Fig. 4: A190 and E386 have a role in AsTSB-like activity.
From: A pseudoenzyme enables indole biosynthesis in eudicot plants
a, Model of the AsTSB-like and AsTSB active site showing A190 and E386. The structure of AsTSB-like was modeled on the crystal structure of Salmonella Typhimurium TSB (Protein Data Bank 7JMQ) in open conformation with PLP (yellow). Indole (gray) was docked in silico. b, Schematic depiction of the role of E190 in tryptophan biosynthesis. c,d, Alignment displaying the conservation of A190 and E386. TSB-like enzymes from A. squarrosa, A. thaliana, L. galeobdolon, N. benthamiana and P. trichocarpa were compared to A. squarrosa and T. maritima TSB. Sequences were aligned using the MUSCLE algorithm and the residues of interest are highlighted in blue for TSB-like and orange for TSB. e, AsTSB-like mutants resulted in a gain of tryptophan biosynthetic activity and reduction in indole biosynthetic activity. Recombinant proteins were assayed on IGP and l-serine. Reaction products were analyzed using LC–MS/MS. Bar graphs represent the mean ± s.d. for three technical replicates (n = 3 assays). Columns labeled with different letters represent statistically significant differences (P < 0.05, Brown–Forsythe and Welch ANOVA with Dunnett test for multiple comparisons). f, The double mutant AsTSB-like-A190E;E386D showed highly reduced indole production in N. benthamiana, as evidenced by monitoring production of the BXD DIBOA-Glc. AsTSB-like mutants were transiently expressed in N. benthamiana along with Z. mays Bx2, Bx3, Bx4, Bx5 and Bx8. Bar graphs represent the mean ± s.d. for four independent biological replicates (n = 4 plants). ***P < 0.0005 (two-tailed t-test; P = 0.0004, t = 6.946, df = 6, 95% CI = −38,675, −18,525) and *P < 0.05 (two-tailed t-test; P = 0.03, t = 2.830, df = 6, 95% CI = −23,181, −1,683).
