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.