Engineering the activity of a newly identified arylalkylamine N‐acetyltransferase in the acetylation of 5‐hydroxytryptamine

Abstract Arylalkylamine N ‐acetyltransferase (AANAT) serves as a key enzyme in the biosynthesis of melatonin by transforming 5‐hydroxytryptamine (5‐HT) to N ‐acetyl‐5‐hydroxytryptamine (NAS), while its low activity may hinder melatonin yield. In this study, a novel AANAT derived from Sus scrofa ( Ss AANAT) was identified through data mining using 5‐HT as a model substrate, and a rational design of Ss AANAT was conducted in the quest to improving its activity. After four rounds of mutagenesis procedures, a triple combinatorial dominant mutant M3 was successfully obtained. Compared to the parent enzyme, the conversion of the whole‐cell reaction bearing the best variant M3 exhibted an increase from 50 % to 99 % in the transformation of 5‐HT into NAS. Additionally, its catalytic efficiency ( k cat / K m ) was enhanced by 2‐fold while retaining the thermostability ( T m >45 °C). In the up‐scaled reaction with a substrate loading of 50 mM, the whole‐cell system incorporating variant M3 achieved a 99 % conversion of 5‐HT in 30 h with an 80 % yield. Molecular dynamics simulations were ultilized to shed light on the origin of improved activity. This study broadens the repertoire of AANAT for the efficient biosynthesis of melatonin.