phosphatase (IP5P), and DNase I was obtained by MUSTANG and slightly altered after visual inspection of superimposed structures. Protein Data Lender codes of corresponding structures are shown in parentheses next to protein names. The consensus collection indicates the conservation of small (s), aliphatic (l), hydrophobic (h), charged (c), positive (+), and unfavorable (?) residues, whereas identical residues are shown as capital letters. Figures in parentheses within the alignment show the residues that were omitted either because of long insertions not shared by all proteins or for.