The pathogenic bacterium responsible for Legionnaires’ disease, Legionella pneumophila, uses SidE family of effectors (such as SdeA) to target several host proteins through a noncanonical ubiquitination mechanism radically different from the ATP-driven, E1-E2-E3 ubiquitination of eukaryotes. This mechanism involves an all-in-one ubiquitination machinery in SdeA which employs, first, mono- ADP-ribosylation (mART) of ubiquitin (Ub) at Arg42, catalyzed by its mART domain, to produce ADP- ribosylated ubiquitin (ADPR-Ub), which is then subjected to an additional catalytic step, executed by the phosphodiesterase (PDE) activity embedded in a separate domain, resulting in phosphoribosyl (PR) ubiquitination of serine residues of host targets. Essential to the pathogen’s intracellular life cycle, SdeA and its orthologs target numerous host proteins involved in a range of processes, from vesicular trafficking to nutrient acquisition and autophagy. While resistant to host deubiquitinases, the PR ubiquitination is regulated at multiple levels at the hands of other effectors: the SidJ effector (and its paralog SdjA) can shut off mART activity by modifying a key catalytic residue though a pseudokinase- based polyglutamylation activity; whereas the DupA and DupB effectors can reverse PRubiquitination by restoring host targets to their native form. This sort of deubiquitination activity, while releasing the native host target, still leaves Ub as a modified derivative, with a phosphoribosyl appendage at Arg42 (PR-Ub). Accumulation of such a Ub derivative, that cannot be used in host ubiquitination pathways, has the effect of poisoning the cellular Ub pool which could be detrimental to Legionella’s replication. In this proposal we explore regeneration of free, functional Ub from PR-Ub through a two-step process involving an unusual AMPylation reaction catalyzed by a novel S-HxxxE motif-containing, actin- activated AMPylator, called LnaB, producing ADPR-Ub, which is then further processed by a macrodomain (ADPribosyl)hydrolase, MavL, returning Ub to its native form. Using single particle cryo- EM we will provide structural basis of actin activation, PR-Ub recognition and the ATP binding site of LnaB. The EM studies will be complemented with x-ray crystallography of apo LnaB and its ATP-bound form. Together with biochemical studies aimed at capturing enzyme intermediates, our work will provide key insights into the novel AMPylation reaction. The MavL effector, while using macrodomain for deADP- ribosylation, features a unique motif which we found was shared by a group of previously uncharacterized proteins in the DUF4804 family of the Pfam database. Such a motif appears to confer residue-level selectivity for arginine de-ADP ribosylation, a novel aspect of macrodomain function. We seek to provide structural basis of ADPR-Ub recognition, while elucidating the basis of arginine selectivity across the newly found MavL-like enzymes.