doi:10.1039/d1sc04065kengGarcía-García A.Hicks T.El Qaidi S.Zhu C.Hardwidge P.R.Angulo J.Hurtado-Guerrero R.NleB/SseK-catalyzed arginine-glycosylation and enteropathogen virulence are finely tuned by a single variable position contiguous to the catalytic machineryART-2021-125795NleB/SseK effectors are arginine-GlcNAc-transferases expressed by enteric bacterial pathogens that modify host cell proteins to disrupt signaling pathways. While the conservedCitrobacter rodentiumNleB andE. coliNleB1 proteins display a broad selectivity towards host proteins, Salmonella entericaSseK1, SseK2, and SseK3 have a narrowed protein substrate selectivity. Here, by combining computational and biophysical experiments, we demonstrate that the broad protein substrate selectivity of NleB relies on Tyr284NleB/NleB1, a second-shell residue contiguous to the catalytic machinery. Tyr284NleB/NleB1is important in coupling protein substrate binding to catalysis. This is exemplified by S286YSseK1and N302YSseK2mutants, which become active towards FADD and DR3 death domains, respectively, and whose kinetic properties match those of enterohemorrhagicE. coliNleB1. The integration of these mutants intoS. entericaincreasesS. entericasurvival in macrophages, suggesting that better enzymatic kinetic parameters lead to enhanced virulence. Our findings provide insights into how these enzymes finely tune arginine-glycosylation and, in turn, bacterial virulence. In addition, our data show how promiscuous glycosyltransferases preferentially glycosylate specific protein substrates.2021http://zaguan.unizar.es/record/15114710.1039/d1sc04065khttp://zaguan.unizar.es/record/151147oai:zaguan.unizar.es:151147info:eu-repo/grantAgreement/ES/DGA-FEDER/E34-R17info:eu-repo/grantAgreement/ES/DGA/LMP58-18info:eu-repo/grantAgreement/ES/MICINN/BFU2016-75633-Pinfo:eu-repo/grantAgreement/ES/MICINN/PID2019-105451GB-I00CHEMICAL SCIENCE 12, 36 (2021), 12181-12191by-nchttps://creativecommons.org/licenses/by-nc/4.0/deed.esinfo:eu-repo/semantics/openAccess