Substituting the catalytic proline of 4-oxalocrotonate tautomerase with non-canonical analogues reveals a finely tuned catalytic system

Scientific Reports
Michael S LukeschBirgit Wiltschi

Abstract

The enzyme 4-oxalocrotonate tautomerase shows remarkable catalytic versatility due to the secondary amine of its N-terminal proline moiety. In this work, we incorporated a range of proline analogues into the enzyme and examined the effects on structure and activity. While the structure of the enzyme remained unperturbed, its promiscuous Michael-type activity was severely affected. This finding demonstrates how atomic changes in a biocatalytic system can abolish its activity. Our work provides a toolbox for successful generation of enzyme variants with non-canonical catalytic proline analogues.

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Citations

Feb 27, 2020·The Journal of Organic Chemistry·Yassin M ElbatrawiJuan R Del Valle
Mar 18, 2021·Beilstein Journal of Organic Chemistry·Vladimir KubyshkinNediljko Budisa
Feb 4, 2021·ACS Central Science·Christoph K WinklerWolfgang Kroutil

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Methods Mentioned

BETA
circular dichroism
NMR
PCR
gel filtration

Software Mentioned

NMRView
- Pipe
MolProbity
GelAnalyzer2010a
XDS
NMR
Coot
PHENIX
TopSpin
Phaser

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