TY - JOUR
T1 - An Energy Optimization Strategy Based on the Perfect Conformation of Prolyl Endopeptidase for Improving Catalytic Efficiency
AU - Li, Xiaolin
AU - Cong, Yalong
AU - Ma, Mingzhe
AU - You, Zhi Neng
AU - Gao, Bei
AU - Zhang, John Z.H.
AU - Zhang, Lujia
N1 - Publisher Copyright:
Copyright © 2020 American Chemical Society.
PY - 2020/5/6
Y1 - 2020/5/6
N2 - Prolyl endopeptidases (PEPs) hydrolyze proteins to yield bioactive peptides and are effective in the treatment of celiac disease. However, the catalytic efficiency of PEPs still has the potential to be improved, which could further strengthen their industrial and therapeutic applications. Herein, a novel rational design strategy based on a "near-attack conformation" of the catalytic state of PEP was adopted. Constrained dynamic simulations were applied, followed by the virtual screening of potentially favorable mutants according to their binding free energy. We redesigned Sphaerobacter thermophiles PEP with high-temperature activity/stability, a wide range of pH stabilities, and high proline specificity. As a result, the kcat value of two PEP mutants (I462W and Q560Y) increased by 208.2 and 150.1%, respectively, and the kcat/KM increased by 32.7 and 6.3%, respectively. These data revealed that the PEP mutants had improved catalytic efficiency and that our strategy can be applied for enzyme engineering.
AB - Prolyl endopeptidases (PEPs) hydrolyze proteins to yield bioactive peptides and are effective in the treatment of celiac disease. However, the catalytic efficiency of PEPs still has the potential to be improved, which could further strengthen their industrial and therapeutic applications. Herein, a novel rational design strategy based on a "near-attack conformation" of the catalytic state of PEP was adopted. Constrained dynamic simulations were applied, followed by the virtual screening of potentially favorable mutants according to their binding free energy. We redesigned Sphaerobacter thermophiles PEP with high-temperature activity/stability, a wide range of pH stabilities, and high proline specificity. As a result, the kcat value of two PEP mutants (I462W and Q560Y) increased by 208.2 and 150.1%, respectively, and the kcat/KM increased by 32.7 and 6.3%, respectively. These data revealed that the PEP mutants had improved catalytic efficiency and that our strategy can be applied for enzyme engineering.
KW - catalytic efficiency
KW - molecular dynamics simulations
KW - prolyl endopeptidase
KW - protein engineering
KW - site-directed mutagenesis
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U2 - 10.1021/acs.jafc.0c00731
DO - 10.1021/acs.jafc.0c00731
M3 - Article
C2 - 32297517
AN - SCOPUS:85084740281
SN - 0021-8561
VL - 68
SP - 5129
EP - 5137
JO - Journal of Agricultural and Food Chemistry
JF - Journal of Agricultural and Food Chemistry
IS - 18
ER -