Fig. 6: Structure of RmlT with bound poly-ribitol phosphate.
a Quantification of phosphate released from TDP generated by RmlT in presence of differently decorated WTAs: containing both GlcNAc and rhamnose modifications (wild-type WTAs), only GlcNAc modification (WTAs-GlcNAc), only rhamnose modification (WTAs-rhamnose) or without any glycosylation (naked WTAs). Reaction mixtures included 5 mM WTAs, 1 mM TDP-rhamnose and 10 μM RmlT incubated during 30 min. Mean± SD (n= 3 refers to biological replicates) and individual measurements are shown; one-way ANOVA, ****p< 0.0001. b Chemical structures of 3-RboP and 4-RboP with ribitol-phosphate unit indicated together with C-2 (light red) and C−4 (light green). c Cartoon representation of RmlT with two bound RboP chains represented as sticks (PDB code 9GZJ chain A). Molecular details of RboP binding sites are shown, with subsites occupied by separate RboP units numbered +1 and +2. Atoms within hydrogen bond distance indicated by dashed lines. C-4 hydroxyl in subsite +1 is highlighted by green circle; C−2 hydroxyl in subsite +2 is highlighted by red circle. d Comparison of active-sites in RmlT/TDP-rhamnose (white with TDP-rhamnose in yellow; PDB code 8BZ7 chain D) and RmlT/3-RboP (light cyan with 3-RboP in cyan; PDB code 9GZJ chain A). Structures are shown in same view after superposition through Cα atom of resides 238–516 (RMSD = 0.364 Å). TDP/rhamnose, 3-RboP, residues D112 and D113 and D198 are shown as sticks. Mg2+ and water molecules are represented by purple and red spheres. Dashed lines indicate atoms within hydrogen bond or coordinating the metal. e TarP/6RboP-(CH2)6NH2 (top image; PDB code 6HNQ chain A) and RmlT/3-RboP (bottom; PDB code 9GZJ chain A) in the same orientation (superposed through Cα atom pairs of RmlT residues 93-100 and 198-204 and TarP residues 75-82 and 181-187 (RMSD 0.672 Å)). Subsites are indicated (+1, +2 and +3). f Quantification of phosphate released from TDP generated by RmlT or RmlTW145C+A472C incubated with naked WTAs and TDP-rhamnose for 30 min in the presence or absence of 100 μM of CdCl2. Mean ± SD (n= 4 refers to biological replicates) and individual measurement are shown; two-way ANOVA; ****p< 0.0001. Source data are provided as a Source Data file.
