title: Scabin, a Novel DNA-acting ADP-ribosyltransferase from Streptomyces scabies.
abstract: A bioinformatics strategy was used to identify Scabin, a novel DNA-targeting enzyme from the plant pathogen 87.22 strain of Streptomyces scabies Scabin shares nearly 40% sequence identity with the Pierisin family of mono-ADP-ribosyltransferase toxins. Scabin was purified to homogeneity as a 22-kDa single-domain enzyme and was shown to possess high NAD(+)-glycohydrolase (Km (NAD) = 68 ± 3 μm; kcat = 94 ± 2 min(-1)) activity with an RSQXE motif; it was also shown to target deoxyguanosine and showed sigmoidal enzyme kinetics (K0.5(deoxyguanosine) = 302 ± 12 μm; kcat = 14 min(-1)). Mass spectrometry analysis revealed that Scabin labels the exocyclic amino group on guanine bases in either single-stranded or double-stranded DNA. Several small molecule inhibitors were identified, and the most potent compounds were found to inhibit the enzyme activity with Ki values ranging from 3 to 24 μm PJ34, a well known inhibitor of poly-ADP-ribosyltransferases, was shown to be the most potent inhibitor of Scabin. Scabin was crystallized, representing the first structure of a DNA-targeting mono-ADP-ribosyltransferase enzyme; the structures of the apo-form (1.45 Å) and with two inhibitors (P6-E, 1.4 Å; PJ34, 1.6 Å) were solved. These x-ray structures are also the first high resolution structures of the Pierisin subgroup of the mono-ADP-ribosyltransferase toxin family. A model of Scabin with its DNA substrate is also proposed.
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada and.
authors.forename: Bronwyn
authors.initials: B
authors.lastname: Lyons
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada and.
authors.forename: Ravikiran
authors.initials: R
authors.lastname: Ravulapalli
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada and.
authors.forename: Jason
authors.initials: J
authors.lastname: Lanoue
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada and.
authors.forename: Miguel R
authors.initials: MR
authors.lastname: Lugo
authors.affiliation: the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2R3, Canada.
authors.forename: Debajyoti
authors.initials: D
authors.lastname: Dutta
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada and.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Carlin
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada and rmerrill@uoguelph.ca.
authors.forename: A Rod
authors.initials: AR
authors.lastname: Merrill
mesh_terms: D036002:ADP Ribose Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D036002:ADP Ribose Transferases
keywords: DNA binding protein; bacterial toxin; bioinformatics; crystallography; enzyme kinetics; fluorescence; molecular modeling