Intracellular TDP-⁠β-L-Rhamnose concentration

Value 2 mM Range: ±0.1 mM
Organism Bacteria Pseudomonas aeruginosa
Reference Krafczyk R et al., 2017. Structural basis for EarP-mediated arginine glycosylation of translation elongation factor EF-P. mBio 8:e01412-17. link 10.1128/mBio.01412-17. p.7 top paragraph PubMed ID28951478
Method P.15 2nd paragraph: "Determination of kinetic parameters: Kinetic parameters were determined by varying TDP-Rha concentrations while keeping concentrations of EarP[Ppu] (0.1 µM) and unmodified EF-PPpu (2.5 µM) constant. A mixture of EarP[Ppu] and unmodified EF-P[Ppu] was equilibrated to 30°C in 100 mM NaPi (pH 7.6). The reaction was started by the addition of TDP-Rha and was stopped after 20 s of incubation at 30°C by the addition of 1 vol of 2× Lämmli buffer (ref 74) and incubation at 95°C for 5 min. Samples were subjected to SDS-PAGE, and rhamnosylated EF-P[Ppu] was detected as described above. Band intensities were quantified using ImageJ (ref 76). Product formation (in nanomoles per milligram) was calculated relative to fully (in vivo) rhamnosylated EF-P[Ppu]. Km and kcat values were determined by fitting reaction rates (in nanomoles per milligram per second) to the Michaelis-Menten equation using SigmaPlot. Time course experiments conducted at a TDP-Rha concentration of 500 µM show that the rhamnosylation reaction is not saturated after 20 s of incubation (Fig. S2A)."
Comments P.6 bottom paragraph: "[Investigators] wondered whether this Km makes sense physiologically and therefore analyzed the cellular TDP-Rha levels in P. putida, P. aeruginosa, and E. coli, which were 3.5 mM, 2.0 mM, and 4.0 mM, respectively (see Materials and Methods and Fig. S6). In good accordance with [their] measurements, the physiological TDP-Rha concentration in Lactococcus lactis was previously determined to be as high as 1 mM (ref 43). Thus, within a bacterial cell, the donor substrate reaches saturating concentrations, according to the WT[EarP] Km measurements." Range/Confidence is given as standard deviation (±)
Entered by R. Krafczyk
ID 114145