Mono-ADP-ribosyltransferase toxins are produced by pathogenic bacteria as virulence factors that target important macromolecules in host cells. In a few cases, the cellular target may be DNA. This family of enzymes transfers an ADP-ribose moiety from NAD+ to the target macromolecule, leading to an altered function of the target and ultimately host-cell death. A bioinformatics strategy was used to identify Scabin, a mono-ADP-ribosyltransferase from the plant pathogen Streptomyces scabies. A detailed kinetic analysis was performed on Scabin, revealing the target as genomic DNA. The crystal structure of Scabin with NADH as a substrate analog was determined, which provided important insights into the active site structure of the enzyme. Residues involved in activity and binding of DNA were identified. Hydrogen-deuterium exchange coupled with mass spectrometry was used to characterize the Scabin:DNA interface, revealing key interacting regions. Understanding the mechanism of Scabin will allow for a more targeted approach in the development of inhibitors against the potentially toxic activity of this enzyme.