Extended Data Fig. 6: Activities of gPRT^KpO7 site-directed alanine-replacement mutants.

a, The chromatograms show the profiles of aliquots of reaction mixtures separated by HPLC on a GlycoSep N column eluted with a normal phase gradient of water. Each reaction was performed with the indicated mutant, PRPP donor and acceptor 1. After 1 h (left column) or 18 h (right column), reactions were stopped by the addition of acetonitrile. R284A and R311A mutant proteins were inactive, while D285A and D417A showed substantially reduced activity. D285A showed no detectable activity at 1 h but retained small amounts of activity evident after 18 h. D417A converted almost half of the substrate after 1-hour reaction and full conversion after 18 hours. The identities of the reaction products are shown on each chromatogram. The black box represents the methoxybenzamide tag in 1. The analysis was performed in triplicate with similar results. b, The identities of the reaction products from 18 h reactions for the active variants were confirmed by mass spectrometry. The extracted ion chromatogram (EIC) traces of m/z 704.345 (1 + Ribf) are shown, together with expanded regions of the corresponding ESI mass-spectrum at the indicated retention time.