Insights into the structure-activity relationships of enzymes implicated in phytate metabolism

Thompson, Emma (2024) Insights into the structure-activity relationships of enzymes implicated in phytate metabolism. Doctoral thesis, University of East Anglia.

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Abstract

Phytases catalyse the stepwise hydrolysis of IP6 (phytate) to release inorganic phosphate along with a set of inositol polyphosphate intermediates. However, the low abundance of endogenous phytases in the gastrointestinal tracts of monogastric livestock limits the bioavailability of dietary phosphate. Exogenous microbial phytases play a key role in monogastric nutrition, where their augmentation to the feed improves phosphorus bioavailability thereby reducing environmental burden arising from excessive inorganic phosphate excretion. However, limitations of commercial phytases – particularly IP4 accumulation and incomplete dephosphorylation - renders this an active area of research.

The overarching aim of this research was to bioengineer phytase variants with superior hydrolysis characteristics, with particular emphasis on improved processing of IP4 to ameliorate this bottleneck. Several approaches were implemented, including building upon previous work on Multiple Inositol Polyphosphate Phosphatases (MINPPs) and investigating novel phytases for structure-activity insights.

Previously shown to be specificity hotspots, 3 different B-pocket residues of BtMINPP (R275, Q276, K280) were mutagenized to generate 7 variants each, which were analysed for their relative IP6 and IP4 activities by measurement of released phosphate and by HPLC of the degradation profiles. Several variants exhibited statistically significant differential activities and/or specificities to the wild-type.

The structure of MβLp01, a postulated novel bacterial phytase, was solved at 1.95A˚ (RFree = 0.23), with the aim of investigating the structure-function relationship. Unexpectedly, whilst limited activity against ADP and ATP suggests this enzyme may be a phosphatase, the IP6 data strongly indicate it is not a phytase. However, phosphodiesterase activity was revealed. Along with the indicated β-lactamase activity (carbapenemase and penicillinase), it is plausible that this enzyme is a promiscuous phosphodiesterase belonging to the metallo β-lactamase (MβL) superfamily. The findings in this chapter illustrate the profound importance of critically assessing published results for their scientific credibility.

This thesis also extends to an enzyme integral to IP6 biosynthesis - ITPK1 from Solanum tuberosum. Attempts to elucidate the structure of StITPK1 as a trapped substrate complex were unfruitful due to the pathologies of the crystals which limited the quality of the X-ray iffraction data.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	10 Jun 2025 11:00
Last Modified:	10 Jun 2025 11:00
URI:	https://ueaeprints.uea.ac.uk/id/eprint/99425
DOI:

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