Kunishima Naoki, Asada Yukuhiko, Sugahara Mayumi, Ishijima Jun, Nodake Yuichi, Sugahara Mitsuaki, Miyano Masashi, Kuramitsu Seiki, Yokoyama Shigeyuki, Sugahara Michihiro
Highthroughput Factory, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Sayo-gun, Hyogo 679-5148, Japan.
J Mol Biol. 2005 Sep 9;352(1):212-28. doi: 10.1016/j.jmb.2005.07.008.
Hot dog fold proteins sharing the characteristic "hot dog" fold are known to involve certain coenzyme A binding enzymes with various oligomeric states. In order to elucidate the oligomerization-function relationship of the hot dog fold proteins, crystal structures of the phenylacetate degradation protein PaaI from Thermus thermophilus HB8 (TtPaaI), a tetrameric acyl-CoA thioesterase with the hot dog fold, have been determined and compared with those of other family members. In the liganded crystal forms with coenzyme A derivatives, only two of four intersubunit catalytic pockets of the TtPaaI tetramer are occupied by the ligands. A detailed structural comparison between several liganded and unliganded forms reveals that a subtle rigid-body rearrangement of subunits within 2 degrees upon binding of the first two ligand molecules can induce a strict negative cooperativity to prevent further binding at the remaining two pockets, indicating that the so-called "half-of-the-sites reactivity" of oligomeric enzymes is visualized for the first time. Considering kinetic and mutational analyses together, a possible reaction mechanism of TtPaaI is proposed; one tetramer binds only two acyl-CoA molecules with a novel asymmetric induced-fit mechanism and carries out the hydrolysis according to a base-catalyzed reaction through activation of a water molecule by Asp48. From a structural comparison with other family members, it is concluded that a subgroup of the hot dog fold protein family, referred to as "asymmetric hot dog thioesterases" including medium chain acyl-CoA thioesterase II from Escherichia coli and human thioesterase III, might share the same oligomerization mode and the asymmetric induced-fit mechanism as observed in TtPaaI.
具有特征性“热狗”折叠的蛋白质已知涉及某些具有不同寡聚状态的辅酶A结合酶。为了阐明“热狗”折叠蛋白的寡聚化与功能的关系，已确定嗜热栖热菌HB8（TtPaaI）的苯乙酸降解蛋白PaaI（一种具有“热狗”折叠的四聚体酰基辅酶A硫酯酶）的晶体结构，并与其他家族成员的晶体结构进行了比较。在与辅酶A衍生物形成的配体结合晶体形式中，TtPaaI四聚体的四个亚基间催化口袋中只有两个被配体占据。对几种配体结合形式和未结合形式的详细结构比较表明，在前两个配体分子结合时，亚基在2度范围内发生细微的刚体重排，可诱导严格的负协同作用，以防止在其余两个口袋进一步结合，这表明首次观察到寡聚酶的所谓“半位点反应性”。综合动力学和突变分析，提出了TtPaaI可能的反应机制；一个四聚体仅以一种新的不对称诱导契合机制结合两个酰基辅酶A分子，并通过Asp48激活水分子，根据碱催化反应进行水解。通过与其他家族成员的结构比较得出结论，“热狗”折叠蛋白家族的一个亚组，称为“不对称热狗硫酯酶”，包括大肠杆菌的中链酰基辅酶A硫酯酶II和人硫酯酶III，可能与TtPaaI具有相同的寡聚化模式和不对称诱导契合机制。
