α-Glucan, water dikinase (GWD):: A plastidic enzyme with redox-regulated and coordinated catalytic activity and binding affinity
α-Glucan, water dikinase (GWD):: A plastidic enzyme with redox-regulated and coordinated catalytic activity and binding affinity
复制标题
DOI：
10.1073/pnas.0406674102
复制
发表时间：
2005-02-01
影响因子：
11.1
通讯作者：
Blennow, A
中科院分区：
文献类型：
--
作者：
Mikkelsen, R;Mutenda, KE;Blennow, A
新近发现的马铃薯块茎(Solanum Tuberosum)α-葡聚糖水二激酶(GWD)(以前称为R1)通过双激酶型反应机制催化淀粉的磷酸化，其中ATP的β-磷酸转移到淀粉的葡萄糖残基的C-6或C-3位置。在目前的研究中，我们发现GWD酶在氧化形式下是不活跃的，这伴随着二硫键连接的多肽图谱所确定的特定分子内二硫键的形成。这种二硫键的调节特性通过定点突变研究得到了证实。菠菜叶片还原态硫氧还蛋白(Trx)f和Trxm在很低浓度下都被氧化GWD还原和活化，其中Trx f的效率更高，S-0.5值为0.4um。有趣的是，GWD表现出与淀粉颗粒的可逆和选择性结合，这取决于植物的光照状态。在这里，我们证明了从暗适应植物中分离出来的淀粉颗粒结合的GWD以非活性的、被氧化的形式存在，它能够在用还原的TRX处理后重新激活。此外，GWD的可溶性形式被发现处于完全还原的状态，这为Trx控制的调节机制提供了证据，该机制将蛋白质的酶活性和特定的结合亲和力连接到细胞内表面。马铃薯GWD的调控位点序列CFATC在其他物种的叶绿体靶向GWD中是保守的，表明GWD酶的整体氧化还原调节。
The recently discovered potato tuber (Solanum tuberosum) a-glucan, water dikinase (GWD) (formerly known as R1) catalyzes the phosphorylation of starch by a dikinase-type reaction mechanism in which the beta-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glucosyl residue of starch. In the present study, we found that the GWD enzyme is inactive in the oxidized form, which is accompanied by the formation of a specific intramolecular disulfide bond as determined by disulfide-linked peptide mapping. The regulatory properties of this disulfide linkage were confirmed by site-directed mutagenesis studies. Both reduced thioredoxin (Trx) f and Trx m from spinach leaves reduced and activated oxidized GWD at very low concentrations, with Trx f being the more efficient, yielding an S-0.5 value of 0.4 muM. Interestingly, GWD displays a reversible and selective binding to starch granules depending on the illumination state of the plant. Here we show that starch granule-bound GWD isolated from dark-adapted plants exists in the inactive, oxidized form, which is capable of reactivation upon treatment with reduced Trx. Furthermore, the soluble form of GWD was found in its fully reduced state, providing evidence of a Trx-controlled regulation mechanism linking enzymatic activity and specific binding affinities of a protein to an intracellular surface. The regulatory site sequence, CFATC, of potato GWD is conserved in chloroplast-targeted GWDs from other species, suggesting an overall redox regulation of the GWD enzyme.