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中最重要的分子。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及其相关结构，它们都含有一个四元β -内酰胺环。-内酰胺不仅是青霉素类的特征，也是其他重要抗生素如头孢菌素的特征。这个环对抗菌活性至关重要，因为它与细菌细胞壁上的酶发生反应，这些酶是这些抗生素的目标。当抗生素中和它们的目标酶时，细菌就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那样，尽管观察到青霉素对许多细菌是致命的，但β -内酰胺实际上是由微生物产生的。事实上，自然产生的双环-内酰胺类抗生素的结构是如此复杂和不寻常，以至于它们不太可能被设计或偶然地通过人类合成发现。除了少数例外，所有使用的β -内酰胺都是通过微生物的直接发酵或通过发酵材料的合成改性来生产的，因为从石化产品中完全合成它们太昂贵了。与所有抗生素家族一样，β -内酰胺类抗生素的持续使用受到耐药性的威胁。这可以采取多种形式，包括伪装目标和分子泵的进化，以驱逐抗生素。一种重要的抗性机制涉及通过加水打破-内酰胺环的酶。这些内酰胺酶已经进化得非常高效，因为它们的活性对细菌来说可能是生死攸关的问题。为了对抗β -内酰胺酶的活性，人类开发了新的抗生素家族，这些家族对β -内酰胺酶介导的水解不太敏感，或者实际上抑制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本身都是β -内酰胺。这些化合物中的一些是非常有效的-内酰胺酶抑制剂，但不足以作为单一使用的抗生素，因此与青霉素抗生素一起配制。随着细菌不断进化出更好的耐药方法，对新型抗生素和β -内酰胺酶抑制剂的需求不断增加。这种发展的一个限制是有用化合物的生产成本。在碳青霉烯类的情况下，这个问题特别严重，因为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制造内酰胺的途径。我们和其他人一起的工作揭示了双环β -内酰胺是由化学上显著的酶的作用产生的。催化四种最重要的内酰胺类——青霉素类、头孢菌素类、克拉霉素类和碳青霉烯类——的两个环的生物合成的酶已经被确定。为了将抗生素与它们的分子靶标结合，这些环必须被其他化学基团修饰或功能化，并且必须改变它们的三维形状（立体化学）。在这项新工作中，我们的目标是试图理解这些不寻常的反应是如何发生的，其中大多数是人们知之甚少的。这项工作在开发新的或更有效的抗生素途径方面具有实际意义；因为所涉及的酶催化非常不寻常的反应，我们设想这项工作将有意想不到的应用。在研究与生产β -内酰胺环有关的酶的工作中就是这种情况，这对研究动物细胞对低氧浓度反应的机制有广泛的影响，并在癌症和心脏病方面有应用。

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.