8XM2

The mutant crystal structure of phytase APPAmut9 from Yersinia intermedia

Summary for 8XM2

• Entry DOI: 10.2210/pdb8xm2/pdb
• Descriptor: Phytase (2 entities in total)
• Functional Keywords: the mutant crystal structure of phytase appamut9 from yersinia intermedia, hydrolase
• Biological source: Yersinia intermedia
• Total number of polymer chains: 1
• Total formula weight: 45700.24

Authors

Tu, T.,Dong, R.Y. (deposition date: 2023-12-27, release date: 2024-07-03, Last modification date: 2025-01-15)

Primary citation

Tu, T.,Wang, Q.,Dong, R.,Liu, X.,Penttinen, L.,Hakulinen, N.,Tian, J.,Zhang, W.,Wang, Y.,Luo, H.,Yao, B.,Huang, H.
Achieving thermostability of a phytase with resistance up to 100 °C.
J.Biol.Chem., 300:107992-107992, 2024

PubMed Abstract: The development of enzymes with high-temperature resistance up to 100 °C is of significant and practical value in advancing the sustainability of industrial production. Phytase, a crucial enzyme in feed industrial applications, encounters challenges due to its limited heat resistance. Herein, we employed rational design strategies involving the introduction of disulfide bonds, free energy calculation, and B-factor analysis based on the crystal structure of phytase APPAmut4 (1.90 Å), a variant with enhanced expression levels derived from Yersinia intermedia, to improve its thermostability. Among the 144 variants experimentally verified, 29 exhibited significantly improved thermostability with higher t values at 65 °C. Further combination and superposition led to APPAmut9 with an accumulation of five additional pairs of disulfide bonds and six single-point mutation sites, leading to an enhancement in its thermostability with a t value of 256.7 min at 65 °C, which was more than 75-fold higher than that of APPAmut4 (3.4 min). APPAmut9 exhibited a T value of 96 °C, representing a substantial increase of 40.9 °C compared to APPAmut4. Notably, approximately 70% of enzyme activity remained intact after exposure to boiling water at 100 °C for a holding period of 5 min. Significantly, these advantageous modifications were strategically positioned away from the catalytic pocket where enzymatic reactions occur to ensure minimal compromise on catalytic efficiency between APPAmut9 (11,500 ± 1100/mM/s) and APPAmut4 (12,300 ± 1600/mM/s). This study demonstrates the feasibility of engineering phytases with resistance to boiling using rational design strategies.

PubMed: 39547510
DOI: 10.1016/j.jbc.2024.107992

Experimental method

X-RAY DIFFRACTION (1.77 Å)