Cloning, expression and characterization of a Nudix hydrolase that catalyzes the hydrolytic breakdown of ADP-glucose linked to starch biosynthesis in Arabidopsis thaliana
Cloning, expression and characterization of a Nudix hydrolase that catalyzes the hydrolytic breakdown of ADP-glucose linked to starch biosynthesis in Arabidopsis thaliana
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DOI：
10.1093/pcp/pcj065
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发表时间：
2006-07-01
影响因子：
4.9
通讯作者：
Pozueta-Romero, Javier
中科院分区：
文献类型：
--
作者：
Munoz, Francisco Jose;Baroja-Fernandez, Edurne;Pozueta-Romero, Javier
“Nutrient”水解酶是广泛分布的核苷酸焦磷酸酶，其具有保守的GX（5）EX（7）REUXEEXGU基序，其中U通常是异亮氨酸、亮氨酸或缬氨酸。其中，大肠杆菌ADP-糖焦磷酸酶（ASPP）已被证明催化ADP-葡萄糖的水解分解，与细菌糖原生物合成有关。将拟南芥基因组的31个不同的Nudix编码序列与已知的细菌和哺乳动物ASPP编码序列进行比较，发现了一个在Nudix基序中具有重要分歧的序列，该序列一旦在E. coli中表达的ASPP蛋白具有ASPP活性。这种蛋白，命名为AtASPP，股票与假设的水稻和马铃薯蛋白的高度同源性，表明ASPP广泛分布在单子叶和双子叶植物。作为第一步，以测试可能参与植物ASPPs在调节细胞内的ADP-葡萄糖连接到淀粉生物合成的水平，我们生产和表征AtASPP-overexpressing拟南芥植物。来自这些植物的源叶表现出ADP葡萄糖和淀粉水平的大幅降低，表明植物ASPP催化与淀粉生物合成相关的相当大的ADP-葡萄糖池的水解分解。在AtASPP过表达的叶片中，与淀粉代谢密切相关的酶的最大催化活性没有多效性变化。总体信息提供了植物营养素水解酶存在的第一个证据，这些水解酶可以进入与淀粉生物合成相关的ADP-葡萄糖的细胞内库。
'Nudix' hydrolases are widely distributed nucleotide pyrophosphatases that possess a conserved GX(5)EX(7)REUXEEXGU motif where U is usually isoleucine, leucine or valine. Among them, Escherichia coli ADP-sugar pyrophosphatase (ASPP) has been shown to catalyze the hydrolytic breakdown of ADP-glucose linked to bacterial glycogen biosynthesis. Comparisons of the 31 different Nudix-encoding sequences of the Arabidopsis genome with those coding for known bacterial and mammalian ASPPs identified one sequence possessing important divergences in the Nudix motif that, once expressed in E. coli, produced a protein with ASPP activity. This protein, designated as AtASPP, shares strong homology with hypothetical rice and potato proteins, indicating that ASPPs are widely distributed in both mono- and dicotyledonous plants. As a first step to test the possible involvement of plant ASPPs in regulating the intracellular levels of ADP-glucose linked to starch biosynthesis, we produced and characterized AtASPP-overexpressing Arabidopsis plants. Source leaves from these plants exhibited a large reduction in the levels of both ADPglucose and starch, indicating that plant ASPPs catalyze the hydrolytic breakdown of a sizable pool of ADP-glucose linked to starch biosynthesis. No pleiotropic changes in maximum catalytic activities of enzymes closely linked to starch metabolism could be detected in AtASPP-overexpressing leaves. The overall information provides the first evidence for the existence of plant Nudix hydrolases that have access to an intracellular pool of ADP-glucose linked to starch biosynthesis.