Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.679145
Title: Structure-function studies of multiple inositol polyphosphate phosphatases from gut commensal bacteria
Author: Li, Arthur
ISNI:       0000 0004 5371 3047
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2014
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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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.679145  DOI: Not available
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