Discovery of the Gut Microbial Enzymes that Drive the Dose-Limiting Toxicity of the Immunosuppressant Mycophenolate Mofetil Marissa M. Bivins1, Lindsay E. Bass2, Amanda L. Graboski1, Michelle E. Fiamingo2, Rebecca L. Johnson3, William G. Walton2, John R. Lee7,8, Matthew R. Redinbo2,4,5,6 Departments of Pharmacology1, Chemistry2, Chemical Biology and Medicinal Chemistry3, Biochemistry & Biophysics4, Microbiology & Immunology5, and the Integrated Program for Biological and Genome Sciences6, University of North Carolina, Chapel Hill, North Carolina, United States; Division of Nephrology and Hypertension7, Department of Medicine, Weill Cornell Medicine, New York, New York, United States; Department of Transplantation Medicine8, New York Presbyterian Hospital - Weill Cornell Medical Center, New York, New York, United States Mycophenolate mofetil (MMF) is an immunosuppressant used chronically by organ transplant and autoimmune patients. Unfortunately, MMF causes dose-limiting, gastrointestinal side effects, including ulcers, weight loss, vomiting, and diarrhea. The active form of MMF, mycophenolic acid (MPA), is inactivated to mycophenolate-glucuronide (MPA-G) via human drug metabolism and sent to the gut for elimination. Bacteria residing within the gut express β-glucuronidase enzymes (GUSs) that convert drug-glucuronides into their active forms and have been subjected to targeted inhibition. Reactivation of MPA-G within the gut is predicted to cause this drug’s intestinal toxicity and drive difficulties in dosing due to enterohepatic recirculation. Here, we show that complex enzyme slurries from MMF-treated patient fecal samples have variable MPA-G processing activities that are not inhibited by previously validated GUS inhibitors. To identify the GUS enzymes that process MPA-G, we kinetically evaluated a panel of GUSs in vitro and discovered two highly efficient isoforms from Bacteroides uniformis and Roseburia hominis. Structural analyses pinpointed specific motifs that enable MPA-G reactivation by these enzymes. Defining the structural basis of efficient MPA-G processing by microbial GUS proteins will facilitate the development of novel inhibitors to prevent MMF-induced gut toxicity.