Abstract: A radical departure from the classical E1-E2-E3 three-enzyme mediated ubiquitination of eukaryotes, the bacterial enzymes of the SidE family of Legionella pneumophila effectors utilize NAD+ to ligate ubiquitin onto target substrate proteins achieved via a two-step mechanism involving (1) ADP-ribosylation of ubiquitin followed by (2) phosphotransfer to a target serine residue. Using fluorescent NAD+ analogues as well as synthetic substrate mimics, a continuous assay system enabling real-time monitoring of both steps of this mechanism is disclosed herein. These assays are amenable to biochemical studies and high-throughput screening of inhibitors of these effectors, and enable the discovery and characterization of putative enzymes similar to the SidE family in other organisms.