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

• Published in: Journal of agricultural and food chemistry Vol. 71; no. 22; pp. 8497 - 8507
• Main Authors: T., Rehka, Li, Xin, Ong, Jing Sen, Esque, Jérémy, Zhang, Congqiang, Lin, Qingsong, André, Isabelle, Chen, Xixian
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 07.06.2023
• Subjects: Biochemistry, Molecular Biology; biosynthesis; Biotechnology and Biological Transformations; biotransformation; food chemistry; Life Sciences; mass spectrometry; methyltransferases; mutagenesis; mutants; irone; enzyme engineering; suicide inactivation; methyltransferase; proteomic analysis; Suicide inactivation; Proteomic analysis; Enzyme engineering; Methyltransferase; Irone
• ISSN: 0021-8561; 1520-5118; 1520-5118
• DOI: 10.1021/acs.jafc.3c01272

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 k cat 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.