TrpF (A) and HisA (B) activities of tHisA variants. (A) The TrpF reaction was monitored essentially as described (18). Each variant (21 μM) was incubated with 100 μM in situ synthesized PRA in the presence of 0.7 μM indoleglycerol-phosphate synthase from E. coli. The buffer conditions were 50 mM Tris⋅HCl, pH 7.5 at 25°C, containing 4 mM MgEDTA and 2 mM DTT. (B) The HisA reaction was monitored essentially as described (19). Each variant, 0.25 μM tHisA, 0.5 μM tHisA_1, 21 μM tHisA_2, or 21 μM tHisA_Asp127Val, was incubated with 20 μM enzymatically synthesized ProFAR (22) in the presence of 1 μM ImGP synthase from T. maritima and of 5 mM glutamine. The buffer conditions were 50 mM Tris⋅HCl, pH 7.5 at 25°C, containing 2 mM EDTA and 1 mM DTT.