Biosynthesis of Beta Lactam Antibiotics

β内酰胺抗生素的生物合成

基本信息

批准号：
10601097
负责人：
CRAIG ARTHUR TOWNSEND
金额：
$ 61.94万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-02-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10601097
关键词：
Acids Active Sites Alkanesulfonates Anabolism Antibiotics Appearance Bacterial Antibiotic Resistance Bacterial Infections Binding Biochemical C-terminal Carbapenems Carbon Catalysis Cephalosporins Chemicals Clavulanic Acids Clinic Clinical Cobalamin Complex Coupled Drug resistance Engineering Enzymatic Biochemistry Enzymes Evolution Family Fermentation Genes Goals Grant Health Healthcare Heart Human Imipenem Immobilization Individual Investigation Isotope Labeling Kinetics Knowledge Lactamase Lactams Libraries Longevity Maps Marketing Medicine Meropenem Methylation Methyltransferase Modeling Modification Monobactams Mutate Mutation Nature Organic Synthesis Orthologous Gene Pantetheine Pathway interactions Penicillins Peptides Pharmaceutical Preparations Physical condensation Process Property Proteins Reaction Resistance Roentgen Rays Role S-Adenosylhomocysteine S-Adenosylmethionine Scheme Series Site Source Stereoisomer Structure System Technology Thienamycins Variant X-Ray Crystallography antibiotic resistant infections azetidinone bacterial resistance beta-Lactamase beta-Lactams cost crosslink economic value enzyme mechanism epimerization experimental study improved in silico inhibitor interest manufacturing cost member methyl group multidisciplinary natural product inspired nocardicin nocardicin A novel therapeutics peptide synthase programs resistance mechanism stoichiometry sulfotransferase tool

项目摘要

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.

项目摘要/摘要将研究B-内酰胺抗生素的四个“非古典”氏族中三个中的三个生物合成。这些药物与第五或“古典”青霉素和头孢菌素一起，占世界上> 60％的占60％抗生素市场，经济价值估计$ 25B/年。它们仍然是人类健康的重要支柱寿命和寿命，但使用它们的宽度是抗生素耐药性感染的必然崛起。结构修饰减慢了这些影响，但对较新的，非古典的保留率增加了家庭；例如，B-内酰胺酶抑制剂克拉维烯酸和有效的，宽光谱的碳青霉烯酸像Imipenem®和MeropeNem一样，灵感来自天然产物硫霉素。 B-内酰胺很有启发性收敛演化的示例，其中五个已知类别的途径显着举例说明不同的生物合成策略，酶功能到新任务的发展以及令人印象深刻的合成效率。在要研究的三种途径中的每一个中将使用从有机合成到酶学，蛋白质X射线的工具进行研究的地方晶体学和计算机建模，以了解其酶机制。这些知识将被使用探索其用于化学酶的功能重编程和靶向突变的潜力实用价值的抗生素变体的合成。关于之间的结构/活动关系知之甚少这些药物可以通过基于工程的发酵技术来降低制造成本生物合成酶和半合成。感兴趣的酶范围从三个钴胺素依赖我们知道的根治性的S-腺苷甲硫代酶是碳青霉生物合成的核心，较大的非核糖体肽合成酶（NRPSS）的进化结构域，这些酶（NRPSS）从肽中产生B-内酰胺环前体以两种明显的不同方式。一个人从胡椒塞里尔居住到内部4元单核B-内酰胺抗生素的环，而另一种是直接赋予单摩酰胺，其独特的n- 磺化的B-内酰胺环完全露出。此结构类别的重新临床兴趣附有其对B类抗性的临床重要特性，或Zn ++ Metallo-B-内乳酶，这甚至可以超越最有效的碳青霉烯。我们最近显示了Monobactam核心的合成 NRP的C末端硫酯酶（TE）结构域。我们具有该域的晶体结构，带有底物模仿结合。提出的是重塑活性位点以适应天然的立体异构体的实验底物以合成不同的替代的单桥酰胺。生化实验的结合，化学交联和X射线晶体学将指导小型域的小型库的工程可能固定和流动技术在更大规模合成中的应用。