UDP-Glo assay

Vmax and Km calculations were performed using data from UDP-Glo assays (Promega) similar to previous studies [28,52]. Purified GST, GST-tagged NleB1, NleB1_G255S, NleB1_DXD, NleB2, NleB1_S252G or NleB2_DXD at 150 nM concentrations were incubated with 1 μM MBP-RIPK1_DD in 1x glycosyltransferase buffer (50 mM Tris-HCL pH 7.4, 100 mM NaCl, 10 mM MgCl₂, 1 mM MnCl₂) with titrated UDP-GlcNAc or UDP-glucose (0–1 mM) for 30 minutes at 30°C. The UDP Glo assay was then performed according to manufacturer instructions, and luminescence was measured using a CLARIOstar microplate reader (BMG Labtech). Readings obtained from GST incubated with MBP-RIPK1_DD in the presence of UDP sugar donors were subtracted from all other readings as background luminescence. Relative light units (RLU) measured at the end of the reaction were divided by 30 to calculate RLU/min produced, and Vmax and Km values were calculated by using the non-linear regression fit Michaelis-Menten equation in GraphPad Prism Version 6.