Figure 4.

In Vitro Reconstitution of the SQ Transaldolase Pathway by Three Recombinant Enzymes

For the organosulfonates, the total-ion chromatograms (TICs) of the MS/MS fragmentation of the quasi-molecular ions ([M-H]^-) are depicted, and for the sugar phosphates F6P and G6P, the characteristic ion traces of the phosphate group ([H₂PO₄]^-, m/z = 97). The reaction mixture initially contained 2 mM ¹³C₆-SQ and 12 mM GAP (0 min). SQ isomerase SftI (protein 1323) was added (100 μg/mL) and the reaction sampled after 10 min. Then, the SF transaldolase SftT (protein 1320) (50 μg/mL) was added and the reaction sampled after 1 min. Finally, SLA dehydrogenase SftD (protein 1325) (100 μg/mL) and 6 mM NAD⁺ was added and the reaction sampled after 17 h. Note that GAP and NAD⁺ were added in excess (12 and 6 mM) because the SLA dehydrogenase oxidizes also GAP in the presence of NAD⁺; therefore, the SQ conversion was incomplete. Furthermore, the reaction mixture converted the [1,2,3-¹³C₃]-F6P to [1,2,3-¹³C₃]-G6P, owing to an activity of the SQ-isomerase also with F6P. The results were replicated twice using independently produced enzyme preparations. A reaction sequence with E4P instead of GAP as the acceptor is shown in the Supplementary files, Figure S8. A chromatogram depicting the HPLC separation of G6P and F6P in more detail is shown in Figure S11, and MS/MS fragmentation mass spectra for G6P and [1,2,3-¹³C₃]-G6P, and for F6P and [1,2,3-¹³C₃]-F6P, are shown in Figures S12 and S13, respectively.