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Exploration of the active site of Staphylococcus aureus Neuraminic acid Lyase using non-canonical amino acids and their effects on substrate specificity.

Smith, Robert Peter (2017) Exploration of the active site of Staphylococcus aureus Neuraminic acid Lyase using non-canonical amino acids and their effects on substrate specificity. PhD thesis, University of Leeds.

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Smith_RP_Molecular_and_Cellular_Biology_PhD_2017.pdf - Final eThesis - complete (pdf)
Restricted until 1 July 2021.

Abstract

The cost of chemical synthesis of pharmaceuticals contributes significantly to their final price and part of this cost is incurred due to use of extreme temperatures and pressures required by some traditional catalysts. The interest in catalysis using enzymes, or biocatalysis, from industry has been growing recently, due to enzymes’ ability to work at room temperature and pressure, and the reduction in toxic solvent waste produced from an enzyme reaction compared to a traditionally catalysed reaction. The specificity of enzymes, while useful in product formation, can make applying them to synthetic chemistry challenging due to the restriction this causes in substrates that each enzyme accepts. This can often be avoided by amino acid mutagenesis, but when this is performed genetically, only 20 different amino acids can be used. Non-canonical amino acids (ncAAs) have the potential to enhance properties of enzymes, such as enzyme stability and substrate specificities, to hitherto unseen extremes, due to the massive diversity of amino acids outside the canonical 20. 900 enzyme-aldehyde pairs were screened for activity, and Y252Lanthionine was found to catalyse the aldol reaction between pyruvate and glucuronolactone better than the wild type enzyme for the same reaction. Upon crystallisation, this enzyme was found to be a mixture of both L- and D-stereoisomers at the protein backbone where the ncAA was inserted. Computational experiments were performed to assess the substrate binding capability of the modified enzyme and the wild-type enzyme. The modified side chain holds the substrate more tightly than the wild-type side chain, contributing to increased residence time in the active site.

Item Type: Thesis (PhD)
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute for Molecular and Cellular Biology (Leeds)
Depositing User: Mr Robert Peter Smith
Date Deposited: 29 Jun 2018 11:53
Last Modified: 29 Jun 2018 11:53
URI: http://etheses.whiterose.ac.uk/id/eprint/20719

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