On the stability of nucleoside diphosphate glucose metabolites: Implications for studies of plant carbohydrate metabolism

Abstract

Nucleoside diphosphate sugars (NDP-sugars) are the substrates for biosynthesis of oligo-and polysaccharides, such as starch and cellulose, and are also required for biosynthesis of nucleotides, ascorbic acid, several cofactors, glycoproteins and many secondary metabolites. A controversial study that questions the generally accepted pathway of ADP-glucose and starch synthesis in plants is based, in part, on claims that NDP-sugars are unstable at alkaline pH in the presence of Mg2+ and that this instability can lead to unreliable results from in vitro assays of enzyme activities. If substantiated, this claim would have far-reaching implications for many published studies that report on the activities of NDP-sugar metabolizing enzymes. To resolve this controversy, we investigated the stability of UDP-and ADP-glucose using biophysical, namely nuclear magnetic resonance (NMR), and highly specifc enzymatic methods. Results obtained with both techniques indicate that NDP-sugars are not as unstable as previously suggested. Moreover, their calculated in vitro half-lives are signifcantly higher than estimates of their in planta turnover times. This indicates that the physico-chemical stability of NDP-sugars has little impact on their concentrations in vivo and that NDP-sugar levels are determined primarily by the relative rates of enzymatic synthesis and consumption. Our results refute one of the main arguments for the controversial pathway of starch synthesis from imported ADPglucose produced by sucrose synthase in the cytosol.