Crystal Structures of T. b. rhodesiense Adenosine Kinase Complexed with Inhibitor and Activator: Implications for Catalysis and Hyperactivation

BackgroundThe essential purine salvage pathway of Trypanosoma brucei bears interesting catalytic enzymes for chemotherapeutic intervention of Human African Trypanosomiasis. Unlike mammalian cells, trypanosomes lack de novo purine synthesis and completely rely on salvage from their hosts. One of the key enzymes is adenosine kinase which catalyzes the phosphorylation of ingested adenosine to form adenosine monophosphate (AMP) utilizing adenosine triphosphate (ATP) as the preferred phosphoryl donor. Methods and FindingsHere, we present the first structures of Trypanosoma brucei rhodesiense adenosine kinase (TbrAK): the structure of TbrAK in complex with the bisubstrate inhibitor P1,P5-di(adenosine-5′)-pentaphosphate (AP5A) at 1.55 Å, and TbrAK complexed with the recently discovered activator 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (compound 1) at 2.8 Å resolution. ConclusionsThe structural details and their comparison give new insights into substrate and activator binding to TbrAK at the molecular level. Further structure-activity relationship analyses of a series of derivatives of compound 1 support the observed binding mode of the activator and provide a possible mechanism of action with respect to their activating effect towards TbrAK.背景 布氏锥虫（Trypanosoma brucei）的核心嘌呤补救通路中存在具备人非洲锥虫病（Human African Trypanosomiasis）化疗干预潜力的关键催化酶。与哺乳动物细胞不同，锥虫自身缺乏从头嘌呤合成途径，完全依赖宿主获取嘌呤补救所需的物质。其中关键酶之一为腺苷激酶（adenosine kinase），其以三磷酸腺苷（adenosine triphosphate, ATP）作为优选磷酸供体，催化摄取的腺苷发生磷酸化反应生成一磷酸腺苷（adenosine monophosphate, AMP）。 方法与结果 本研究首次解析了罗得西亚布氏锥虫腺苷激酶（Trypanosoma brucei rhodesiense adenosine kinase, TbrAK）的晶体结构：其一为TbrAK与双底物抑制剂P1,P5-二(腺苷-5′)-五磷酸（AP5A）结合的复合物结构，分辨率达1.55 Å；其二为TbrAK与最新发现的激活剂4-[5-(4-苯氧基苯基)-2H-吡唑-3-基]吗啉（化合物1）结合的复合物结构，分辨率为2.8 Å。 结论 上述结构细节及其对比分析，从分子层面为阐明TbrAK与底物、激活剂的结合机制提供了全新认知。后续针对化合物1的一系列衍生物开展的构效关系分析，验证了该激活剂的结合模式，并为其激活TbrAK的潜在作用机制提供了理论依据。