title: Shiga toxin 1 acting on DNA in vitro is a heat-stable enzyme not requiring proteolytic activation.
abstract: Shiga toxin 1 (Stx1) catalyses the removal of a specific adenine from 28S rRNA within ribosomes (RNA-N-glycosylase activity) and the removal of multiple adenines from DNA (DNA-glycosylase activity). For the in vitro activity the toxin requires activation by trypsin, urea and DTT which releases the enzymatically active A1 fragment. We show that activated Stx1 acts on DNA as a heat-stable enzyme. Moreover, heat-treatment of the pro-enzyme at acidic pH turns it into an enzymatically active species which efficiently depurinates DNA. Although the effect of this treatment is centred on the enzyme and not on DNA, we found no evidence for covalent modification of the holotoxin. We suggest that high temperatures and acidic buffer induce unfolding of the holotoxin allowing the substrate to gain access to the active site. Possible practical applications (rapid assay for Stx1 detection, use of the toxin for DNA sequencing) are discussed.
authors.affiliation: Dipartimento di Patologia Sperimentale dell'Università degli Studi di Bologna, Via San Giacomo 14, 40126 Bologna, Italy. brigotti@alma.unibo.it
authors.forename: Maurizio
authors.initials: M
authors.lastname: Brigotti
authors.forename: Domenica
authors.initials: D
authors.lastname: Carnicelli
authors.forename: Antonio González
authors.initials: AG
authors.lastname: Vara
mesh_terms: D000225:Adenine / Q000378:metabolism*; D001665:Binding Sites; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D004795:Enzyme Stability; D006358:Hot Temperature; D066298:In Vitro Techniques; D022622:Shiga Toxin 1 / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity*; D014357:Trypsin / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
chemical_list: D022622:Shiga Toxin 1; D014508:Urea; D004247:DNA; D014357:Trypsin; D000225:Adenine; D004229:Dithiothreitol