Identification of Features for Enzymatic Catalysis and Their Application Towards Enzyme Engineering

• NCJ Number: 309930
• Author(s): Timothy A. Coulther
• Date Published: 2019
• Length: 162 pages
• Grant Number(s): 2015-R2-CX-0011
• Agencies: NIJ-Sponsored
• Publication Type: Thesis/Dissertation; Research (Applied/Empirical)

Annotation

This study explored the identification of features for enzymatic catalysis and their application towards enzyme engineering.

Abstract

This dissertation explored natural and designed enzyme active sites, with a particular focus on understanding features essential for catalysis. 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. 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. Two engineering projects, both utilizing MultiPOOL, were undertaken. The first involves engineering of a natural DNA polymerase for accurate bypass of a DNA damage. In the second project, MultiPOOL was used to help understand the origins of the increased rate enhancements that de novo designed retro-aldolases gain during evolution. 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. Enzyme engineering alters 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. Due to limitations in 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 and de novo enzyme design is used.