STRUCTURE-FUNCTION STUDIES OF MULTIPLE INOSITOL POLYPHOSPHATE PHOSPHATASES FROM GUT COMMENSAL BACTERIA

Li, Arthur (2014) STRUCTURE-FUNCTION STUDIES OF MULTIPLE INOSITOL POLYPHOSPHATE PHOSPHATASES FROM GUT COMMENSAL BACTERIA. Doctoral thesis, University of East Anglia.

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Abstract

Inositol hexakisphosphate (InsP6) is the main storage form of phosphorous in
animal feeds. Phytases are enzymes (myo-inositol hexakisphosphate phosphohydrolases)
that break down InsP6 by hydrolysis to release inorganic phosphate. Non-ruminant animals
do not produce phytases needed to digest dietary InsP6, instead relying on enzymes
produced by their gut microbiota. A similar situation is found in humans where several
members of the commensal bacteria have been found to produce multiple inositol
polyphosphate phosphatases (Minpp) which display phytase activity.
In this study, high resolution X-ray crystal structures of Minpps from two human
commensal gut bacteria, Bacteroides thetaiotaomicron (BtMinpp) and Bifidobacterium
longum (BlMinpp), were solved and refined. High performance liquid chromatography
was employed to analyse the products of InsP6 hydrolysis, revealing that Minpps attack
InsP6 with high positional promiscuity, unlike bacterial and fungal phytases which display
high catalytic specificity.
Site-directed mutagenesis was employed to further investigate the catalytic
promiscuity of BtMinpp, mutagenizing its active site to mimic that of PhyA, a specific 3-
phytase from Aspergillus niger. Further experiments introduced active site residues from
human Minpp. The results of these studies reveal that by altering key active site residues,
the positional specificity and the ratios of the InsP5 products generated by BtMinpp action
can be altered, opening the possibility of engineering catalytic flexibility into phytases
used as commercial animal feed additives.
Disulfide bridges were engineered into BtMinpp with the aim of enhancing its
thermostability – an attractive characteristic for animal feed enzymes. Sites for potential
disulfide bridges were identified and a one such mutant was produced. However, the
engineered protein did not show a significant enhancement in thermostability.
The results of experiments described in this thesis provide novel insights into the
hydrolysis of InsP6 by Minpps that suggest a role as precursors for a new generation of
phytases for the animal feed industry.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Users 2259 not found.
Date Deposited:	29 Jan 2016 13:00
Last Modified:	30 Sep 2017 00:38
URI:	https://ueaeprints.uea.ac.uk/id/eprint/56898
DOI:

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