Structure and function of GDP-mannose-3',5'-epimerase: an enzyme which performs three chemical reactions at the same active site.
Structure and function of GDP-mannose-3',5'-epimerase: an enzyme which performs three chemical reactions at the same active site.
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DOI：
10.1021/ja056490i
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发表时间：
2005-12-28
影响因子：
15
通讯作者：
Naismith, JH
中科院分区：
文献类型：
--
作者：
Major, LL;Wolucka, BA;Naismith, JH
来自拟南芥的GDP-甘露糖-3′，5′-外聚酶（GME）催化GDP-α-d-甘露糖的3′和5′位置的外聚反应生成GDP-β-l-半乳糖。GDP-α-d-甘露糖的C5 '外聚物，GDP-β-l-葡糖的生产也有报道。该反应是植物中维生素C生物合成的一部分。我们测定了GME与GDP-α-d-甘露糖、GDP-β-l-半乳糖以及GDP-β-l-葡糖与GDP-β-l-4-酮-葡糖混合物的配合物结构，分辨率从2.0 Å到1.4 Å不等。该酶具有典型的扩展短链脱水酶/还原酶（SDR）折叠。我们已经证实GME在GDP-β-l-半乳糖和GDP-β-l-葡萄糖两种产物之间建立了平衡。C4′氧化GDP-α-d-甘露糖，C5′位外聚得到GDP-β-l-4-酮糖。该中间体要么被还原为GDP-β-l-葡萄糖，要么C3 ‘位置被外映得到GDP-β-l-4-酮半乳糖，然后C4 ’被还原为GDP-β-l- l-半乳糖。氧化、外聚和还原在单一活性位点的结合是不寻常的。结合位点定向诱变的结构分析表明，C145和K217是导致这两种异聚化的酸碱对。根据GDP-β-l-葡聚糖/GDP-β-l-4-酮-葡聚糖共络合物的结构，我们预测在第一次外聚过程中会发生环翻转，而在第二次外聚过程中可能会出现船状中间体。将GME与其他已知的能够将蛋白质α提取为碳水化合物的酮功能的SDR酶进行比较，确定了关键的共同特征。
GDP-mannose-3′,5′-epimerase (GME) from Arabidopsis thaliana catalyses the epimerization of both the 3′ and 5′ positions of GDP-α-d-mannose to yield GDP-β-l-galactose. Production of the C5′ epimer of GDP-α-d-mannose, GDP-β-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-α-d-mannose, GDP-β-l-galactose and a mixture of GDP-β-l-gulose with GDP-β-l-4-keto-gulose, to resolutions varying from 2.0 Å to 1.4 Å. The enzyme has the classical extended short chain dehydratase/reductase (SDR) fold. We have confirmed that GME establishes an equilibrium between two products, GDP-β-l-galactose and GDP-β-l-gulose. The reaction proceeds by C4′ oxidation of GDP-α-d-mannose followed by epimerization of the C5′ position to give GDP-β-l-4-keto-gulose. This intermediate is either reduced to give GDP-β-l-gulose or the C3′ position is epimerized to give GDP-β-l-4-keto-galactose, then C4′ is reduced to GDP-β-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. Based on the structure of the GDP-β-l-gulose/GDP-β-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 α to the keto function of a carbohydrate identifies key common features.
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影响因子：
5.7
作者：
Dong, CJ;Major, LL;Naismith, JH
通讯作者：
Naismith, JH
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