7Z2T

Escherichia coli periplasmic phytase AppA D304A mutant, complex with myo-inositol hexakissulfate

7Z2T の概要

• エントリーDOI: 10.2210/pdb7z2t/pdb
• 関連するPDBエントリー: 7Z1J 7Z2S
• 分子名称: Acidphosphatase, D-MYO-INOSITOL-HEXASULPHATE, GLYCEROL, ... (6 entities in total)
• 機能のキーワード: histidine acid phosphatase, phytase, hydrolase
• 由来する生物種: Escherichia coli
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 45886.65

構造登録者

Acquistapace, I.M.,Brearley, C.A.,Hemmings, A.M. (登録日: 2022-02-28, 公開日: 2022-03-16, 最終更新日: 2024-10-16)

主引用文献

Acquistapace, I.M.,Thompson, E.J.,Kuhn, I.,Bedford, M.R.,Brearley, C.A.,Hemmings, A.M.
Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase, AppA.
Int J Mol Sci, 23:-, 2022

PubMed Abstract: AppA, the 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--Ins(2,3,4,5)P 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.

PubMed: 35683026
DOI: 10.3390/ijms23116346

実験手法

X-RAY DIFFRACTION (1.41 Å)