Figure 3.

HPLC Mass Spectrometry Confirmed a Transaldolase Reaction in Cell Extracts of SQ-grown B. aryabhattai SOS1 Using Fully ¹³C-Labeled SQ as Substrate and Erythrose-4-Phosphate as Acceptor Molecule

In the presence of unlabeled erythrose-4-phosphate (E4P) and of ¹³C₆-SQ, a transient formation of ¹³C₆-SF and ¹³C₃-SLA, and an accumulation of [1,2,3-¹³C₃]-sedoheptulose-7-phosphate (S7P) and of ¹³C₃-SL, was detected. The ¹³C₃-SL formation resulted from an SLA dehydrogenase reaction because of the additional presence of NAD⁺ in the reaction mixture. The reaction contained 2 mM ¹³C₆-SQ, 6 mM E4P (with G6P as impurity; not shown), and 6 mM NAD⁺ and was sampled before addition of the gel-filtered cell extract (exclusion, >5 kDa) and after 10, 30, and 240 min of incubation. For the organosulfonates, the total-ion chromatograms (TIC) of the MS/MS-fragmentation of the quasi-molecular ions ([M-H]^-) are shown. For the sugar phosphates E4P and [1,2,3-¹³C₃]-S7P, the MS/MS-ion traces of the phosphate group ([H₂PO₄]^-, m/z = 97) are shown. Note that ¹³C₃-SLA was separated by HPLC as trident peak. The results were replicated twice when starting from independent growth experiments. When E4P was exchanged by GAP as the acceptor, [1,2,3-¹³C₃]-F6P/G6P accumulation was detected (see Figure S3). MS/MS fragmentation mass spectra are shown as Supplementary files for [1,2,3-¹³C₃]-S7P (Figure S2), ¹³C₆-SF and ¹³C₆-SQ (Figure S5), ¹³C₃-SLA (Figure S6), ¹³C₃-SL (Figure S7), and E4P (Figure S13).