Extended Data Fig. 8: Tse8 is not active on a substrate of GatA.
a, Amidase reaction catalysed by GatA and b, MS analysis of Tse8 enzymatic assay using glutamine as substrate. Signal of the expected product (glutamate) was also found as a contaminant in the blank and product stock and therefore subtracted from the reaction incubation. The graph on the left shows relative differences (%Δ) of glutamate and glutamine in reaction incubation and blank samples. The green-shaded area indicates the zone in which the observed differences could indicate enzymatic reaction. (%ΔProduct = 100 * ([product in incubation] – [product in blank]/ [product in incubation]; %ΔSubstrate = 100 * ([substrate in incubation] – [substrate in blank]/ [substrate in incubation]). The graph on the right shows the ratios between substrate and product (Rsp) in the blank (red) and reaction (Tse8) incubation (blue) samples. The product signal (contaminant) was ca. 100 times lower than that of the substrate. (Rsp = (Signal substrate / Signal product)). c, Glutaminase assays of lysates of E. coli cells expressing GatA, Tse8 or empty vector demonstrate that Tse8 does not have the same substrate (L-glutamine) as GatA as measured by relative NADPH levels/(CFU/mLx108) and here normalized to empty vector (EV) mean. Mean ± SEM of six biological replicates performed in triplicate (n=6). Two-tailed student’s t-test, ** p<0.005 for empty vector compared to pET41a:gatA; ns for empty vector compared to pET41A:tse8 (p=0.621).
