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Repurposing a bacterial prolidase for organophosphorus hydrolysis: Reshaped catalytic cavity switches substrate selectivity
Author(s) -
Yang Jian,
Xiao YunZhu,
Li Ru,
Liu Yu,
Long LiJuan
Publication year - 2020
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27455
Subject(s) - paraoxon , selectivity , chemistry , substrate (aquarium) , catalysis , hydrolysis , adaptive evolution , in silico , peptide , directed evolution , biophysics , biochemistry , enzyme , combinatorial chemistry , stereochemistry , biology , gene , mutant , ecology , acetylcholinesterase
Enzyme promiscuity is critical to the acquisition of evolutionary plasticity in cells and can be recruited for high‐value chemical synthesis or xenobiotic degradation. The molecular determinants of substrate ambiguity are essential to this activity; however, these details remain unknown. Here, we performed the directed evolution of a prolidase to enhance its initially weak paraoxonase activity. The in vitro evolution led to an unexpected 1,000,000‐fold switch in substrate selectivity, with a 30‐fold increase in paraoxon hydrolysis and 40,000‐fold decrease in peptide hydrolysis. Structural and in silico analyses revealed enlarged catalytic cavities and substrate repositioning as responsible for rapid catalytic transitions between distinct chemical reactions.