Keppetipola Niroshika, Shuman Stewart
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
J Biol Chem. 2006 Jul 14;281(28):19251-9. doi: 10.1074/jbc.M602549200. Epub 2006 May 4.
The central phosphatase domain of Clostridium thermocellum polynucleotide kinase/phosphatase (CthPnkp) belongs to the dinuclear metallophosphoesterase superfamily. Prior mutational studies of CthPnkp identified 7 individual active site side chains (Asp-187, His-189, Asp-233, Asn-263, His-323, His-376, and Asp-392) required for Ni2+-dependent hydrolysis of p-nitrophenyl phosphate. Here we find that Mn2+-dependent phosphomonoesterase activity requires two additional residues, Arg-237 and His-264. We report that CthPnkp also converts bis-p-nitrophenyl phosphate to p-nitrophenol and inorganic phosphate via a processive two-step mechanism. The Ni2+-dependent phosphodiesterase activity of CthPnkp requires the same seven side chains as the Ni2+-dependent phosphomonoesterase. However, the Mn2+-dependent phosphodiesterase activity does not require His-189, Arg-237, or His-264, each of which is critical for the Mn2+-dependent phosphomonoesterase. Mutations H189A, H189D, and D392N transform the metal and substrate specificity of CthPnkp such that it becomes a Mn2+-dependent phosphodiesterase. The H189E change results in a Mn2+/Ni2+-dependent phosphodiesterase. Mutations H376N, H376D, and D392E convert the enzyme into a Mn2+-dependent phosphodiesterase-monoesterase. The phosphodiesterase activity is strongly stimulated compared with wild-type CthPnkp when His-189 is changed to Asp, Arg-237 is replaced by Ala or Gln, and His-264 is replaced by Ala, Asn, or Gln. Steady-state kinetic analysis of wild-type and mutated enzymes illuminates the structural features that affect substrate affinity and kcat. Our results highlight CthPnkp as an "undifferentiated" diesterase-monoesterase that can evolve toward narrower metal and substrate specificities via alterations of the active site milieu.
嗜热栖热放线菌多核苷酸激酶/磷酸酶（CthPnkp）的中央磷酸酶结构域属于双核金属磷酸二酯酶超家族。先前对CthPnkp的突变研究确定了7个单独的活性位点侧链（Asp-187、His-189、Asp-233、Asn-263、His-323、His-376和Asp-392），它们是镍离子依赖性水解对硝基苯磷酸酯所必需的。在此，我们发现锰离子依赖性磷酸单酯酶活性还需要另外两个残基，即Arg-237和His-264。我们报告称，CthPnkp还通过一种连续的两步机制将双对硝基苯磷酸酯转化为对硝基苯酚和无机磷酸。CthPnkp的镍离子依赖性磷酸二酯酶活性所需的七个侧链与镍离子依赖性磷酸单酯酶相同。然而，锰离子依赖性磷酸二酯酶活性并不需要His-189、Arg-237或His-264，而这三个残基对锰离子依赖性磷酸单酯酶而言都是至关重要的。H189A、H189D和D392N突变改变了CthPnkp的金属和底物特异性，使其成为一种锰离子依赖性磷酸二酯酶。H189E变化导致产生一种锰离子/镍离子依赖性磷酸二酯酶。H376N、H376D和D392E突变将该酶转变为一种锰离子依赖性磷酸二酯酶 - 单酯酶。当His-189变为Asp、Arg-237被Ala或Gln取代以及His-264被Ala、Asn或Gln取代时，与野生型CthPnkp相比，磷酸二酯酶活性受到强烈刺激。对野生型和突变型酶的稳态动力学分析揭示了影响底物亲和力和催化常数（kcat）的结构特征。我们的结果突出了CthPnkp作为一种“未分化”的二酯酶 - 单酯酶，它可以通过改变活性位点环境朝着更窄的金属和底物特异性方向进化。
