幽门螺杆菌嘌呤核苷磷酸化酶呈现出新的开放和关闭活性部位构象的分布模式和不寻常的生化特征。

Helicobacter pylori purine nucleoside phosphorylase shows new distribution patterns of open and closed active site conformations and unusual biochemical features.

机构信息

Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Poland.

Department of Chemistry, Faculty of Science, University of Zagreb, Croatia.

出版信息

FEBS J. 2018 Apr;285(7):1305-1325. doi: 10.1111/febs.14403. Epub 2018 Feb 26.

DOI:10.1111/febs.14403

PMID:29430816

Abstract

UNLABELLED

Even with decades of research, purine nucleoside phosphorylases (PNPs) are enzymes whose mechanism is yet to be fully understood. This is especially true in the case of hexameric PNPs, and is probably, in part, due to their complex oligomeric nature and a whole spectrum of active site conformations related to interactions with different ligands. Here we report an extensive structural characterization of the apo forms of hexameric PNP from Helicobacter pylori (HpPNP), as well as its complexes with phosphate (P ) and an inhibitor, formycin A (FA), together with kinetic, binding, docking and molecular dynamics studies. X-ray structures show previously unseen distributions of open and closed active sites. Microscale thermophoresis results indicate that a two-site model describes P binding, while a three-site model is needed to characterize FA binding, irrespective of P presence. The latter may be related to the newly found nonstandard mode of FA binding. The ternary complex of the enzyme with P and FA shows, however, that P binding stabilizes the standard mode of FA binding. Surprisingly, HpPNP has low affinity towards the natural substrate adenosine. Molecular dynamics simulations show that P moves out of most active sites, in accordance with its weak binding. Conformational changes between nonstandard and standard binding modes of nucleoside are observed during the simulations. Altogether, these findings show some unique features of HpPNP and provide new insights into the functioning of the active sites, with implications for understanding the complex mechanism of catalysis of this enzyme.

DATABASES

The atomic coordinates and structure factors have been deposited in the Protein Data Bank: with accession codes 6F52 (HpPNPapo_1), 6F5A (HpPNPapo_2), 6F5I (HpPNPapo_3), 5LU0 (HpPNP_PO4), 6F4W (HpPNP_FA) and 6F4X (HpPNP_PO4_FA).

ENZYMES

Purine nucleoside orthophosphate ribosyl transferase, EC2.4.2.1, UniProtID: P56463.

摘要

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即使经过几十年的研究，嘌呤核苷磷酸化酶（PNP）仍然是一种其机制尚未完全了解的酶。对于六聚体 PNP 来说尤其如此，这可能部分是由于其复杂的寡聚性质以及与不同配体相互作用相关的一系列活性部位构象。在这里，我们报告了来自幽门螺杆菌（HpPNP）的六聚体 PNP 的 apo 形式的广泛结构特征，以及其与磷酸盐（P ）和抑制剂，形式霉素 A（FA）的复合物，以及动力学、结合、对接和分子动力学研究。X 射线结构显示了以前未见的开放和封闭活性部位的分布。微量热泳结果表明，二位点模型描述了 P 的结合，而无论 P 是否存在，三位点模型都需要描述 FA 的结合。后一种情况可能与新发现的 FA 结合的非标准模式有关。然而，酶与 P 和 FA 的三元复合物表明，P 的结合稳定了 FA 结合的标准模式。令人惊讶的是，HpPNP 对天然底物腺苷的亲和力较低。分子动力学模拟表明，P 从大多数活性部位移出，与它的弱结合一致。在模拟过程中观察到核苷的非标准和标准结合模式之间的构象变化。总的来说，这些发现显示了 HpPNP 的一些独特特征，并为活性部位的功能提供了新的见解，这对理解该酶复杂的催化机制具有重要意义。