Accelerated evolution of enzyme activity to determine functional and structural tolerance

由于酶的高催化能力和酶工程改造的潜力，酶的工业应用在世界范围内越来越重要。我们的目标是通过确定特定酶对密集的靶向突变努力的耐受性来增加酶工程的范围和效率。不同的酶似乎对活性位点突变表现出不同的耐受性。在这里，我们建议研究两种不同的，不相关的酶系统。在使用强大的分子生物学方法进行广泛的修饰后，我们将表征功能变体，以确定其对密集的靶向突变的耐受性。研究的第一个系统由临床相关的β-内酰胺酶组成，其水解抗生素如青霉素。迫切需要更好地定义这种快速发展的抗生素耐药性的分子性质。将通过动力学、圆二色性和蛋白质NMR来表征β-内酰胺酶的生物工程化的、催化活性的嵌合体。这些实验室产生的β-内酰胺酶是用于观察对高突变负荷的耐受性的原始且高效的系统。深入了解这些人工进化的功能酶的结构-功能关系将为耐药性的快速发展提供深入了解，同时为有效的酶工程建立更大的知识基础。第二个系统是R67二氢叶酸还原酶，一种提供细菌对抗生素甲氧苄啶抗性的酶。我们将确定抑制剂开发的结合支架，以这种最近演变的药物靶点，朝着发现新的抗生素。同时，我们将修改这种酶的活性位点，以开发新的生物催化剂，用于还原合成有用的前手性亚胺和酮。比较这些系统的突变耐受性和由此产生的结构-功能效应将有助于提高我们在酶工程方面的能力。沿着新的生物催化剂的开发，这一知识将服务于重要的工业需求。

The industrial application of enzymes is gaining in importance world-wide, as a result of their high catalytic power and the potential for modification by enzyme engineering. Our goal is to increase the scope and efficiency of enzyme engineering, by determining the tolerance of specific enzymes to intensive, targeted mutational efforts. Different enzymes appear to show different tolerance to active-site mutations. Here, we propose to study two different, unrelated enzyme systems. Following extensive modification using powerful molecular biological methodologies, we will characterize the functional variants to establish their tolerance to intensive, targeted mutations. The first system under study consists of clinically-relevant ß-lactamases which hydrolyze antibiotics such as penicillins. It is urgent to better define the molecular nature of such rapidly evolving antibiotic resistance. Artificially engineered, catalytically active chimeras of ß-lactamases will be characterized by kinetics, circular dichroism and protein NMR. These laboratory-generated ß-lactamases are an original and highly effective system for observing tolerance to high mutation load. Insight into the structure-function relationship of these artificially evolved yet functional enzymes will provide insight into the rapid development of drug resistance, while establishing a greater knowledge base for effective enzyme engineering. The second system is R67 dihydrofolate reductase, an enzyme that provides bacterial resistance to the antibiotic trimethoprim. We will identify binding scaffolds for inhibitor development to this recently evolved drug target, toward discovery of new antibiotics. In parallel, we will modify the active site of this enzyme to develop new biocatalysts for reduction of synthetically-useful prochiral imines and ketones. Comparison of the mutational tolerance and the resulting structure-function effects of these systems will contribute to advancing our capacity in enzyme engineering. Along with the development of new biocatalysts, this knowledge will serve an important industrial need.