Biosynthesis of Beta-Lactam Antibiotics

青霉素和相关的β-内酰胺类抗生素50年来一直是治疗感染的中流砥柱。 然而，与其他已知的抗生素一样，它们的有效性也受到了越来越多的挑战 来自多重耐药病原菌的兴起。已加紧努力了解 抗药性的机制并克服它们。通过遗传操作进行的结构修饰 它们的生物合成途径是一种有希望的方法，通过经济有效的方式生产已知抗生素的变种。 发酵法和半合成法。继续开展研究β-内酰胺类抗生素的计划 生物合成就是在这一应用中提出的。已知的四类抗生素中的三类将是 研究：(1)克拉维酸，一种有效的β-内酰胺酶抑制剂/失活剂，广泛存在于 耐药性的来源，(2)诺卡菌素，一类单环β-内酰胺类药物，以及与代谢有关的 单内酰胺类，以及(3)碳青霉烯类抗生素，临床上以硫代霉素及其衍生物为代表，但大多数 简单地说就是碳烯-2-EM-3-羧酸。至少一个生物合成基因簇的特征 这些主要群体中的每一个成员在当前赠款期间取得了快速进展，使用 从有机合成和酶学到分子生物学和大分子的技术 结构方法。建议通过机械和结构研究来追求这些发现 N_2-(羧乙基)-L-精氨酸合成酶定点突变的研究及工程机理 β-内酰胺合成酶的功能，合作研究以表征克拉维酸酯的铁中心 合酶，底物类似物和定点突变实验，以研究其机制， 并研究在克拉维酸生物合成的倒数第二步中发生的“对映异构化”。 诺卡菌素生物合成基因的干扰和过度表达将被用于描绘该途径， 了解单环β-内酰胺的形成机制并确定两种不寻常的作用 非核糖体多肽合成酶。将对生物合成相关的单丁内酰胺进行研究 已启动。构成碳青霉烯核的三种关键蛋白质的特性将在 对这一组的研究，以及对硫霉素的新实验将开始了解更复杂的情况 这个家族的成员及其合成的工程学。

Penicillin and related beta-lactam antibiotics have been a mainstay in the treatment of infections for 50 years. Their effectiveness, however, like other known classes of antibiotics has come under increasing challenge from the rise of multiply drug-resistant pathogenic bacteria. Efforts have intensified to understand the mechanisms of resistance and to overcome them. Structural modification through genetic manipulation of their biosynthetic pathways is a promising approach to produce variants of known antibiotics by cost-effective fermentation and semi-synthetic methods. Continuation of a program to investigate beta-lactam antibiotic biosynthesis is proposed in this application. Three of the four known classes of these antibiotics will be studied: (1) clavulanic acid, a potent inhibitor/inactivator of beta-lactamase enzymes and a wide-spread source of resistance, (2) the nocardicins, a family of monocyclic beta-lactams, and the metabolically related monobactams, and (3) the carbapenems, represented clinically by thienamycin and its derivatives, but most simply by carbapen-2-em-3-carboxylic acid. Characterization of the biosynthetic gene clusters for at least one member of each of these principal groups has led to rapid advances in the current grant period using techniques ranging from organic synthesis and enzymology to molecular biology and macromolecular structural methods. It is proposed to pursue these discoveries through mechanistic and structural studies of N2-(carboxyethyl)-L-arginine synthase, site-specific mutatagenesis to examine the mechanism and engineer the function of beta-lactam synthetase, collaborative studies to characterize the iron center of clavaminate synthase, and substrate analogue and site-directed mutatagenesis experiments to examine its mechanism, and to investigate the "enantiomerization that occurs in the penultimate step of clavulanic acid biosynthesis. Disruption and over-expression of nocardicin biosynthetic genes will be undertaken to delineate the pathway, understand the mechanism of monocyclic beta-lactam formation and determine the roles of two unusual non-ribosomal peptide synthetases. Investigation of the biosynthetically related monobactams will be initiated. Characterization of three key proteins that form the carbapenem nucleus will figure prominantly in studies of this group, and new experiments with thienamycin will be begun to understand the more complex members of this family and engineering of their synthesis.