Pathways of ubiquitin-like (UBL) molecule transfer regulate a myriad of cellular cascades. Here we report a high-throughput assay that correlates catalytic human-NEDD8 transfer to bacterial survival. The assay was utilized to screen mutant NEDD8 and NEDD8-activating enzyme (NAE) libraries to engineer a more stable NEDD8 and redesign the NEDD8-NAE interface. This approach will be useful in understanding the specificities underlying UBL pathways
