Nucleoside Phosphorylases make N7-xanthosine

Abstract

Modern, highly evolved nucleoside-processing enzymes are known to exhibit perfect regioselectivity over the glycosylation of purine nucleobases at N9. We herein report an exception to this paradigm. Wild-type nucleoside phosphorylases also furnish N7-xanthosine, a "non-native" ribosylation regioisomer of xanthosine. This unusual nucleoside possesses several atypical physicochemical properties such as redshifted absorption spectra, a high equilibrium constant of phosphorolysis and low acidity. Ultimately, the biosynthesis of this previously unknown natural product illustrates how even highly evolved, essential enzymes from primary metabolism are imperfect catalysts.

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 pK_a 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 k_obs 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 data.