The Exquisite Structure and Reaction Mechanism of Bacterial Pz-peptidase A toward Collagenous Peptides X-RAY CRYSTALLOGRAPHIC STRUCTURE ANALYSIS OF PZ-PEPTIDASE A REVEALS DIFFERENCES FROM MAMMALIAN THIMET OLIGOPEPTIDASE
The Exquisite Structure and Reaction Mechanism of Bacterial Pz-peptidase A toward Collagenous Peptides X-RAY CRYSTALLOGRAPHIC STRUCTURE ANALYSIS OF PZ-PEPTIDASE A REVEALS DIFFERENCES FROM MAMMALIAN THIMET OLIGOPEPTIDASE
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DOI：
10.1074/jbc.m110.141838
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发表时间：
2010-11-05
影响因子：
4.8
通讯作者：
Watanabe, Kunihiko
中科院分区：
文献类型：
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作者：
Kawasaki, Akio;Nakano, Hiroaki;Watanabe, Kunihiko
来自嗜热细菌Geobacillus collagenovorans MO-1的Pz-肽酶A水解合成肽底物4-苯基偶氮苄氧基羰基-Pro-Leu-Gly-ProD-Arg（Pz-PLGPR），其含有胶原特异性三肽序列-Gly-Pro-X-，但不作用于胶原蛋白本身。哺乳动物的酶，thimet寡肽酶（TOP），它具有可比的功能与细菌的PZ-肽酶，但有限的身份在一级序列水平，最近已受到X-射线晶体学分析，但是，没有晶体结构尚未被报道的TOP与底物类似物的复合物。在这里，我们报告的结晶重组Pz-肽酶A复合物与两个膦肽抑制剂（PPI），也作为抑制剂的TOP和这些复合物的晶体结构的测定在1.80-2.00埃分辨率。Pz-肽酶A和TOP之间最显著的区别是没有通道贯穿细菌蛋白的长度。TOP的结构类似于开放的双壳类，而Pz-肽酶A的结构是封闭的球形。这表明胶原肽底物在封闭的Pz-肽酶A分子的顶部通道处进入隧道，并且反应物肽在位于隧道中心的活性位点处裂解后从底部通道释放。PPI-2是两种PPI中的一种，它含有胶原蛋白特异性序列，有助于阐明Pz-肽酶A对胶原蛋白肽的精细结构和反应机制。本研究描述了哺乳动物酶的底物结合模式及其意义。
Pz-peptidase A, from the thermophilic bacterium Geobacillus collagenovorans MO-1, hydrolyzes a synthetic peptide substrate, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-ProD-Arg (Pz-PLGPR), which contains a collagen-specific tripeptide sequence, -Gly-Pro-X-, but does not act on collagen proteins themselves. The mammalian enzyme, thimet oligopeptidase (TOP), which has comparable functions with bacterial Pz-peptidases but limited identity at the primary sequence level, has recently been subjected to x-ray crystallographic analysis; however, no crystal structure has yet been reported for complexes of TOP with substrate analogues. Here, we report crystallization of recombinant Pz-peptidase A in complex with two phosphinic peptide inhibitors (PPIs) that also function as inhibitors of TOP and determination of the crystal structure of these complexes at 1.80-2.00 angstrom resolution. The most striking difference between Pz-peptidase A and TOP is that there is no channel running the length of bacterial protein. Whereas the structure of TOP resembles an open bivalve, that of Pz-peptidase A is closed and globular. This suggests that collagenous peptide substrates enter the tunnel at the top gateway of the closed Pz-peptidase A molecule, and reactant peptides are released from the bottom gateway after cleavage at the active site located in the center of the tunnel. One of the two PPIs, PPI-2, which contains the collagen-specific sequence, helped to clarify the exquisite structure and reaction mechanism of Pz-peptidase A toward collagenous peptides. This study describes the mode of substrate binding and its implication for the mammalian enzymes.