Investigation of metal ion binding in phosphonoacetaldehyde hydrolase identifies sequence markers for metal-activated enzymes of the HAD enzyme superfamily.
Investigation of metal ion binding in phosphonoacetaldehyde hydrolase identifies sequence markers for metal-activated enzymes of the HAD enzyme superfamily.
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DOI：
10.1021/bi036309n
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发表时间：
2004-05
期刊：
影响因子：
2.9
通讯作者：
Guofeng Zhang;M. Morais;Jianying Dai;Wenhai Zhang;D. Dunaway-Mariano;Karen N Allen-
中科院分区：
文献类型：
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作者：
Guofeng Zhang;M. Morais;Jianying Dai;Wenhai Zhang;D. Dunaway-Mariano;Karen N Allen-
2-卤代烷酸脱卤酶（HAD）家族是已知的最大的酶超家族之一，其包含碳和磷酰基转移酶。HAD成员保留了一个alpha，beta核心结构域，该结构域构成了四环活性位点平台。每个环贡献一个或多个催化基团，其用于介导核心化学（即，组转移）。在本文中，我们提供的证据表明，环4上的羧酸残基的数量和它们的位置（站）的循环是决定因素，因此可靠的序列标记，金属离子激活HAD家族成员之间。使用这个预测器，我们得出结论，绝大多数的HAD成员利用金属辅因子。金属辅因子结合的最低要求进行了分析，使用镁（II）激活蜡状芽孢杆菌膦酰乙醛水解酶（磷酸酶）作为金属激活的HAD成员的实验模型。Mg（II）结合通过与环1 Asp 12羧酸和Thr 14骨架羰基以及环4 Asp 186羧酸连接而发生。环4 Asp 190与Mg（II）水配体形成氢键。X-射线结构测定的D12 A突变体的存在下的底物膦酰乙醛表明，更换的环1天冬氨酸，共同的所有HAD家族成员，与丙氨酸转移的位置的镁（II），从而使内层协调Asp 190，并导致转移的位置的基板。4环突变体的动力学分析表明，Asp 186是必不可少的辅因子结合，而Asp 190只是增强它。在磷酸酯酶亚家族，Asp 186是严格保守的，而位置185或位置190是用来定位第二环4天冬氨酸残基。在G185 D/D190 G磷酸酶突变体中保留高水平的催化活性证明了金属结合环的可塑性，这反映在沿着所检查的2700个潜在HAD序列的七个残基序定位的两个或三个Asp残基沿着的各种组合中。
The 2-haloalkanoic acid dehalogenase (HAD) family, which contains both carbon and phosphoryl transferases, is one of the largest known enzyme superfamilies. HAD members conserve an alpha,beta-core domain that frames the four-loop active-site platform. Each loop contributes one or more catalytic groups, which function in mediating the core chemistry (i.e., group transfer). In this paper, we provide evidence that the number of carboxylate residues on loop 4 and their positions (stations) on the loop are determinants, and therefore reliable sequence markers, for metal ion activation among HAD family members. Using this predictor, we conclude that the vast majority of the HAD members utilize a metal cofactor. Analysis of the minimum requirements for metal cofactor binding was carried out using Mg(II)-activated Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) as an experimental model for metal-activated HAD members. Mg(II) binding occurs via ligation to the loop 1 Asp12 carboxylate and Thr14 backbone carbonyl and to the loop 4 Asp186 carboxylate. The loop 4 Asp190 forms a hydrogen bond to the Mg(II) water ligand. X-ray structure determination of the D12A mutant in the presence of the substrate phosphonoacetaldehyde showed that replacement of the loop 1 Asp, common to all HAD family members, with Ala shifts the position of Mg(II), thereby allowing innersphere coordination to Asp190 and causing a shift in the position of the substrate. Kinetic analysis of the loop 4 mutants showed that Asp186 is essential to cofactor binding while Asp190 simply enhances it. Within the phosphonatase subfamily, Asp186 is stringently conserved, while either position 185 or position 190 is used to position the second loop 4 Asp residue. Retention of a high level of catalytic activity in the G185D/D190G phosphonatase mutant demonstrated the plasticity of the metal binding loop, reflected in the variety of combinations in positioning of two or three Asp residues along the seven-residue motif of the 2700 potential HAD sequences that were examined.