Assignment of pterin deaminase activity to an enzyme of unknown function guided by homology modeling and docking.
Assignment of pterin deaminase activity to an enzyme of unknown function guided by homology modeling and docking.
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DOI：
10.1021/ja309680b
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发表时间：
2013-01-16
影响因子：
15
通讯作者：
Raushel, Frank M.
中科院分区：
文献类型：
--
作者：
Fan, Hao;Hitchcock, Daniel S.;Seidel, Ronald D., II;Hillerich, Brandan;Lin, Henry;Almo, Steven C.;Sali, Andrej;Shoichet, Brian K.;Raushel, Frank M.
在非冗余 TrEMBL 数据库中超过 2200 万个蛋白质序列中，只有不到 1% 具有经过实验证实的功能。基于结构的方法已用于根据实验确定的结构预测酶活性；然而，对于绝大多数蛋白质来说，没有这样的结构。在此，基于远程模板结构计算了来自放射农杆菌 K84 (Arad3529) 的功能未表征的酰胺水解酶的同源模型。活性位点附近两个环的蛋白质主链被重塑，产生四种不同的活性位点构象。通过将 6440 个代谢物的 57672 个高能中间体 (HEI) 形式与这四种同源模型对接来预测 Arad3529 的底物。根据对接等级和几何形状，建议将一组修饰的蝶呤作为 Arad3529 的候选底物。通过酶学实验验证了预测结果，Arad3529 使许多蝶呤代谢物脱氨基（底物，kcat/Km [M−1s−1]）：甲酰蝶呤，5.2 × 106；蝶呤-6-羧酸酯，4.0×106；蝶呤-7-羧酸酯，3.7 × 106；蝶呤，3.3×106；羟甲基蝶呤，1.2×106；生物蝶呤，1.0×106； D-(+)-新蝶呤，3.1×105；异黄蝶呤，2.8×105；墨蝶呤，1.3×105；叶酸，1.3×105，黄蝶呤，1.17×105； 7,8-二氢羟甲基蝶呤，3.3 × 104。虽然蝶呤是叶酸降解过程中普遍存在的氧化产物，但基因组分析表明，未描述的蝶呤降解途径的第一步是由 Arad3529 催化的。基于同源模型的虚拟筛选，特别是蛋白质骨架灵活性的建模，可能广泛用于酶功能注释和发现新的途径和药物靶点。
Of the over 22 million protein sequences in the nonredundant TrEMBL database, fewer than 1% have experimentally confirmed functions. Structure-based methods have been used to predict enzyme activities from experimentally determined structures; however, for the vast majority of proteins, no such structures are available. Here, homology models of a functionally uncharacterized amidohydrolase from Agrobacterium radiobacter K84 (Arad3529) were computed based on a remote template structure. The protein backbone of two loops near the active site was remodeled, resulting in four distinct active site conformations. Substrates of Arad3529 were predicted by docking of 57672 high-energy intermediate (HEI) forms of 6440 metabolites against these four homology models. Based on docking ranks and geometries, a set of modified pterins were suggested as candidate substrates for Arad3529. The predictions were tested by enzymology experiments, and Arad3529 deaminated many pterin metabolites (substrate, kcat/Km [M−1s−1]): formylpterin, 5.2 × 106; pterin-6-carboxylate, 4.0 × 106; pterin-7-carboxylate, 3.7 × 106; pterin, 3.3 × 106; hydroxymethylpterin, 1.2 × 106; biopterin, 1.0 × 106; D-(+)-neopterin, 3.1 × 105; isoxanthopterin, 2.8 × 105; sepiapterin, 1.3 × 105; folate, 1.3 × 105, xanthopterin, 1.17 × 105; 7,8-dihydrohydroxymethylpterin, 3.3 × 104. While pterin is a ubiquitous oxidative product of folate degradation, genomic analysis suggests that the first step of an undescribed pterin degradation pathway is catalyzed by Arad3529. Homology model-based virtual screening, especially with modeling of protein backbone flexibility, may be broadly useful for enzyme function annotation and discovering new pathways and drug targets.
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