Mechanistic Studies on the Remarkable Epimerisations of Clavam and Carbapenem Biosynthesis

尽管存在耐药性问题，但抗生素可以说是医学上使用的最重要的分子。在世界范围内，以商业或医药标准衡量，最重要的抗生素是青霉素类和相关结构，所有这些结构都含有一个四元β-内酰胺环。β-内酰胺不仅具有青霉素的特征，而且还具有其他重要抗生素的特征，如头孢菌素。这个环对抗菌活性至关重要，因为它与细菌细胞壁中的酶发生反应，这些酶是这些抗生素的靶标。当抗生素中和它们的目标酶时，细菌不能正确地合成它们的细胞壁，因此无法生存。正如弗莱明所观察到的，尽管观察到青霉素对许多细菌是致命的，但β-内酰胺类药物实际上是由微生物产生的。事实上，天然产生的双环β-内酰胺类抗生素的结构是如此复杂和不寻常，以至于极不可能通过人工合成来发现，无论是故意的还是偶然的。几乎没有例外，所有正在使用的β-内酰胺类药物都是通过微生物直接发酵或通过发酵材料的合成修饰来生产的，因为它们从石化产品中合成的总成本太高。与所有抗生素家族一样，β-内酰胺类药物的持续使用受到耐药性的威胁。这可以采取各种形式，包括伪装目标和进化分子泵来驱逐抗生素。一种重要的抗性机制涉及到通过加水来打破β-内酰胺环的酶。这些β-内酰胺酶已经进化成高效的，因为它们的活性可能是细菌的生死攸关的问题。为了对抗β-内酰胺酶的活性，人类已经开发出新的抗生素家族，这些抗生素不太容易受到β-内酰胺酶介导的水解或实际上抑制β-内酰胺酶的活性。这些家族包括头孢菌素类和碳青霉烯类。值得注意的是，临床上有用的抑制剂都被证明是β-内酰胺类药物。其中一些化合物是非常有效的β-内酰胺酶抑制剂，但不足以单独使用抗生素，因此使用青霉素抗生素。随着细菌不断进化出更好的耐药方法，对新的抗生素和β-内酰胺酶抑制剂的需求持续存在。这种发展的一个限制是有用化合物的生产成本。在碳青霉烯的情况下，这个问题尤其严重，因为还没有开发出商业上可行的发酵方法，所以它们是通过昂贵的全合成来制备的。我们一直在研究微生物产生β-内酰胺类药物的途径。我们与其他人的工作揭示了双环β-内酰胺是通过化学上显著的酶的作用而产生的。催化四种最重要的β-内酰胺类抗生素-青霉素类、头孢菌素类、克拉维类和碳青霉烯类-两环生物合成的酶已被鉴定。为了将抗生素结合到它们的分子靶标上，这些环必须与其他化学基团一起修饰或功能化，并且它们的三维形状(立体化学)必须改变。在这项新的工作中，我们的目标是试图理解这些不寻常的反应是如何发生的，其中大多数是鲜为人知的。这项工作对于开发新的或更有效的抗生素路线具有实际意义；因为所涉及的酶催化非常不寻常的反应，我们可以预见这项工作将具有意想不到的应用。在涉及β-内酰胺环产生的酶的研究中就是这样的情况，事实证明，这对动物细胞对低氧浓度做出反应的机制以及在癌症和心脏病中的应用具有广泛的影响。

Despite problems of resistance, antibiotics are arguably the most important molecules used in medicine. Worldwide the most important antibiotics, by commercial or medicinal standards, are the penicillins and related structures all of which contain a four membered beta-lactam ring. The beta-lactam characterises not only the penicillins but also other important antibiotics such as the cephalosporins. This ring is vital for antibacterial activity since it reacts with the enzymes located in the bacterial cell wall that are the targets of these antibiotics. When the antibiotics neutralise their target enzymes the bacteria cannot properly synthesise their cell walls and so cannot survive. As Fleming observed, despite the observations that penicillins are lethal to many bacteria, beta-lactams are actually produced by microorganisms. In fact the structures of the naturally occurring bicyclic beta-lactam antibiotics are so complex and unusual that it is highly improbable they would be discovered by human synthesis either by design or chance. With few exceptions all the beta-lactams in use are produced either by direct fermentation of microorganisms or by synthetic modification of fermented materials, as their total synthesis from petrochemicals is too expensive. As with all antibiotic families the continued use of beta-lactams is threatened by resistance. This can take various forms including camouflage of the target and the evolution of molecular pumps to expel the antibiotics. One important resistance mechanism involves enzymes that break the beta-lactam ring by addition of water. These beta-lactamases have evolved to be highly efficient as their activity can be a matter of life or death for bacteria. To counter the activity of beta-lactamases humans have developed new families of antibiotics that are less susceptible to beta-lactamase mediated hydrolysis or actually inhibit beta-lactmase activity. Such families include the cephalosporins and the carbapenems. Remarkably the clinically useful inhibitors have themselves all turned out to be beta-lactams. Some of these compounds were very potent beta-lactamase inhibitors but were not powerful enough antibiotics for sole use, so are formulated with a penicillin antibiotic. There is an ongoing need for new antibiotics and beta-lactamase inhibitors as bacteria continually evolve better methods of resistance. A limitation in this development is the production costs of useful compounds. In the case of the carbapenems this problem is particularly acute as no method for their commercially viable fermentation has been developed, so they are prepared by expensive total synthesis. We have been studying the routes by which microorganisms make beta-lactams. Our work together with that of others has revealed that bicyclic beta-lactams are produced by the action of chemically remarkable enzymes. The enzymes that catalyse the biosynthesis of the two rings of the four most important groups of beta-lactams, the penicillins, the cephalosporins, the clavams and the carbapenems, have been identified. In order to bind the antibiotics to their molecular targets these rings have to be both modified or functionalised with other chemical groups and their three dimensional shape (stereochemistry) has to be changed. In the new work we aim to attempt to understand how these unusual reactions, most of which are poorly understood, occur. The work is of practical significance in terms of developing new or more efficient routes to antibiotics; because the enzymes involved catalyse highly unusual reactions we envisage that the work will have unenvisaged applications. This was the case in work on the enzymes involved in the production of the beta-lactam rings which has turned out to have widespread implications for work on the mechanism by which animal cells respond to low oxygen concentrations and has applications in cancer and heart disease.