Fig. 5: Donor substrate selectivity in RmlT.
a Purified recombinant RmlT (2 µM) was incubated with 1 mM WTAs and 200 µM TDP-rhamnose, L-rhamnose, TDP-glucose, UDP-glucose or UDP-GlcNAc. RmlT activity was measured after 30 min by quantifying released phosphate. b ITC measurement of RmlT/donor substrate interactions. Upper panels show the titration of RmlT with TDP-rhamnose, TDP-glucose, TDP or UDP-glucose. The lower panel shows heat released when substrate binds to RmlT as a function of relative concentrations. Dilution heat was corrected. Solid lines correspond to the best fit of data using a bimolecular interaction model. Table shows N-value (commonly referred as the stoichiometry), binding constant KD (μM) and reaction enthalpy (ΔH) extracted from model fitting. c Superposition of active-sites of RmlT/UDP-glucose (green with UDP-glucose in pink, PDB code: 8BZ6; chain D) and RmlT/TDP-rhamnose (wheat with TDP-rhamnose in yellow, PDB code: 8BZ7; chain D) through residues 467−477 (Cα RMSD: 0.114 Å) with the substrate-interacting residues shown as sticks. Hydrogen bonds between RmlT residues and UDP-glucose are represented as black dashed lines. Water and Mg2+ are shown as red and purple spheres, respectively. d Chemical structure of TDP (left) and UDP (right) with differences highlighted by red circles. e Quantification of phosphate released over time for RmlT incubated with different donor substrates. Reactions contained 1 mM WTAs, 200 µM of each nucleotide and 2 µM RmlT. Mean ± SD (n = 3 refers to biological replicates, except for the curve of TDP-glucose in which n = 4) are shown for a and e; individual values shown in a; two-way ANOVA; *p < 0.05, ****p < 0.0001. Source data are provided as a Source Data file.
