Mutagenesis of Dimer Interfacial Residues Improves the Activity and Specificity of Methyltransferase for cis -α-Irone Biosynthesis

Rehka T. (1) , Xin Li (2) , Jing Sen Ong (1) , Jérémy Esque (3) , Congqiang Zhang (1) , Qingsong Lin (2) , Isabelle André (3) , Xixian Chen (1)

1 SIFBI - Singapore Institute of Food and Biotechnology Innovation
2 Department of Biological Sciences [Singapore]
3 TBI - Toulouse Biotechnology Institute

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 ∼1.6–4.8-fold higher kcat than the previously reported best mutant, pMT10, and increased the cis-α-irone percentage from ∼70 to ∼83%. By one-step biotransformation, ∼121.8 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.

Keywords: Methyltransferase; Irone; Enzyme engineering; Suicide inactivation; Proteomic analysis

Domains: Life Sciences [q-bio]; Biochemistry, Molecular Biology

Rehka T., Xin Li, Jing Sen Ong, Jérémy Esque, Congqiang Zhang, et al.. Mutagenesis of Dimer Interfacial Residues Improves the Activity and Specificity of Methyltransferase for cis -α-Irone Biosynthesis. Journal of Agricultural and Food Chemistry, 2023, 71 (22), pp.8497-8507. ⟨10.1021/acs.jafc.3c01272⟩. ⟨hal-04251978⟩