FIGURE 1.

RmlA, an important enzyme for L-rhamnose formation, is phosphorylated by PknB. (A) The biosynthesis pathway of cell wall linker-L-rhamnose. dTDP-rhamnose is the precursor of L-rhamnose. The biosynthetic pathway of dTDP-rhamnose consists of four steps from a-D-glucose-1-phosphate and TTP to dTDP-rhamnose. (B) In vitro phosphorylation of M. tuberculosis RmlA by STPKs. The three recombinant M. tuberculosis STPKs (PknB/D/H) were expressed and purified as His-tagged fusions and incubated with purified His-tagged RmlA and [γ−³²P] ATP. Samples were separated by SDS-PAGE, stained with Coomassie blue (upper panel), and visualized by autoradiography after overnight exposure to a film (lower panel). (C) In E. coli phosphorylation of M. tuberculosis RmlA by STPKs. rmlA gene was cloned into pDEST15 vector with a GST tag using the Gateway system, whereas the kinases (pknB, pknD, and pknH) were cloned into pET28a vector with His tag. The two plasmids were cotransformed into E. coli BL21 (DE3), and the RmlA and kinases were enriched with GST and Ni²⁺ agarose beads, respectively. After immunoblotting, the phosphorylated signals were detected with an antithreonine phosphorylation antibody.