Fig. 1: Discovery and characterization of N7X.
a Formation of N7X and/or N9X catalyzed by nucleoside phosphorylases. b Serendipitous discovery of N7X in an enzymatic cascade through (c) its unprecedented redshift compared to the free nucleobase. d–g Orthogonal data confirming N7X as the major product of the glycosylation of X by Geobacillus thermoglucosidasius purine NP (GtPNP) at pH 9. f Energy-minimized structure of N7X and key 2D NMR correlations. h UV absorption spectra of X and N7X at pH 10, where both species exist as monoanions. i pKa values as determined by UV spectroscopy. j, k Continuous monitoring of the X → N7X glycosylation at pH 10 (with 0.4 g L−1 GtPNP) to establish its equilibrium constant. By convention, these equilibrium constants are given for the phosphorolytic direction. l Michaelis-Menten kinetics for N7X formation by GtPNP. The relationship between kobs and [X] is non-linear (as supported by the Akaike information criterion, AIC), allowing us to estimate a Michaelis-Menten constant. m Formation of N9X and N7X by various wild-type nucleoside phosphorylases (with 500 µM X and 1 g L−1 enzyme). The bars in this panel each display the isomer distribution obtained in a single experiment (n = 1). Please see the Supplementary Information for experimental details and the externally hosted supplementary material for all raw data38.
