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. 2021 Aug 19;28(8):1180–1191.e20. doi: 10.1016/j.chembiol.2021.02.023

Figure 6.

Figure 6

TrpB converts 4-AI to 4-amino-L-Trp in vitro and in Mtb

(A) Representative chromatograms of synthetic 4-aTrp (red trace), the enzymatic reaction mixture obtained by incubating TrpAB with 4-AI (blue trace), and the enzyme reaction spiked with synthetic 4-aTrp (black trace).

(B and C) Fragmentation pattern of synthetic 4-aTrp (B) and the enzyme reaction mixture (C) in a triple quadrupole mass spectrometer.

(D) 4-AI was incubated with WT or mutant TrpAB in the presence of saturating L-Ser concentrations. The resulting 4-aTrp was quantified using LC-MS. Data shown are mean ± SEM (n = 2).

(E) Calculated catalytic efficiency, kcat, of the WT and mutant TrpAB in (D). Bars represent mean ± SEM (n = 2). ∗p < 0.05, ∗∗p < 0.01 when compared with the WT kcat using one-way ANOVA.

(F) Intracellular 4-AI incorporation into 4-aTrp was measured by extracting the metabolites using 1:1 ACN:MeOH. The concentration of 4-aTrp produced within the indicated C1R mutants were normalized against that of the WT. Bars represent mean ± SEM (n = 2). ∗p < 0.05, ∗∗p < 0.01 when compared with the WT using one-way ANOVA.