Identification of features for enzymatic catalysis and their application towards enzyme engineering

Abstract

Enzymes are biological catalysts, without which life would not be possible. Most important reactions in nature proceed at rates many orders of magnitude greater than if they were uncatalyzed. Their ability to perform specific reactions at such great speeds makes them suitable for a number of purposes, especially in the biotechnology field. However, since enzymes are so finely tuned for their natural functions, they do not always have the desired properties for industrial applications. While there are special cases where natural enzymes are suitable, such as nitrile hydratase for the production of acrylamide, this is not typical. Enzyme engineering seeks to fill this gap, by either altering the activity of natural enzymes or the de novo design of new enzymes. In both general processes, selection of variants or mutations and the ability to screen for desired functions can be challenging and yet will be essential for success. Altering the natural activity of an enzyme is most often done through mutations at or near the active site, which is where the reaction occurs. While these mutations are more likely to influence the function, this can often be detrimental to the enzyme's activity by disrupting the catalytic or functional residues. This is only exacerbated by the often extensive nature of many active sites where distal residues are essential for catalysis. Due to limitations in our understanding of enzyme active sites and how they perform catalysis, the development of new functions often requires random mutagenesis and screening of large numbers of variants. Altering the function of natural proteins expands their use, but there are some reactions for which there are no known natural enzymes. In these cases, de novo enzyme design is used to build enzymes from the inside out, starting with a few catalytic residues termed the theozyme and building out a protein scaffold. While some enzyme designs do show activity, they are very poor enzymes, with rate enhancements orders of magnitude below their natural counterparts. In this dissertation, both natural and designed enzyme active sites will be explored, with a particular focus on understanding features essential for catalysis. First, the active site residues of a natural DNA polymerase were predicted through computation and then verified with laboratory experiments. The computational methods used, including POOL and THEMATICS, place a focus on electrostatic features of the residues. While the protocol employed is suitable for prediction of active residues of a single protein, its use is limited for enzyme engineering, where the screening of large numbers of variants is required. The development of a system for high-throughput use of the aforementioned methods will be discussed, entitled MultiPOOL. Previous steps that required user input have been automated and the methods have been parallelized for running on the Discovery Cluster, a high-performance computing cluster. This enables the running of thousands of proteins on the scale of hours; the applications of this development, both realized and potential, are discussed. Two engineering projects, both utilizing MultiPOOL, follow in the remaining chapters. The first will be the engineering of a natural DNA polymerase for accurate bypass of a DNA damage which often results in nucleotide misinsertion. Computational screening was employed for both affinity and activity. The metrics from THEMATICS were used to identify mutations that are likely to negatively impact catalysis and have therefore been avoided. In the second project, MultiPOOL is used to help understand the origins of the increased rate enhancements that de novo designed retro-aldolases gain during evolution. These identified properties have been utilized in attempts for computational optimization, and their potential use in further engineering applications will also be described. The computational methodology developed and employed in this dissertation reveal features of enzymes that are important for their rate enhancement and offers additions to current protocols in the field of enzyme design and engineering.