Fig. 3: TSB-like binds to TSA and triggers its allosteric activation, resulting in indole formation.
From: A pseudoenzyme enables indole biosynthesis in eudicot plants
a,b, AsTSB-like promotes indole formation by TSA but does not produce tryptophan in vitro. Recombinant proteins were assayed with the substrates IGP and l-serine. Accumulation of indole (a) and tryptophan (b) was measured by LC–MS/MS. Bar graphs represent the mean ± s.d. for three technical replicates (n = 3, assays). c, TSB-like forms a complex with TSA. Copurification of C-terminal His-tagged and untagged AsTSA and AsTSB-like through affinity purification. Untagged TSA or TSB-like could be copurified with the corresponding tagged partner as shown in SDS–PAGE and western blot. d, AsTSB and AsTSB-like compete for AsTSA in vitro. Recombinant proteins were assayed with the substrates IGP and l-serine. Left: equimolar concentrations of TSA and TSB-like were incubated with increasing concentrations of TSB, resulting in increased accumulation of tryptophan and reduced accumulation of indole. Right: equimolar concentrations of TSA and TSB were incubated with increasing concentrations of TSB-like, resulting in increased accumulation of indole and tryptophan. The x axis shows the molar ratio of the indicated protein. Points represent the mean ± s.d. for three technical replicates (n = 3 assays). e, AsTSB and AsTSB-like compete for TSA in N. benthamiana. Coexpression of Z. mays Bx2, Bx3, Bx4 and Bx8 with AsTSB and AsTSB-like compared to AsTSB-like resulted in a ~50% decrease in HBOA-Glc produced. Bar graphs represent the mean ± s.d. for four independent biological replicates (n = 4 plants). ***P < 0.0005 (two-tailed t-test; P = 0.0002, t = 8.219, df = 6, 95% confidence interval (CI) = −608,573, −329,332). f, Schematic of how TSB-like and TSA produce indole (1). For visual clarity, only one αβ-like dimer is shown.
