Fig. 4: Impact of active-site residues in RmlT activity.
a Quantification of the amount of phosphate released from the product generated by RmlT, RmlTW89A, RmlTE197A, RmlTD198A and RmlTW223A. Phosphate was quantified after 30 min reaction from mixtures containing 1 mM naked WTAs, 200 μM TDP-rhamnose and 2 μM protein. b Superposition of active-sites of RmlT (light yellow, PDB code: 8BZ7; chain A) and RmlTD198A (grey, PDB code: 8BZ8; chain F) in complex with TDP-rhamnose through Cα of residues 110, 112–113 and 197–198 (Cα RMSD: 0.245 Å). Apparent change in the position of rhamnose moiety is indicated by arrow. c Impact of mutations in the active site of RmlT on L. monocytogenes surface rhamnosylation. Fluorescence microscopy analysis of L. monocytogenes strains EGDe wild-type, EGDe ΔrmlT and EGDe ΔrmlT complemented with rmlT, rmlTW89A, rmlTE197A, rmlTD198A or rmlTW223A. Bacterial cells and the rhamnose at the bacterial surface were stained with wheat germ agglutinin (WGA, red) and gp17-GFP rhamnose binding protein (green), respectively. d Quantification of the fluorescence associated to gp17-GFP bound to rhamnose at the surface of the strains in c. Mean ± SD (n = 3 refers to biological replicates) and individual measurements are shown; one-way ANOVA; **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.
