Actin cross-linking domains (ACDs) are distinct domains found in several bacterial toxins, including the Vibrio cholerae MARTX toxin. The ACD of V. cholerae (ACDVc) catalyses the formation of an irreversible iso-peptide bond between lysine 50 and glutamic acid 270 on two actin molecules in an ATP- and Mg/Mn2+-dependent manner. In vivo, cross-linking depletes the cellular pool of G-actin leading to actin cytoskeleton depolymerization. While the actin cross-linking reaction performed by these effector domains has been significantly characterized, the ACDVc catalytic site has remained elusive due to lack of significant homology to known proteins. Using multiple genetic approaches, we have identified regions and amino acids of ACDVc required for full actin cross-linking activity. Then, using these functional data and structural homology predictions, it was determined that several residues demonstrated to be important for ACDVc activity are conserved with active-site residues of the glutamine synthetase family of enzymes. Thus, the ACDs are a family of bacterial toxin effectors that may be evolutionarily related to ligases involved in amino acid biosynthesis.