RESUMEN
Promiscuous enzymes show great potential to establish new-to-nature pathways and expand chemical diversity. Enzyme engineering strategies are often employed to tailor such enzymes to improve their activity or specificity. It is paramount to identify the target residues to be mutated. Here, by exploring the inactivation mechanism with the aid of mass spectrometry, we have identified and mutated critical residues at the dimer interface region of the promiscuous methyltransferase (pMT) that converts psi-ionone to irone. The optimized pMT12 mutant showed â¼1.6-4.8-fold higher kcat than the previously reported best mutant, pMT10, and increased the cis-α-irone percentage from â¼70 to â¼83%. By one-step biotransformation, â¼121.8 mg L-1 cis-α-irone was produced from psi-ionone by the pMT12 mutant. The study offers new opportunities to engineer enzymes with enhanced activity and specificity.