Fig. 2: SccN and SccP synthesize alternative stereoisomers of Glc-P-Und.

A Kinetic analysis of Glc-lipid synthases in membrane fractions from the E. coli JW2347 strain expressing sccN or sccP on a plasmid. Membrane fractions were assayed for the formation of [³H]Glc-lipids in the presence of increasing amounts of UDP-[³H]Glc or Und-P (undecaprenol phosphate), added as a dispersion in 1% CHAPS. Median average velocity and standard errors for three separate experiments are plotted versus substrate concentrations. Apparent kinetic parameters for UDP-Glc and Und-P were calculated using GraphPad Prism 9.3 software, Michaelis-Menten enzyme kinetics tool. Km and Vmax for UDP-Glc and Und-P are presented in Table 1. The data are derived from three separate experiments. B Thin layer chromatography (TLC) of alkali-stable [³H]Glc-lipids from in vitro incubations of the membranes of S. mutans mutants with UDP-[³H]Glc. Membrane fractions from S. mutans WT, ΔsccN, ΔsccP, or ΔsccNΔsccP were incubated with UDP-[³H]Glc and analyzed for [³H]Glc-lipid synthesis by TLC following mild-alkaline methanolysis as described in Methods. [³H]Glc-lipids were detected by scanning with an AR2000 Bioscan Radiochromatoscanner. The results are representative of three independent experiments. C Reverse reactions of SccN and SccP to reform UDP-Glc from their respective enzymatic products following incubation (37 °C, 40 min) with UDP. [³H]Glc-P-Und_SccN, synthesized by SccN, and [³H]Glc-P-Und_SccP, synthesized by SccP, were tested as substrates in the discharge reactions, containing solubilized, partially-purified SccN or SccP, as described in Methods. Columns and error bars represent the mean and S.D., respectively (three biologically independent replicates). P values were calculated by two-way ANOVA with Šídák’s multiple comparison test. D Origin of diagnostic ion fragments used to deduce the anomeric configuration of Glc-P-Und. If the stereochemistries of the phosphodiester at C1 of glucose and the 2-OH are configured trans (β-anomeric configuration) then [Und-HPO₄]^- (m/z = 845.7) is the primary high molecular weight ion detected in the mass spectrum. However, if the stereochemistries of glucose at C1 and the 2-OH group are configured cis (α-anomeric configuration) then an additional ion fragment with the composition of [Und-PO₄-C₂H₃O]^- (m/z = 887.7), formed by ‘cross-ring’ fragmentation as shown, is found⁴². Source data for a and c are provided as a Source Data file.