Structure-function studies of a purple acid phytase

Faba Rodriguez, Raquel (2018) Structure-function studies of a purple acid phytase. Doctoral thesis, University of East Anglia.

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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.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Gillian Aldus
Date Deposited: 11 Jan 2019 10:11
Last Modified: 22 Mar 2019 01:44
URI: https://ueaeprints.uea.ac.uk/id/eprint/69554
DOI:

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