Major Louise L, Wolucka Beata A, Naismith James H
Centre for Biomolecular Sciences, University of St. Andrews, North Haugh, St. Andrews, Fife, Scotland KY16 9ST, United Kingdom.
J Am Chem Soc. 2005 Dec 28;127(51):18309-20. doi: 10.1021/ja056490i.
GDP-mannose-3',5'-epimerase (GME) from Arabidopsis thaliana catalyzes the epimerization of both the 3' and 5' positions of GDP-alpha-D-mannose to yield GDP-beta-L-galactose. Production of the C5' epimer of GDP-alpha-D-mannose, GDP-beta-L-gulose, has also been reported. The reaction occurs as part of vitamin C biosynthesis in plants. We have determined structures of complexes of GME with GDP-alpha-D-mannose, GDP-beta-L-galactose, and a mixture of GDP-beta-L-gulose with GDP-beta-L-4-keto-gulose to resolutions varying from 2.0 to 1.4 A. The enzyme has the classical extended short-chain dehydratase/reductase (SDR) fold. We have confirmed that GME establishes an equilibrium between two products, GDP-beta-L-galactose and GDP-beta-L-gulose. The reaction proceeds by C4' oxidation of GDP-alpha-D-mannose followed by epimerization of the C5' position to give GDP-beta-L-4-keto-gulose. This intermediate is either reduced to give GDP-beta-L-gulose or the C3' position is epimerized to give GDP-beta-L-4-keto-galactose, then C4' is reduced to GDP-beta-L-galactose. The combination of oxidation, epimerization, and reduction in a single active site is unusual. Structural analysis coupled to site-directed mutagenesis suggests C145 and K217 as the acid/base pair responsible for both epimerizations. On the basis of the structure of the GDP-beta-L-gulose/GDP-beta-L-4-keto-gulose co-complex, we predict that a ring flip occurs during the first epimerization and that a boat intermediate is likely for the second epimerization. Comparison of GME with other SDR enzymes known to abstract a protein alpha to the keto function of a carbohydrate identifies key common features.
来自拟南芥的GDP - 甘露糖 - 3',5'-表异构酶（GME）催化GDP-α-D-甘露糖3'和5'位的表异构化反应，生成GDP-β-L-半乳糖。也有报道称会生成GDP-α-D-甘露糖的C5'差向异构体GDP-β-L-古洛糖。该反应是植物中维生素C生物合成的一部分。我们已经确定了GME与GDP-α-D-甘露糖、GDP-β-L-半乳糖以及GDP-β-L-古洛糖与GDP-β-L-4-酮古洛糖混合物形成的复合物结构，分辨率在2.0至1.4埃之间。该酶具有经典的延伸短链脱水酶/还原酶（SDR）折叠结构。我们已经证实GME在两种产物GDP-β-L-半乳糖和GDP-β-L-古洛糖之间建立了平衡。反应通过GDP-α-D-甘露糖的C4'氧化，随后C5'位表异构化生成GDP-β-L-4-酮古洛糖。该中间体要么被还原生成GDP-β-L-古洛糖，要么C3'位表异构化生成GDP-β-L-4-酮半乳糖，然后C4'被还原为GDP-β-L-半乳糖。在单个活性位点中同时发生氧化、表异构化和还原反应的情况并不常见。结构分析结合定点诱变表明，C145和K217是负责两次表异构化反应的酸碱对。基于GDP-β-L-古洛糖/GDP-β-L-4-酮古洛糖共复合物的结构，我们预测在第一次表异构化过程中会发生环翻转，并且第二次表异构化可能存在船型中间体。将GME与其他已知可从碳水化合物的酮官能团中提取蛋白质α的SDR酶进行比较，确定了关键的共同特征。
