Significantly improved catalytic efficiency of caffeic acid O-methyltransferase towards N-acetylserotonin by strengthening its interactions with the unnatural substrate’s terminal structure

Publication date: Available online 14 February 2019Source: Enzyme and Microbial TechnologyAuthor(s): Wenya Wang, Sisi Su, Shizhuo Wang, Lidan Ye, Hongwei YuAbstractO-methylation of N-acetylserotonin (NAS) has been identified as the bottleneck in melatonin biosynthesis pathway. In the present paper, caffeic acid O-methyltransferase from Arabidopsis thaliana (AtCOMT) was engineered by rational design to improve its catalytic efficiency in conversion of NAS to melatonin. Based on the notable difference in the terminal structure of caffeic acid and NAS, mutants were designed to strengthen the interactions between the substrate binding pocket of the enzyme and the terminal structure of the unnatural substrate NAS. The final triple mutant (C296F-Q310L-V314 T) showed 9.5-fold activity improvement in O-methylation of NAS. Molecular dynamics simulations and binding free energy analysis attributed the increased activity to the higher affinity between the substrate terminal structure and AtCOMT, resulting from the introduction of N—H···π interaction by Phe296 substitution, hydrophobic interaction by Thr314 substitution and elimination of electrostatic repulsion by substitution of Gln310 with Leu310. This work provides hints for O-methyltransferase engineering and meanwhile lays foundation for biotechnological production of melatonin.
Source: Enzyme and Microbial Technology - Category: Biotechnology Source Type: research