A bacterial family of fatty acid acyltransferases related to the <i>Shigella</i> effector IcsB

N^ε-lysine-protein fatty acid acyltransferase (k-FAT) is a new class of enzyme found in bacteria that express either a MARTX toxin with a Rho-interacting domain (RID) or a type III secretion system (T3SS). The prototype k-FAT is the T3SS effector IcsB, which enables enteropathogenic <i>Shigella</i> species to evade destruction by autophagy in host cells. We mined protein databases using the acyltransferase domain of IcsB to expand this family. In addition to the known BopA homolog from <i>Burkholderia pseudomallei</i>, we identified several additional IcsB-like k-FATs in β- and γ-proteobacteria. These new proteins are poorly characterized and are primarily found in bacteria harboring a T3SS. Despite high sequence diversity, structure predictions indicate a highly conserved spatial disposition of catalytic residues. Furthermore, yeast cytotoxicity and in-gel acyltransferase activity depend on the integrity of the catalytic site, which supports the idea that most of the IcsB homologs identified are also acyltransferases. Finally, we used mass spectrometry to identify the yeast proteins acylated by seven of these novel enzymes. These proteins share a variety of substrates with IcsB, including membrane- and vesicle-associated proteins, such as SNAREs and small GTPases of the Rho, Rab, and Ras families. Taken together, this study provides evidence of a family of k-FAT related to IcsB.IMPORTANCEIcsB-like k-FATs are found in the related Pseudomonadota and Thermodesulfobacteriota phyla, suggesting that they are a recent biochemical innovation. Like IcsB, new k-FATs are primarily found in proteobacterial species with a T3SS. This leaves open the possibility that they may play a role in the colonization of plants or animals. However, we characterized one k-FAT from an environmental bacterium that is unlikely to possess a T3SS. Additionally, measurable fatty acid acyltransferase activity was not detected in approximately 25% of the proteins tested. These results imply that the IcsB-like k-FAT family has undergone functional diversification and may have a more complex evolutionary origin than previously thought. In summary, this study describes the properties of the IcsB-like k-FAT family and presents yeast-based assays for characterizing new family members and unrelated proteins with similar fatty acid acyltransferase activity.