An Investigation into the Structural Determinants of the Positional Specificity of Hydrolysis of myo-Inositol Hexakisphosphate by HAP Phytases

Acquistapace, Isabella (2018) An Investigation into the Structural Determinants of the Positional Specificity of Hydrolysis of myo-Inositol Hexakisphosphate by HAP Phytases. Doctoral thesis, University of East Anglia.

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Abstract

Phytases are phosphatases which catalyse the stepwise hydrolysis of the phosphomonoester bonds of myo-inositol hexakisphosphate (IP6). IP6 is the major storage form of phosphorous in grains, seeds and beans found in animal feeds, but also can act as an antinutrient. Feed conversion ratios for monogastric animals are improved by addition of phytases to feed preparations in a market with an annual worldwide turnover of more than $500m. New phytases are continually sought to satisfy this demand. An alternative approach is to reengineer known phytases, tuning their positional stereospecificity with the aim of generating more efficient IP6 hydrolysis.

To help shed light on the structural basis for differences in positional stereospecificity between phytases of clade 2 of the family of histidine phosphatase superfamily (HP2), the role of the catalytic proton donor in the Escherichia coli phytase, AppA, was investigated in comparison with that observed for multiple inositol polyphosphate phosphatases (MINPPs). Four AppA active site mutants were generated by site-directed mutagenesis of the wild type “HDT” motif to replace it with the MINPPs-like “HAE”. Shifts in the ratio of IP5 intermediates were detected. In the process, the 6-phytase AppA, was converted to a 1/3- or 1/3/4/6-phytase. High resolution X-ray structures of the mutants revealed the primary roles played by size and polarity of specificity pockets in determining positional stereospecificity traits.

The first structural evidence for a large α-domain closure motion and its involvement in catalysis in a HP2 phytase is also reported, the MINPP of Bifidobacterium longum susp. infantis. Ligand binding-driven conformational changes correlate with the presence of a unique active site loop insertion. This loop significantly reduces the size of the active site and contributes to the coordination of the substrate.

Many MINPPs lack the positional stereospecificity displayed by other HP2 phytases. In principle, this property could be utilized for the more efficient dephosphorylation of IP6. To identify sequence determinants of this characteristic, 15 MINPPs were selected by genome mining. The resulting recombinant enzymes were tested and classified as either positionally or non-positionally stereospecific. X-ray crystal structures of an enzyme from each group revealed conserved active site residues which were mutated in silico, and the IP6 binding poses for each mutant predicted by molecular docking. This process revealed hotspot residues which can potentially direct positional stereospecificity in this family of phytases.

The work reported in this thesis has led to the identification of potential residue determinants of positional stereospecificity in HP2 phytases and has prepared the ground for future engineering studies possibly leading to more efficient animal feed enzymes.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Users 9280 not found.
Date Deposited:	26 Nov 2019 11:55
Last Modified:	05 Mar 2024 08:31
URI:	https://ueaeprints.uea.ac.uk/id/eprint/73011
DOI:

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