White Rose University Consortium logo
University of Leeds logo University of Sheffield logo York University logo

Biotransformations of Proline by 2-oxoglutarate-dependent hydroxylases

Omar, Muhiadin (2017) Biotransformations of Proline by 2-oxoglutarate-dependent hydroxylases. PhD thesis, University of York.

This is the latest version of this item.

[img] Text
Muhiadin Omar_PhD Thesis_White Rose_corrected.pdf - Examined Thesis (PDF)
Restricted until 22 February 2023.

Request a copy

Abstract

Hydroxylases introduce hydroxyl groups with excellent regio- and enantioselectivity making them of significant interest for use in the production of pharmaceutical intermediates and drug metabolites. 2-oxoglutarate dependent oxygenases (2OGDOs) are non-haem dependent Fe(II) containing enzymes that catalyse various oxidation reactions, including the hydroxylation of free amino acids. Unlike the more studied cytochromes P450, these enzymes only require molecular oxygen, Fe(II) and 2-oxoglutarate for catalysis, circumventing the need for a costly cofactor regeneration system. The targets of this work were three proline hydroxylases: a trans-4-proline hydroxylase from Dactylsporangium sp. RH1 (DOGDH), a cis-3-proline-hydoxylase from Streptomyces sp. (StP3H) and a cis-4-proline-hydroxylase from Mesorhizobium loti (MlC4H). Genes encoding all three were cloned into the pET-YSBLIC3C (and pET22b for DOGDH) expression vectors, expressed in Escherichia coli, and produced and purified by chromatography for use in crystallisation studies and enzymatic transformations. Extensive crystallisation trials were attempted for DOGDH including enzymatic, chemical and mutagenic modification with little success. A homology model was therefore constructed in order to identify catalytic residues within the active site that could be manipulated for enhancing the function of DOGDH. A precolumn derivatisation assay using FMOC-Cl was developed for the analysis of proline and its hydroxylated equivalents by HPLC and LC-MS. Biotransformations were performed with L-proline using the three hydroxylases with whole cell reaction conditions deemed optimal due to the multi-component nature of the enzymes, with the cell providing machinery for the recycling of cofactors. Reactions were scaled from shake flasks to stirred tank vessels with the flow of air into the vessel and stirring rate deemed key parameters for optimal function. Finally, a high-throughput substrate screening method using a BioLecter micro-bioreactor was successfully developed and trialled with the three hydroxylases with a panel of substrates providing a platform for future investigations.

Item Type: Thesis (PhD)
Academic Units: The University of York > Chemistry (York)
Depositing User: Mr Muhiadin Omar
Date Deposited: 07 Mar 2018 13:46
Last Modified: 07 Mar 2018 13:46
URI: http://etheses.whiterose.ac.uk/id/eprint/19532

Available Versions of this Item

Please use the 'Request a copy' link(s) above to request this thesis. This will be sent directly to someone who may authorise access.
You can contact us about this thesis. If you need to make a general enquiry, please see the Contact us page.

Actions (repository staff only: login required)