Department of Chemistry, Rutgers University, Newark, New Jersey 07102, United States.
Biochemistry. 2012 Feb 28;51(8):1648-57. doi: 10.1021/bi201625w. Epub 2012 Feb 13.
The opportunistic pathogen Burkholderia thailandensis produces a number of structurally similar unsaturated quinolones involved in quorum sensing. However, little is known about the biosynthesis of these unsaturated quinolones. In this study, we have characterized the starting point of the biosynthesis of unsaturated quinolone molecules produced in B. thailandensis. We have shown by using in vitro enzymology, liquid chromatography, and mass spectrometry that protein HmqF is involved in the biosynthesis of unsaturated quinolones produced by B. thailandensis. HmqF consists of three domains: an adenylation domain (A domain), a dehydrogenase domain (DH domain), and an acyl carrier domain (ACP). The three domains (A, DH, and ACP) were cloned and expressed individually in Escherichia coli, and their reactivity was studied using high-performance liquid chromatography (HPLC) and mass spectrometry (MS) based assays. Our in vitro studies show that the A domain catalyzes ATP-dependent activation of medium chain (C6-C14) fatty acids without activation by coenzyme A (CoA). Results from competition assays are consistent with decanoic acid being the preferred substrate. Incubation of the ACP domain with 4'-phosphopantetheine transferase and CoA led to the formation of phosphopantetheinylated ACP (Ppant-ACP). In a Ppant ejection assay using tandem MS (MS/MS), a mass consistent with the mass of a cyclic variant of dephosphorylated Ppant was detected. We further demonstrated that Ppant-ACP could be loaded with medium chain fatty acids in the presence of ATP and the A domain. MS analysis was consistent with the formation of Ppant-ACP thiol esters of the fatty acids. MS/MS Ppant ejection experiments confirmed the loss of 2H in samples of fatty acid-loaded Ppant-ACP in the presence of the DH domain. HPLC analysis of benzyl amide ligation products allowed us to conclude that dehydrogenation produced trans-β,γ-unsaturation in the fatty acid chains. Our results are in good agreement with naturally observed quinolone molecules produced by B. thailandensis, which predominately produce nine-carbon trans-β,γ-unsaturated alkyl chain quinolone molecules.
机会性病原体伯克霍尔德菌产生了许多结构相似的不饱和喹诺酮类物质，这些物质参与了群体感应。然而，关于这些不饱和喹诺酮类物质的生物合成知之甚少。在本研究中，我们已经确定了伯克霍尔德菌产生的不饱和喹诺酮类分子生物合成的起始点。通过使用体外酶学、液相色谱和质谱，我们证明蛋白 HmqF 参与了伯克霍尔德菌产生的不饱和喹诺酮类物质的生物合成。HmqF 由三个结构域组成：一个腺苷酰化结构域 (A 结构域)、一个脱氢结构域 (DH 结构域) 和一个酰基载体结构域 (ACP)。这三个结构域 (A、DH 和 ACP) 分别在大肠杆菌中进行了克隆和表达，并使用高效液相色谱 (HPLC) 和基于质谱 (MS) 的测定方法研究了它们的反应性。我们的体外研究表明，A 结构域催化 ATP 依赖性激活中链 (C6-C14) 脂肪酸，而无需辅酶 A (CoA) 激活。竞争测定的结果与癸酸是首选底物一致。将 ACP 结构域与 4'-磷酸泛酰巯基乙胺转移酶和 CoA 孵育，导致磷酸泛酰巯基乙胺化 ACP (Ppant-ACP) 的形成。在使用串联 MS (MS/MS) 的 Ppant 逐出测定中，检测到与去磷酸化 Ppant 的环状变体质量一致的物质。我们进一步证明，在 ATP 和 A 结构域的存在下，Ppant-ACP 可以与中链脂肪酸结合。MS 分析与脂肪酸负载的 Ppant-ACP 的 Ppant-ACP 硫酯的形成一致。MS/MS Ppant 逐出实验证实，在 DH 结构域存在下，负载脂肪酸的 Ppant-ACP 样品中丢失了 2H。苄基酰胺连接产物的 HPLC 分析使我们能够得出结论，脱氢反应在脂肪酸链中产生了 trans-β,γ-不饱和。我们的结果与伯克霍尔德菌产生的天然观察到的喹诺酮类分子非常吻合，这些分子主要产生九碳 trans-β,γ-不饱和烷基链喹诺酮类分子。
