Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase, AppA

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.

Stable Isotopomers of myo-Inositol Uncover a Complex MINPP1-Dependent Inositol Phosphate Network

“… Another interesting observation, which runs counter to assumptions on MINPP1 activity, ,,− is that the dephosphorylation sequences for InsP 6 and InsP 5 [2OH] do not share any overlap (compare Figures S10, S11, S20, S26, and S27) because MINPP1 seems to be incapable of removing the phosphoryl group at the 2-position. Likely, the charged phosphoryl group on the only axial position of the myo- inositol scaffold plays a role in positioning the InsPs inside MINPP1’s catalytic pocket …”

“…The question if or how InsP metabolism of the host cell and the parasite influences each other might lead to new therapeutic avenues for these parasitoses. The dissection of InsP degradation in an extracellular context, namely, how InsPs contained in food are converted by digestive processes or the gut microbiome and if the resulting metabolites might have beneficial or detrimental effects on health, is also a fascinating question. ,, With the tools and methods reported here, these topics now become addressable.…”

Stable isotopomers of myo-inositol to uncover the complex MINPP1-dependent inositol phosphate network

“…Likely, the charged phosphoryl group on the only axial position of the myo- inositol scaffold plays a role in positioning the InsPs inside MINPP1’s catalytic pocket. [43]…”

“…The dissection of InsP degradation in an extracellular context, namely how InsPs contained in food are converted by digestive processes or the gut microbiome and if the resulting metabolites might have beneficial or detrimental effects on health, are also fascinating questions. [43,60,61] With the tools and methods reported here these topics now become addressable.…”

Sequence diversity and catalytic properties of phytases

“…The association between the structure and the biochemical properties of enzymes can provide relevant information about factors that affect stability and catalytic activity. Several methods have been used for this purpose, including comparative sequence, structure analysis, molecular dynamics studies, or even the evaluation of random or rational mutations on enzyme activity (Zhang 2007;Liao, et al, 2013;Fei et al, 2013;Hesampour et al, 2015;Kumar et al, 2015;Tan et al, 2016;Han et al, 2018;Li et al, 2019;Zhang et al, 2020;Acquistapace et al, 2022). In this study, we report a comparative approach between sequences, threedimensional structures and biochemical characteristics of phytases already characterized and described in the literature, adopting factors such as: hydrogen, ionic and Van der Waals (VdW) interactions and disulfide bonds.…”