Biosynthesis of Beta Lactam Antibiotics

项目摘要/摘要 将对四种非经典的β-内酰胺类抗生素中的三种的生物合成进行研究。 这些药物与第五种或“经典”青霉素和头孢菌素类药物一起，构成了世界60%的份额 抗生素市场，每年创造250亿美元的经济价值。它们仍然是人类健康的重要支柱 和长寿，但随着它们的广泛使用，不可避免地会出现耐药感染的增加。 结构修改减缓了这些影响，但人们更多地依赖于较新的、非经典的 家族；例如，β-内酰胺酶抑制剂克拉维酸和强效广谱碳青霉烯类 像亚胺培南®和美罗培南，®的灵感来自天然产品硫那霉素。β-内酰胺类药物很有启发性 收敛进化的例子，其中通向五个已知类别的路径显著地例证了 不同的生物合成策略，酶功能向新任务的进化和令人印象深刻的合成 效率。在要调查的三条道路中的每一条中，都有值得注意的，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 结晶学和电子计算机建模，以了解它们的酶机制。这些知识将被用来 探索它们在化学-酶功能重编程和靶向突变方面的潜力 具有实用价值的抗生素变种的合成。许多关于结构/活性关系的知识都在 这些药物的生产成本可以通过基于工程技术的发酵技术来降低 生物合成酶和半合成。感兴趣的酶包括三种依赖钴胺的酶 我们现在所知的自由基S-腺苷甲硫氨酸酶位于碳青霉烯生物合成的核心，以 更大的非核糖体多肽合成酶(NRPS)的进化结构域，从多肽产生b-内酰胺环 以两种截然不同的方式传播。一个从多肽丝氨基残基通向内部4元 一种是单环β-内酰胺类抗生素，另一种是直接给药，具有独特的N-内酰胺。 磺化β-内酰胺环完全成熟。这一结构类别重新引起了临床的兴趣，因为它 临床上对B类或锌++金属β-内酰胺酶耐药的重要特性，这种酶甚至可以超过 最有效的碳青霉烯类抗生素。我们最近表明，单菌素核心的合成是在 NRPS的C-末端硫代酯酶(TE)结构域。我们有一个带有衬底的这个区域的晶体结构 模仿的界限。建议进行实验以重塑活性部位以适应天然的立体异构体 底物合成不同取代的单内酰胺。生物化学实验的组合， 化学交联法和x射线结晶学将指导TE结构域小库的设计 可能的固定化和流动技术的应用，以实现更大规模的合成。

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.