Date of Award

2021

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Area of Concentration

Biochemistry

Abstract

Amino acids have many important roles in biology. For example, amino acids are linked together by peptide bonds to form proteins. Also, in metabolism, amino acids can be broken down via the process of transamination, in which they lose their amine group and are converted into keto acids. Keto acids can then be converted into precursors for many other biological pathways, such as to produce energy, fat or glucose. 3-Hydroxyisobutyrate dehydrogenase is an enzyme that plays a role in amino acid metabolism, specifically the catabolism of L-valine. In humans, this is encoded by the HIBADH gene and is responsible for catalyzing the oxidation reaction of 3-hydroxyisobutyrate to methylmalonate semialdehyde. One goal of this study was to characterize the kinetics of the B0250.5 gene found on chromosome 5 of Caenorhabditis elegans. While information on or studies of this gene are limited, it is expected to encode a protein very similar to the human 3-hydroxyisobutyrate dehydrogenase enzyme and as such, is also expected to have a similar role in the catabolism of L-valine. Another goal of this study was to model the crystal structure of the B0250.5 protein in C. elegans and study how different substrates bind to the protein. The B0250.5 plasmid was successfully cloned into the expression vector pET303/CT-His. The vector plasmid was then used to transform T7 Express Competent E. coli cells (C3566H). However, there was no sign of successful overexpression of C. elegans 3-hydroxyisobutyrate dehydrogenase. A solution to this expression issue may be to attempt protein expression using a different cell type, such as E. coli strain BL21 (DE3) pLysS or Rosetta2, in future studies. The crystal structure of C. elegans 3-hydroxyisobutyrate dehydrogenase was modeled using SWISS-MODEL software. The docking of 3-hydroxyisobutyrate and NAD+ was performed by the AutoDock Vina program and was visualized in Pymol 2.0. The active site for 3-hydroxyisobutyrate was predicted to contain Gly123, Gly124 and Asn176 resides. Additionally, NAD+ was found to be very close to the active site and was surrounded by Thr128. Thr132, Thr134, Asn158, Asn176, Trp215, Thr219 and Asp286 residues. While AutoDock Vina was able to perform the docking, there were a few limitations. In future studies, a more sophisticated ligand-protein docking software may be able to provide better results and allow for more complex and accurate interactions.

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