Matsui Takashi, Kodama Takeshi, Mori Takahiro, Tadakoshi Tetsuhiro, Noguchi Hiroshi, Abe Ikuro, Morita Hiroyuki
From the Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194.
the Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, and.
J Biol Chem. 2017 Jun 2;292(22):9117-9135. doi: 10.1074/jbc.M117.778977. Epub 2017 Apr 14.
2-Alkylquinolone (2AQ) alkaloids are pharmaceutically and biologically important natural products produced by both bacteria and plants, with a wide range of biological effects, including antibacterial, cytotoxic, anticholinesterase, and quorum-sensing signaling activities. These diverse activities and 2AQ occurrence in vastly different phyla have raised much interest in the biosynthesis pathways leading to their production. Previous studies in plants have suggested that type III polyketide synthases (PKSs) might be involved in 2AQ biosynthesis, but this hypothesis is untested. To this end, we cloned two novel type III PKSs, alkyldiketide-CoA synthase (ADS) and alkylquinolone synthase (AQS), from the 2AQ-producing medicinal plant, (Rutaceae). Functional analyses revealed that collaboration of ADS and AQS produces 2AQ via condensations of -methylanthraniloyl-CoA, a fatty acyl-CoA, with malonyl-CoA. We show that ADS efficiently catalyzes the decarboxylative condensation of malonyl-CoA with a fatty acyl-CoA to produce an alkyldiketide-CoA, whereas AQS specifically catalyzes the decarboxylative condensation of an alkyldiketide acid with -methylanthraniloyl-CoA to generate the 2AQ scaffold via C-C/C-N bond formations. Remarkably, the ADS and AQS crystal structures at 1.80 and 2.20 Å resolutions, respectively, indicated that the unique active-site architecture with Trp-332 and Cys-191 and the novel CoA-binding tunnel with Tyr-215 principally control the substrate and product specificities of ADS and AQS, respectively. These results provide additional insights into the catalytic versatility of the type III PKSs and their functional and evolutionary implications for 2AQ biosynthesis in plants and bacteria.
2-烷基喹诺酮（2AQ）生物碱是细菌和植物产生的具有药学和生物学重要性的天然产物，具有广泛的生物学效应，包括抗菌、细胞毒性、抗胆碱酯酶和群体感应信号活性。这些多样的活性以及2AQ在截然不同的生物门类中的存在，引发了人们对其生物合成途径的浓厚兴趣。此前在植物中的研究表明，III型聚酮合酶（PKSs）可能参与2AQ的生物合成，但这一假设尚未得到验证。为此，我们从产2AQ的药用植物（芸香科）中克隆了两个新型III型PKSs，即烷基二酮酰辅酶A合酶（ADS）和烷基喹诺酮合酶（AQS）。功能分析表明，ADS和AQS协同作用，通过将脂肪酰辅酶A -甲基邻氨基苯甲酰辅酶A与丙二酰辅酶A缩合产生2AQ。我们发现ADS能高效催化丙二酰辅酶A与脂肪酰辅酶A的脱羧缩合反应，生成烷基二酮酰辅酶A，而AQS则特异性催化烷基二酮酸与 -甲基邻氨基苯甲酰辅酶A的脱羧缩合反应，通过形成碳-碳/碳-氮键生成2AQ骨架。值得注意的是，分别在1.80 Å和2.20 Å分辨率下解析得到的ADS和AQS晶体结构表明，具有色氨酸-332和半胱氨酸-191的独特活性位点结构以及具有酪氨酸-215的新型辅酶A结合通道，分别主要控制着ADS和AQS的底物和产物特异性。这些结果为III型PKSs的催化多样性及其对植物和细菌中2AQ生物合成的功能和进化意义提供了更多见解。
