Hill Benjamin L, Figueroa Carlos M, Asencion Diez Matías D, Lunn John E, Iglesias Alberto A, Ballicora Miguel A
Department of Chemistry and Biochemistry, Loyola University Chicago, 1068 West Sheridan Road, 60660 Chicago, IL, USA.
Instituto de Agrobiotecnología del Litoral, UNL, CONICET, FBCB, Colectora Ruta Nacional 168 km 0, 3000 Santa Fe, Argentina.
J Exp Bot. 2017 Jun 15;68(13):3331-3337. doi: 10.1093/jxb/erx190.
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 specific 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 significantly 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 ADP-glucose produced by sucrose synthase in the cytosol.
核苷二磷酸糖（NDP-糖）是寡糖和多糖（如淀粉和纤维素）生物合成的底物，也是核苷酸、抗坏血酸、几种辅酶、糖蛋白和许多次生代谢产物生物合成所必需的。一项有争议的研究对植物中普遍接受的ADP-葡萄糖和淀粉合成途径提出了质疑，该研究部分基于这样的说法：在Mg2+存在的情况下，NDP-糖在碱性pH条件下不稳定，这种不稳定性会导致体外酶活性测定结果不可靠。如果这一说法得到证实，将对许多已发表的关于NDP-糖代谢酶活性的研究产生深远影响。为了解决这一争议，我们使用生物物理方法，即核磁共振（NMR）和高特异性酶法，研究了UDP-葡萄糖和ADP-葡萄糖的稳定性。两种技术获得的结果表明，NDP-糖并不像之前认为的那样不稳定。此外，它们计算出的体外半衰期明显高于对其在植物体内周转时间的估计。这表明NDP-糖的物理化学稳定性对其体内浓度影响很小，NDP-糖水平主要由酶促合成和消耗的相对速率决定。我们的结果驳斥了关于由蔗糖合酶在细胞质中产生的输入ADP-葡萄糖合成淀粉的争议途径的主要论点之一。
