The crystal structure of benzoylformate decarboxylase at 1.6 Å resolution:: Diversity of catalytic residues in thiamin diphosphate-dependent enzymes
The crystal structure of benzoylformate decarboxylase at 1.6 Å resolution:: Diversity of catalytic residues in thiamin diphosphate-dependent enzymes
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DOI：
10.1021/bi973047e
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发表时间：
1998-07-14
期刊：
影响因子：
2.9
通讯作者：
Ringe, D
中科院分区：
文献类型：
--
作者：
Hasson, MS;Muscate, A;Ringe, D
二磷酸硫胺素（ThDP）依赖性酶苯甲酰甲酸脱羧酶（BFD），在恶臭假单胞菌的扁桃酸途径的第三种酶的晶体结构，已解决了在1.6埃分辨率的多个同晶置换，并细化到15.0%的R-因子（游离R = 18.6%）。BFD的结构已与其他ThDP依赖性酶（包括丙酮酸脱羧酶）的结构进行了比较。BFD的整体结构类似于其他家族成员的结构，并且辅因子和金属结合残基很好地保守。令人惊讶的是，不直接结合到辅因子的活性位点残基没有保守。然而，活性位点中官能团的位置可以是保守的。在BFD晶体结构中已经确定了三类金属离子：Ca 2+与每个亚基中的辅因子结合，Mg 2+在四聚体的2倍轴上，Ca 2+在晶体接触处。该结构包括一个非脯氨酸顺式肽键和一个异常长且规则的聚脯氨酸II型螺旋，该螺旋介导晶体中四聚体之间的主要接触。高质量的电子密度图允许校正总计超过10%的氨基酸序列的错误，这是从mdlC基因的报道序列预测的。BFD结构的分析表明，活化的辅因子，反应中间体的性质，和建筑的考虑有关的蛋白质折叠的要求一直占主导地位的力量在ThDP依赖性酶的进化。
The crystal structure of the thiamin diphosphate (ThDP)-dependent enzyme benzoylformate decarboxylase (BFD), the third enzyme in the mandelate pathway of Pseudomonas putida, has been solved by multiple isomorphous replacement at 1.6 Angstrom resolution and refined to an R-factor of 15.0% (free R = 18.6%). The structure of BFD has been compared to that of other ThDP-dependent enzymes, including pyruvate decarboxylase. The overall architecture of BFD resembles that of the other family members, and cofactor- and metal-binding residues are well conserved, Surprisingly, there is no conservation of active-site residues not directly bound to the cofactor. The position of functional groups in the active site may be conserved, however. Three classes of metal ions have been identified in the BFD crystal structure: Ca2+ bound to the cofactor in each subunit, Mg2+ on a 2-fold axis of the tetramer, and Ca2+ at a crystal contact. The structure includes a non-proline cis-peptide bond and an unusually long and regular polyproline type II helix that mediates the main contact between tetramers in the crystal. The high-quality electron-density map allowed the correction of errors totaling more than 10% of the amino acid sequence, which had been predicted from the reported sequence of the mdlC gene. Analysis of the BFD structure suggests that requirements for activation of the cofactor, the nature of the reaction intermediates, and architectural considerations relating to the protein fold have been dominant forces in the evolution of ThDP-dependent enzymes.