Studies on Structurally Mobile Regions of the Varicella-Zoster Virus Thymidylate Synthase

Deursen, Frederick John van (1997) Studies on Structurally Mobile Regions of the Varicella-Zoster Virus Thymidylate Synthase. PhD thesis, University of Glasgow.

Abstract

Thymidylate synthase (EC 2.1.1.45) is one of the most highly conserved enzymes. It is the sole de novo source of 2'-deoxythymidylate (dTMP) in a diverse range of organisms. Thymidylate synthase (TS) catalyses the conversion of 2'-deoxyuridylate (dUMP) and N5, N10-methylene-tetrahydrofolate to dTMP and dihydrofolate. To date, sequences are known from 36 different species of TS. These include the human enzyme which is a drug target for anti-cancer agents, thymidylate synthases from protozoa, which are generally bifunctional with TS and dihydrofolate reductase present in a single protein, and TS from bacterial and viral species. TS is an essential enzyme to almost all living species and is encoded by several large DNA viruses, presumably to support an increased need for DNA synthesis during the viral life cycle. There are only a few organisms that adequately scavenge thymidine from their environment and do not require TS. The essential requirement for TS activity makes TS an important drug target for the development of anti-parasitic and possibly antiviral agents. The work presented in this thesis identifies a mobile surface loop region of TS (the DRTG loop) and shows that it is important for substrate turnover. It is implicated in packing against the C-terminus of VZV TS facilitating efficient closing of the active site upon ligand binding. This loop is highly conserved throughout the TS family with the phage ?3T and Llactis being the most divergent species. Site-directed mutagenesis was used to substitute the VZV residues in the DRTG loop for their L. Iactis counterparts. The resultant variant proteins were dysfunctional in their ability to complete the catalytic reaction. With the exception of G40M they could still bind the substrate, dUMP. The development of a region-directed mutagenesis protocol is also described. Such an approach was used to select for covariant residues of the DRTG loop mutants that might restore activity. In this study, no covariant residues were identified. The validity of the protocol was however, shown by generation of several temperature sensitive mutants. The C-terminal region of TS is also mobile and plays a significant role in catalysis by closing down the active site upon binding of ligands. Previous studies proposed a unique interaction at the C-terminus of VZV TS and this has been investigated further. Mutations of the C-terminus of both VZV and human TS suggest that VZV is not unique in this respect. Temperature sensitive mutants allow the role of amino acid residues in protein stability to be investigated. Several temperature sensitive mutants were generated to different regions of VZV TS and showed that the enzyme can tolerate many changes even within residues that are fully conserved within the TS family.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: John H Subak-Sharpe
• Keywords: Virology
• Date of Award: 1997
• Depositing User: Enlighten Team
• Unique ID: glathesis:1997-75275
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 21:22
• Last Modified: 19 Nov 2019 21:22
• URI: https://theses.gla.ac.uk/id/eprint/75275