Biosynthesis of Beta Lactam Antibiotics

项目总结/摘要 将研究四种“非经典”b-内酰胺抗生素族中的三种的生物合成。 加上第五类，即“经典的”青霉素类和头孢菌素类，这些药物占世界的60%以上。 抗生素市场和占超过250亿美元/年的经济价值。它们仍然是人类健康的重要支柱 但随着它们的广泛使用，不可避免地出现了抗药性感染。 结构上的改变减缓了这些影响，但人们越来越依赖于更新的、非经典的 例如，b-内酰胺酶抑制剂克拉维酸和强效广谱碳青霉烯类 如亚胺培南®和美罗培南®，灵感来自天然产物硫霉素。β-内酰胺类药物是有益的 趋同进化的例子，其中五个已知类别的途径显著重叠， 不同的生物合成策略，酶功能进化到新的任务和令人印象深刻的合成 效率在这三条待研究的途径中，每一条都发生了显著的、往往是前所未有的反应， 将使用从有机合成到酶学，蛋白质X射线等工具进行研究的地方 晶体学和计算机建模来了解它们的酶机制。这些知识将用于 探索它们在化学酶促功能重编程和靶向突变方面的潜力， 合成具有实用价值的抗生素变体。关于结构/活性之间的关系， 这些药物的生产成本可以通过基于工程化的发酵技术来降低， 生物合成酶和半合成酶。感兴趣的酶范围从三种钴胺素依赖性 我们现在知道，自由基S-腺苷甲硫氨酸酶是碳青霉烯生物合成的核心， 由肽生成β-内酰胺环的较大非核糖体肽合成酶（NRPS）的进化结构域 两种截然不同的方式。一个从肽丝氨酰残基通向内部的4元 环的单环b-内酰胺类抗生素，而另一种则直接与其独特的N- 磺化β-内酰胺环完全成熟。重新引起临床兴趣的是这种结构类型， 对B类或Zn++金属-B-内酰胺酶的耐药性的临床重要性质，其甚至可以超过 最有效的碳青霉烯类我们最近已经表明，单内酰胺核心的合成是在 NRPS的C末端硫酯酶（TE）结构域。我们有一个这个域的晶体结构， 模仿束缚提出了改造活性位点以容纳天然化合物的立体异构体的实验。 底物来合成不同取代的单环内酰胺。一系列生化实验， 化学交联和X射线晶体学将指导小型TE域库的工程设计 用于可能的固定化和流动技术在更大规模合成中的应用。

Project Summary/Abstract The biosynthesis of three of the four “non-classical” clans of the b-lactam antibiotics will be investigated. Together with the fifth, or “classical” penicillins and cephalosporins, these drugs constitute >60% of the world antibiotic market and account for >$25B/yr in economic value. They remain vital mainstays of human health and longevity, but with their widespread use has come the inevitable rise of antibiotic-resistant infections. Structural modifications have slowed these effects, but there is increased reliance on the newer, non-classical families; for example, the b-lactamase inhibitor clavulanic acid and the potent, broad-spectrum carbapenems like Imipenem® and Meropenem,® inspired by the natural product thienamycin. The b-lactams are instructive examples of convergent evolution where the pathways to the five known classes exemplify remarkably different biosynthetic strategies, evolution of enzyme function to new tasks and impressive synthetic efficiency. In each of the three pathways to be investigated, remarkable, often unprecedented reactions, take place that will be studied using tools ranging from organic synthesis to enzymology, protein X-ray crystallography and in silico modeling to understand their enzyme mechanisms. This knowledge will be used to explore their potential for functional reprogramming and targeted mutation for the chemo-enzymatic synthesis of antibiotic variants of practical value. Much is known about structure/activity relationships among these drugs where manufacturing costs might be reduced by fermentation technology based on engineered biosynthetic enzymes and semi-synthesis. The enzymes of interest range from three cobalamin-dependent radical S-adenosylmethionine enzymes that we know now lie at the heart of carbapenem biosynthesis, to evolved domains of larger non-ribosomal peptide synthetases (NRPSs) that create b-lactam rings from peptide precursors in two strikingly distinct ways. One leads from a peptide seryl residue to the internal 4-membered ring of monocyclic b-lactam antibiotics while the other gives monobactams directly with their distinctive N- sulfonated b-lactam rings fully fledged. Renewed clinical interest attaches to this structural class for its clinically important property of resistance to Class B, or Zn++ metallo-b-lactamases, which can overmatch even the most potent carbapenems. We have recently shown the synthesis of the monobactam core is carried out in the C-terminal thioesterase (TE) domain of a NRPS. We have a crystal structure of this domain with a substrate mimic bound. Proposed are experiments to remodel the active site to accommodate stereoisomers of the native substrate to synthesize differently substituted monobactams. A combination of biochemical experiments, chemical crosslinking and x-ray crystallography will guide the engineering of a small library of TE domains for possible immobilization and the application of flow technology for larger scale synthesis.