Accelerated evolution of enzyme activity to determine functional and structural tolerance

由于酶的高催化能力和酶工程修饰的潜力，酶的工业应用在世界范围内正变得越来越重要。我们的目标是通过确定特定酶对密集的、有针对性的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部位突变表现出不同的耐受性。在这里，我们建议研究两个不同的、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之后，我们将表征功能变体，以建立它们对密集的靶向突变的耐受性。 正在研究的第一个系统由临床相关的β-内酰胺酶组成，这种酶能分解青霉素等抗生素。当务之急是更好地定义这种快速演变的抗生素耐药性的分子性质。人工设计的具有催化活性的内酰胺酶嵌合体将通过动力学、圆二色谱和蛋白质核磁共振进行表征。这些实验室产生的β-内酰胺酶是一种观察对高突变负荷耐受性的原创和高效系统。深入了解这些人工进化而又具有功能的酶的结构-功能关系将为了解耐药性的快速发展提供洞察，同时为有效的酶工程建立更大的知识库。第二个系统是R67二氢叶酸还原酶，这是一种使细菌对抗生素甲氧普林产生抗药性的酶。我们将确定结合支架，用于开发这种最近进化的药物靶点，以发现新的抗生素。同时，我们将对该酶的活性部位进行修饰，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。随着新型生物催化剂的发展，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.