Repurposing a bacterial prolidase for organophosphorus hydrolysis: reshaped catalytic cavity switches substrate selectivity.
BIOTECHNOLOGY AND BIOENGINEERING(2020)
摘要
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.
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关键词
active-site reshaping,catalytic selectivity,enzyme promiscuity,protein engineering,substrate repositioning
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