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Title: An investigation into the catalytic mechanism of vitamin B6 biosynthesis using X-ray crystallography and UV-Vis spectroscopy
Author: Rodrigues, Matthew
ISNI:       0000 0004 6348 9421
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2017
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The catalytic mechanism of the Pdx1 subunit of the pyridoxal 5’-phosphate (PLP) syn-thase enzyme complex has been the subject of intense study since its discovery in 1999. Pdx1 uses two active sites (P1 and P2) to perform a complex reaction combining ribose 5-phosphate, ammonia and glyceraldehyde 3-phosphate to form PLP. While some aspects of the Pdx1 mechanism are now understood, several questions re-main, in particular how the enzyme transfers the reaction from one active site to the next and how the reactions in the two sites are coordinated. In this investigation, X-ray crystallography and UV-Vis spectrophotometry have been used to determine the struc-ture of the protein in various intermediate states and elucidate the catalytic mechanism. The role of specific active site residues in catalysis of PLP biosynthesis by the Arabidop-sis thaliana Pdx1 protein is investigated using site-directed mutagenesis. The crystal structures presented in this thesis demonstrate that the Pdx1 enzyme uses a novel relay mechanism to covalently transfer intermediates between active sites and to coordinate substrate binding, intermediate shuttling and catalysis. Chapter 1 provides an introduction to the biological role of pyridoxal 5’-phosphate and its biosynthesis by the PLP synthase enzyme complex. Chapter 1 also describes the use of X-ray crystallography to understand how proteins function and how complementary methods such as UV-Vis spectrophotometry can be used to track the effects of site-specific radiation damage during collection of X-ray diffraction data. The methods used to produce, purify and investigate the Pdx1 enzyme are described in Chapter 2. Chapter 3 provides an analysis of the activity of wild type AtPdx1.3 using UV-Vis spectropho-tometry and X-ray crystallography to monitor the accumulation of chromophoric inter-mediates and the product, PLP. Chapter 4 describes the use of site-directed mutagenesis to trap the Pdx1 protein in additional intermediate states and the characterisation of these intermediate states using UV-Vis spectroscopy and crystallography. UV-Vis spectra of Pdx1 crystals were collected to ensure that the Pdx1 enzyme was in the desired intermediate state before collection of X-ray diffraction data. It became clear that the UV-Vis spectra of the Pdx1 crystals in some intermediate states were changing during X-ray data collection. Chapter 5 describes the experiments that were performed to check that the Pdx1 structures obtained were not affected by site-specific radiation damage. The use of multi-crystal data collection strategies and UV-Vis spectroscopy showed that changes in the spectra were instead caused by radiolysis of the solvent surrounding the crystals.
Supervisor: Tews, Ivo Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available