Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.768422
Title: Structure-function studies of a purple acid phytase
Author: Faba Rodriguez, Raquel
ISNI:       0000 0004 7654 1081
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2018
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Abstract:
The enzymatic cleavage of phosphate monoesters of myo-inositol hexakisphosphate (InsP6) or phytate is the property of a group of enzymes collectively known as phytases. These enzymes adopt a variety of protein folds and utilise a number of different reaction mechanisms and may be classified accordingly. Among these, the purple acid phytases (PAPhy), a subclass of the purple acid phosphatases (PAP), are the least well characterised. The aim of this thesis is a biochemical and structural characterisation of cereal PAPhy with the additional purpose of the identification of structural features that distinguish PAPhy from PAP. In this project, the partial enzymatic deglycosylation of a recombinant PAPhy from wheat yielded high quality crystals that allowed the solution of the high-resolution X-ray crystallographic structure of the first PAPhy, with inorganic phosphate bound in different poses and in complex with the inhibitor myo-inositol hexakissulfate. Molecular dynamics simulations of the enzyme-substrate complex allowed the identification of key protein-substrate interactions, leading to the proposal of six phytate specificity pockets for the wheat PAPhy isoform b2 (TaPAPhy_b2). A characterisation of TaPAPhy_b2 allowed the estimation of its kinetic parameters, revealed optimum phytase activity at pH 5.5 and 37°C, with denaturation and subsequent inactivation over 50°C, and the determination of the D-4/6-phosphate as preferred initiation site of InsP6 hydrolysis. A conservation of the pathway of phytate hydrolysis identified in TaPAPhy_b2 was observed in other cereal PAPhy, while the soybean PAPhy displayed higher positional promiscuity. Structure-function relationships of TaPAPhy_b2 were elucidated by site-directed mutagenesis and mutant characterisation alongside the wild type enzyme. Two amino acid residues critical for phytase activity were identified, His229 and Lys410, while a third, Lys348, was shown to influence substrate affinity more subtly. The work described in this thesis provides novel insights into the structure and phytase activity of the purple acid phytases.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.768422  DOI: Not available
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