Insights to the structural basis for the stereospecificity of the Escherichia coli phytase, AppA

Acquistapace, Isabella, Thompson, Emma ORCID: https://orcid.org/0000-0002-7579-3215, Kuhn, Imke, Bedford, Mike, Brearley, Charles ORCID: https://orcid.org/0000-0001-6179-9109 and Hemmings, Andrew ORCID: https://orcid.org/0000-0003-3053-3134 (2022) Insights to the structural basis for the stereospecificity of the Escherichia coli phytase, AppA. International Journal of Molecular Sciences, 23 (11). ISSN 1661-6596

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Abstract

AppA, the Escherichia coli periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed supplement. AppA is a 1D-6-phytase and highly stereospecific but transiently accumulates 1D-myo-Ins(2,3,4,5)P4 and other lower phosphorylated intermediates. If this bottleneck in liberation of orthophosphate is to be obviated through protein engineering, an explanation of its rather rigid preference for the initial site and subsequent cleavage of phytic acid is required. To help explain this behaviour, the role of the catalytic proton donor residue in determining AppA stereospecificity was investigated. Four variants were generated by site-directed mutagenesis of the active site HDT amino acid sequence motif containing the catalytic proton donor, D304. The identity and position of the prospective proton donor residue was found to strongly influence stereospecificity. While the wild-type enzyme has a strong preference for 1D-6-phytase activity, a marked reduction in stereospecificity was observed for a D304E variant, while a proton donor-less mutant (D304A) displayed exclusive 1D-1/3-phytase activity. High-resolution X-ray crystal structures of complexes of the mutants with a non-hydrolysable substrate analogue inhibitor point to a crucial role played by D304 in stereospecificity by influencing the size and polarity of specificity pockets A and B. Taken together, these results provide the first evidence for the involvement of the proton donor residue in determining the stereospecificity of HP2 phytases and prepares the ground for structure-informed engineering studies targeting the production of animal feed enzymes capable of the efficient and complete dephosphorylation of dietary phytic acid.

Item Type: Article
Additional Information: Funding Information: This work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and AB Vista Feed Ingredients Ltd. through IPA award BB/M022978/1. I.M.A. was supported through a joint UEA‐AB Vista Feed Ingredients Ltd. PhD studentship and E.J.T. was supported by a BBSRC‐funded Norwich Research Park Doctoral Training Partnership CASE studentship with AB Vista Feed Ingredients Ltd.
Uncontrolled Keywords: appa,x‐ray crystallography,catalytic mechanism,enzyme structure,enzymology,phytase,stereospecificity,catalysis,molecular biology,spectroscopy,computer science applications,physical and theoretical chemistry,organic chemistry,inorganic chemistry ,/dk/atira/pure/subjectarea/asjc/1500/1503
Faculty \ School: Faculty of Science > School of Biological Sciences
Faculty of Science > School of Chemistry
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Depositing User: LivePure Connector
Date Deposited: 20 Jul 2022 12:30
Last Modified: 01 Aug 2022 10:30
URI: https://ueaeprints.uea.ac.uk/id/eprint/86728
DOI: 10.3390/ijms23116346

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