Biosynthesis of Beta-Lactam Antibiotics

50年来，青霉素和相关的β-内酰胺类抗生素一直是治疗感染的主要药物。 然而，与其他已知抗生素类一样，它们的有效性受到越来越多的挑战 多重耐药致病菌的增加。加强了努力以了解 抵抗机制并克服它们。通过基因操作进行结构修饰 它们的生物合成途径是一种有前途的方法， 发酵和半合成方法。β-内酰胺类抗生素研究计划的继续 在本申请中提出了生物合成。这些抗生素的四种已知类别中的三种将是 研究：（1）克拉维酸，一种有效的β-内酰胺酶抑制剂/灭活剂， 耐药来源，（2）诺卡菌素，一个单环β-内酰胺家族，以及代谢相关的 单环内酰胺类，和（3）碳青霉烯类，临床上以硫霉素及其衍生物为代表，但大多数 简单地通过碳青霉烯-2-烯-3-羧酸。表征至少一种的生物合成基因簇 这些主要群体中的每一个成员都导致了本赠款期内的快速进展， 从有机合成和酶学到分子生物学和大分子 结构方法建议通过对以下物质的机理和结构研究来追求这些发现： N2-（羧乙基）-L-精氨酸合酶，位点特异性突变以检查机制和工程 β-内酰胺合成酶的功能，合作研究克拉维胺酯铁中心的特征 合成酶，底物类似物和定点突变实验来研究其机制， 并研究克拉维酸生物合成的倒数第二步中发生的“对映异构化”。 将进行诺卡氏菌素生物合成基因的破坏和过表达以描绘该途径， 了解单环β-内酰胺形成的机制，并确定两种不寻常的 非核糖体肽合成酶。生物合成相关的单环内酰胺类抗生素的研究将是 启动。形成碳青霉烯核的三种关键蛋白的表征将在本研究中得到重要的体现。 这一组的研究，和新的实验与thienamycin将开始了解更复杂的 这个家族的成员和他们的合成工程。

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.