Fig. 3: Mtb-FPGS-dependent glutamylation of folate-2 with 15N-glutamate monitored by NMR spectroscopy.
a Reaction scheme for Mtb-FPGS/Tm-DHFR coupled conversion of folate-2 to tetrahydrofolate-3 and subsequent oxidation to N-(4-aminobenzoyl)-(L-Glu)3. The orange asterisk indicates the 15N-enriched site. Panels b to e show the amide region of 1D 1H and 2D 1H-15N HSQC spectra for samples as follows. b Reaction sample containing folate-2 and 15N-glutamate prior to addition of enzyme. c Reaction sample following a 2 h preincubation with Tm-DHFR, followed by incubation with both Tm-DHFR and Mtb-FPGS for 2 h and d 20 h. e Folate-3 standard. Panels f to i show the results of 1H-1H correlation experiments confirming addition of 15N-glutamate at the terminus of the Mtb-FPGS/Tm-DHFR-catalyzed reaction product. f Arrows indicate through-bond (solid line) and through-space (dashed line) correlations observed between the 15N-labelled amide and other 1H nuclei in the polyglutamate chain, based on folate-3 assignments. g Comparison of the chemical shifts of 1H-1H correlations observed for the 15N-enriched amide and the 3′ glutamyl amide of folate-3. h Overlay of 15N-coupled (blue), and 15N-decoupled (pink) 1H-1H TOCSY experiments for the 15N-enriched amide highlighting correlations with the 3′α, 3′β, and 3′γ protons. i Overlay of 15N-coupled (purple), and 15N-decoupled (green) 1H-1H NOESY experiments for the 15N-enriched amide highlighting a correlation with the 2′γ proton.
