Romier C, Dominguez R, Lahm A, Dahl O, Suck D
European Molecular Biology Laboratory, Heidelberg, Germany.
Proteins. 1998 Sep 1;32(4):414-24.
The reaction mechanism of nuclease P1 from Penicillium citrinum has been investigated using single-stranded dithiophosphorylated di-, tetra-, and hexanucleotides as substrate analogs. The complexes crystallize in tetragonal and orthorhombic space groups and have been solved by molecular replacement. The high resolution structures give a clear picture of base recognition by P1 nuclease at its two nucleotide-binding sites, especially the 1.8 A structure of a P1-tetranucleotide complex which can be considered a P1-product complex. The observed binding modes are in agreement with a catalytic mechanism where the two closely spaced zinc ions activate the attacking water while the third, more exposed zinc ion stabilizes the leaving 03' oxyanion. Stacking as well as hydrogen bonding interactions with the base 5' to the cleaved phosphodiester bond are important elements of substrate binding and recognition. Modelling of a productive P1-substrate complex based on the solved structures suggests steric hindrance as the likely reason for the resistance of Rp-phosphorothioates and phosphorodithioates. Differences with the highly homologous nuclease S1 from Aspergillus oryzae are discussed.
利用单链二硫代磷酸化的二核苷酸、四核苷酸和六核苷酸作为底物类似物，对桔青霉核酸酶P1的反应机制进行了研究。这些复合物在四方晶系和正交晶系空间群中结晶，并通过分子置换法解析了结构。高分辨率结构清晰地展示了P1核酸酶在其两个核苷酸结合位点对碱基的识别，特别是P1 - 四核苷酸复合物的1.8 Å结构，该结构可被视为P1 - 产物复合物。观察到的结合模式与催化机制一致，即两个紧密间隔的锌离子激活进攻水，而第三个更暴露的锌离子稳定离去的03'氧阴离子。与切割的磷酸二酯键5'端碱基的堆积以及氢键相互作用是底物结合和识别的重要因素。基于解析结构对有活性的P1 - 底物复合物进行建模表明，空间位阻可能是Rp - 硫代磷酸酯和二硫代磷酸酯抗性的原因。还讨论了与米曲霉高度同源的核酸酶S1的差异。
