Mechanistic enzymology of beta-lactam antibiotic resistance mechanisms and target proteins

丝氨酸β-内酰胺酶(SBLS)是一种细菌酶，可降解β-内酰胺类抗生素(如青霉素和头孢菌素)，导致抗生素耐药性。我们的研究项目致力于了解SBLS的催化机制，并表征SBLS如何进化来降解新的β-内酰胺类药物。大多数SBL不能降解碳青霉烯类抗生素，碳青霉烯类抗生素是一种重要的β-内酰胺类抗生素，碳青霉烯类结构的几个特征被认为干扰了SBL的催化作用。然而，近年来，人们已经发现了能有效降解碳青霉烯类抗生素的新的SBL。 我们将研究这些SBL碳青霉烯酶如何降解碳青霉烯类抗生素，重点是涉及碳青霉烯羟乙基侧链和吡咯环的相互作用。结合分子生物学和生物化学技术，我们将使用19F核磁共振波谱来研究这些酶的催化机理的动态方面。基于我们最近的工作，我们已经确定了可能抑制SBL碳青霉烯酶的碳青霉烯类衍生物。我们将测试这些衍生物作为SBL抑制剂，使用19F核磁共振来表征这些相互作用的机理细节。除了拟议的机制和抑制研究外，我们还将研究SBLS可能是如何进化成碳青霉烯酶活性的，并测试微生物组和环境样品中可能编码的SBL是否具有碳青霉烯酶活性。 本研究旨在明确SBL碳青霉烯酶的催化机理，为开发新型耐SBL催化降解的β-内酰胺类抗生素奠定基础。我们在这项工作中开发的19F核磁共振方法将使未来对SBLS降解其他β-内酰胺类药物(例如头孢菌素)的机理研究成为可能，并且可以很容易地应用于其他酶-底物系统。更广泛地说，我们提议的研究计划为研究酶如何进化以克服抑制剂提供了一个极好的模型，这是农业和兽医中具有重要经济意义的领域(例如，抗除草剂杂草的进化，感染牲畜的抗生素耐药细菌的进化)。详细的机理研究，如本文提出的那些，被证明是未来缓蚀剂开发的重要考虑因素。 除了使专注于抗生素和抑制剂开发的学术和工业努力受益外，该研究项目还将通过为8名高素质人员(1名博士、2名硕士和5名USRA学生)提供宝贵和抢手的化学、生物化学和微生物学研究技能，使加拿大科学界受益。这将为未来的加拿大科学领袖提供一个强大的研究基础，他们可以在此基础上促进未来的自然科学研究和发展。

Serine beta-lactamases (SBLs) are bacterial enzymes that degrade beta-lactam antibiotics (e.g., penicillins and cephalosporins), contributing to antibiotic resistance. Our research program is dedicated to understanding the catalytic mechanisms of the SBLs, and characterizing how SBLs evolve to degrade new beta-lactams. Most SBLs cannot degrade carbapenems, an important class of beta-lactam antibiotic, and several features of the carbapenem structure are thought to interfere with SBL catalysis. However, in recent years, new SBLs have been identified that efficiently degrade carbapenems. We will investigate how these SBL carbapenemases degrade carbapenems, focusing on interactions involving the carbapenem hydroxyethyl side chain and pyrroline ring. In combination with molecular biology and biochemistry techniques, we will use 19F NMR spectroscopy to study dynamic aspects of the catalytic mechanisms of these enzymes. Based on our recent work, we have identified carbapenem derivatives that are likely to inhibit SBL carbapenemases. We will test these derivatives as SBL inhibitors, using 19F NMR to characterize the mechanistic details of these interactions. In addition to the proposed mechanistic and inhibition studies, we will examine how SBLs might have evolved to develop carbapenemase activity, and test whether putative SBLs encoded in microbiome and environmental samples possess carbapenemase activity. Our research aims to define the mechanisms of SBL carbapenemase catalysis, which will assist the development of new beta-lactam antibiotics that are resistant to SBL-catalyzed degradation. The 19F NMR methods that we develop during the course of this work will enable future mechanistic studies on the degradation of other beta-lactams (e.g., cephalosporins) by SBLs, and can be readily applied to other enzyme-substrate systems. More generally, our proposed research program provides an excellent model for investigating how enzymes evolve to overcome inhibitors, an area of great economic importance in agriculture and veterinary medicine (e.g., evolution of herbicide-resistant weeds, antibiotic-resistant bacteria infecting livestock). Detailed mechanistic studies, such as those proposed herein, are proving to be an essential consideration for future inhibitor development. In addition to benefitting academic and industrial efforts focused on antibiotic and inhibitor development, this research program will benefit the Canadian scientific community by providing 8 highly qualified personnel (1 PhD, 2 MSc, and 5 USRA students) with valuable and sought-after research skills in chemistry, biochemistry and microbiology. This will provide future Canadian scientific leaders with a strong research base from which they can promote future research and development in the natural sciences.