Fig. 4: STM0725 and STM0726 are required for polymerization of T1 Ribf-PS.

a AlphaFold models of STM0725 (Uniprot: Q8ZQU9) and STM0726 (Uniprot: Q8ZQU8) resemble the crystal structures of a prototypical GT-A-fold GT enzyme, Bacillus subtilis SpsA (PDB ID: 1h7q), and the Thermobacillus compostii Ribf-transferase (PDB ID: 7shg), respectively. STM0725 shares 15% identity with SpsA, and the structural comparison shows a Z-score of 17.9 and an rmsd of 3.2 Å. STM0726 shares 16% identity with the prototypical Ribf-transferase structure, and the structures have a Z-score of 21.4 with an rmsd of 6.5 Å. The lower level of three-dimensional similarity in this STM0726 comparison arises primarily from differences in the relative orientation of the PRP and gPRT domains in the two proteins. b HPLC traces of in vitro reactions containing STM0725 and STM0726 PRPP donor substrate and acceptor compound 2. Reactions were incubated for 18 h at 4 °C to prevent protein precipitation, which occurred rapidly at warmer temperatures. Reactions were stopped by the addition of an equal volume of acetonitrile and the mixture analyzed by HPLC, which detected the methoxybenzamide tag in the acceptor. Products indicated on the chromatograms were confirmed by MS (Supplementary Fig. 8) and the results from triplicate reactions were identical. c NMR analysis of the purified STM0725/STM0726 product from a scaled-up reaction. The determined structure, including the reducing terminal synthetic acceptor, is shown above the spectra and assignments are shown on the HSQC spectrum. Linkage points of the monosaccharides were determined by an HMBC experiment and are shown. Chemical shifts are reported in Supplementary Table 3.