REFINED CRYSTAL-STRUCTURE OF THE CATALYTIC DOMAIN OF DIHYDROLIPOYL TRANSACETYLASE (E2P) FROM AZOTOBACTER-VINELANDII AT 2.6-ANGSTROM RESOLUTION
REFINED CRYSTAL-STRUCTURE OF THE CATALYTIC DOMAIN OF DIHYDROLIPOYL TRANSACETYLASE (E2P) FROM AZOTOBACTER-VINELANDII AT 2.6-ANGSTROM RESOLUTION
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DOI：
10.1006/jmbi.1993.1235
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发表时间：
1993-04-20
影响因子：
5.6
通讯作者：
HOL, WGJ
中科院分区：
文献类型：
--
作者：
MATTEVI, A;OBMOLOVA, G;HOL, WGJ
二氢脂酰基转乙酰化酶（E2p）在结构上和功能上都是丙酮酸脱氢酶多酶复合物的中心酶。采用多重同构置换法求解了偶氮细菌vinelandiiE2p （E2pCD）催化域382 ~ 637残基的晶体结构，并采用能量最小化方法对其进行了细化。最终的模型包含2182个蛋白质原子和37个有序的水分子。在10.0 ~ 2.6 Å分辨率范围内，10344次反射的反射系数为18.7%。键长与理想值的均方根位移偏差为0·017 Å，键角为3·3°。n端残基382 ~ 394是无序的，在电子密度图中不可见，否则所有残基都有明确的密度。催化结构域形成由24个亚基组成的低聚物，具有432八面体对称。在E2pCD晶体中，24个亚基之间存在着晶体对称关系。亚单位的立方排列产生了一个大的中空立方体，边缘为120 Å。立方体的表面有直径为30 Å的孔。这个立方体真正的构建块是E2p三聚体，其中八个占据了立方体的四个角。分子间接触可以区分为两个层次：(1)3倍相关亚基之间的广泛相互作用导致紧密相关的三聚体；(2)导致三聚体组装成立方24聚体的2折轴相互作用。每个亚基具有类似于氯霉素乙酰转移酶（CAT）的拓扑结构，并包括一个由五个α-螺旋包围的中心β-片。两种蛋白的比较表明，E2pCD的n端残基395 ~ 426发生了较大的旋转，重塑了底物结合位点，扩展了三个相关亚基之间的相互作用。催化中心由一个30 Å长的通道组成，从“内”侧延伸到“外”侧，其中“内”和“外”指的是丙酮酸脱氢酶复合物在24立方核中的位置，分别对应于辅酶a和脂酰胺结合位点。活性位点由原子因子表明的迁移率最低的残基形成。五个脯氨酸残基围绕在活性位点周围。His610的侧链，与CAT类似，最有可能参与催化，通过Asp609和Arg611之间的盐桥，以及与Val435， Tyr608, Leu425 ‘和Ile571 ’侧链的接触，稳定在其不寻常的构象中，后两个残基位于三个相关亚基上。在蛋白质的N端，残基395至402形成一个延伸臂。由于它们是连接催化剂与E1/E3结合域的连接体的一部分，因此它们的构象表明，连接体可能由相当不灵活的延伸多肽链片段组成，这些片段通过更灵活的“铰链”残基相互连接。
Dihydrolipoyl transacetylase (E2p) is both structurally and functionally the central enzyme of the pyruvate dehydrogenase multienzyme complex. The crystal structure of the catalytic domain, i.e. residues 382 to 637, ofAzotobacter vinelandiiE2p (E2pCD) was solved by multiple isomorphous replacement and refined by energy minimization procedures. The final model contains 2182 protein atoms and 37 ordered water molecules. TheR-factor is 18·7% for 10,344 reflections between 10·0 and 2·6 Å resolution. The root-mean-square shift deviation from the ideal values is 0·017 Å for bond lengths and 3·3° for bond angles. The N-terminal residues 382 to 394 are disordered and not visible in the electron density map, otherwise all residues have well-defined density. The catalytic domain forms an oligomer of 24 subunits, having octahedral 432 symmetry. In the E2pCD crystals, the 24 subunits are related by the crystallographic symmetry. The cubic arrangement of subunits gives rise to a large hollow cube with edges of 120 Å. The faces of the cube have pores of diameter of 30 Å. The true building block of the cube is the E2p trimer, eight of which occupy the corners of the cube. Two levels of intermolecular contacts can be distinguished: (1) the extensive interactions between 3-fold related subunits leading to a tightly associated trimer; and (2) the interactions along the 2-fold axis leading to the assembly of the trimers into the cubic 24-mer. Each subunit has a topology similar to chloramphenicol acetyltransferase (CAT) and comprises a central β-sheet surrounded by five α-helices. The comparison of the two proteins indicates a large rotation of the N-terminal residues 395 to 426 of E2pCD, which reshapes the substrate binding site and extends the interaction between threefold related subunits. The catalytic centre consists of a 30 Å long channel extending from the "inner" side of the trimmer to the "outer" side, where inner and outer refer to the position in the 24-meric cubic core of the pyruvate dehydrogenase complex and correspond with CoA and lipoamide binding sites, respectively. The active site is formed by the residues with the lowest mobility as indicated by the atomicB-factors. Five proline residues surround the active site. The side-chain of His610, which, by analogy with CAT, is most likely involved in catalysis, is stabilized in its unusual conformation by the salt-bridge between Asp609 and Arg611, and by contacts with the side-chains of Val435, Tyr608, Leu425′ and Ile571′, the latter two residues being located on a threefold related subunit. At the N terminus of the protein, residues 395 to 402 form an extended arm. Since they are part of the linker connecting the catalytic to the E1/E3 binding domain, their conformation is suggestive that the linker might consist of segments of rather inflexible extended polypeptide chain connected to each other by more flexible "hinge" residues.