Novel enzymology in bacterial secondary metabolic pathways

二级代谢是不寻常酶学的真正宝库，黄素依赖酶催化了一系列令人惊讶的氧化还原反应，从而促进了天然产物丰富的结构多样性。然而，尽管经过了几十年的深入研究，我们对黄素辅助因子的化学多样性和反应性还只是部分了解。我的研究项目旨在阐明海洋链霉菌细菌中高度不寻常的抗生素灰黄素A的黄素依赖生物合成。与红霉素多酮家族的其他成员一样，灰黄素A对临床重要的酶HIV逆转录酶和人类端粒酶表现出显著的生物活性。因此，红霉素被认为是医学应用药物工程的潜在先导结构。值得注意的是，一个对生物活性至关重要的独特螺旋形部分引起了平面红霉素结构的剧烈扭曲。依赖黄素的酶最有可能通过高度复杂的碳主链重排来组装这些螺旋状药物载体。为了研究这一生物合成壮举，我将使用异种生产的酶在体外完全重建灰黄素A的生物合成途径，这将允许识别所有的酶反应、途径中间体和未表征的灰黄素类似物。在完成功能分配后，将对关键酶进行机械和结构表征。对这种前所未有的氧化还原化学的深入了解可能会使生物合成途径的生物工程成为可能，从而开发具有改进药理特性的灰芹素类似物。我预计我的研究将对扩展我们对化学反应如何在自然界中被控制的机制的理解产生深远的影响，这些知识对合成生物学的新兴领域至关重要。由于无数的生命过程依赖于黄素辅助因子的催化作用，新的黄素生物化学的发现可能最终揭示细胞生物学和生理学的各个方面。

Secondary metabolism is a true treasure trove for unusual enzymology as exemplified by flavin-dependent enzymes that catalyze a wide array of astonishing redox reactions and thereby contribute to the rich structural diversity of natural products. Yet, despite decades of intensive studies, we only partially comprehend the chemical versatility and reactivity of the flavin cofactor. My research program aims to elucidate the flavin-dependent biosynthesis of the highly unusual antibiotic griseorhodin A from marine Streptomyces bacteria. Like other members of the rubromycin family of polyketides, griseorhodin A exhibits significant bioactivity against the clinically important enzymes HIV reverse transcriptase and human telomerase. Rubromycins were consequently suggested as potential lead structures for the engineering of drugs for medical application. Notably, a drastic distortion of the otherwise planar rubromycin structure is caused by a unique spiroketal moiety that is crucial for the bioactivity. Flavin-dependent enzymes most likely assemble these spiroketal pharmacophores through highly complex rearrangements of the carbon backbone. To investigate this biosynthetic feat, I will employ heterologously produced enzymes to fully reconstitute the griseorhodin A biosynthetic pathway in vitro, which should allow for the identification of all enzymatic reactions, pathway intermediates, and uncharacterized griseorhodin analogues. Subsequent to their functional assignment, the key enzymes will be mechanistically and structurally characterized. An in-depth knowledge of this unprecedented redox-chemistry may then enable the bioengineering of the biosynthetic pathway in order to develop griseorhodin analogues with improved pharmacological features. I anticipate that my studies will have a profound impact on extending our mechanistic understanding of how chemical reactivity is controlled in nature, knowledge that is paramount to the emerging field of synthetic biology. As countless life processes depend on flavin cofactor catalysis, discovery of novel flavin biochemistry may ultimately shed light into various aspects of cell biology and physiology.