Structural and Molecular Dynamics Analysis of Plant Serotonin N‐Acetyltransferase Reveal an Acid/Base‐Assisted Catalysis in Melatonin Biosynthesis

Abstract

Serotonin N‐acetyltransferase (SNAT) is the key rate‐limiting enzyme in melatonin biosynthesis. It mediates melatonin biosynthesis in plants by using serotonin and 5‐methoxytryptamine (5‐MT), but little is known of its underlying mechanisms. Herein, we present a detailed reaction mechanism of a SNAT from Oryza sativa through combined structural and molecular dynamics (MD) analysis. We report the crystal structures of plant SNAT in the apo and binary/ternary complex forms with acetyl‐CoA (AcCoA), serotonin, and 5‐MT. OsSNAT exhibits a unique enzymatically active dimeric fold not found in the known structures of arylalkylamine N‐acetyltransferase (AANAT) family. The key residues W188, D189, D226, N220, and Y233 located around the active pocket are important in catalysis, confirmed by site‐directed mutagenesis. Combined with MD simulations, we hypothesize a novel plausible catalytic mechanism in which D226 and Y233 function as catalytic base and acid during the acetyl‐transfer reaction.

Functional Characterization of Arylalkylamine N-Acetyltransferase, a Pivotal Gene in Antioxidant Melatonin Biosynthesis from Chlamydomonas reinhardtii

“…Recombinant Trx-CrAANAT-His6 exhibited serotonin N -acetyltransferase (SNAT) activity of 10.1 pkat/mg protein at 45 °C and pH 8.8, compared to 18.7 pkat/mg protein for GST-CrAANAT-His6 ( Figure 3 C). Therefore, CrAANAT transfers an acetyl group from acetyl-CoA to serotonin to produce N -acetylserotonin, the final substrate in melatonin biosynthesis [ 4 ].…”

“…The final two genes in melatonin biosynthesis are AANAT (or SNAT ) and ASMT [ 4 ]. AANAT was first cloned and characterized in sheep and rats [ 40 , 41 ].…”

“…Moreover, the increase in BR levels caused by endogenous melatonin overproduction induces melatonin biosynthesis by activating the expression of melatonin biosynthetic genes such as tryptophan decarboxylase 1 ( TDC1 ), TDC3 , and tryptamine 5-hydroxylase ( T5H ) [ 64 ] ( Figure 9 ). In sum, the effect of melatonin on seed size and yield [ 17 , 61 ] and stress tolerance [ 65 , 66 ] suggests that the generation of melatonin-rich rice plants by exogenous application [ 64 ] or transgenic approaches [ 4 ] would increase yields and resistance to many biotic and abiotic stresses and enable the production of melatonin-rich foods with health benefits [ 67 ].…”

“…Both animal AANAT and plant SNAT proteins belong to the GCN5-related N -acetyltransferase superfamily, which transfer an acetyl group from acetyl-coenzyme A (CoA) [ 3 ]. However, there is no significant amino sequence homology between animal AANATs and plant SNATs except for a few amino acids in the acetyl-CoA-binding domain [ 4 ]. Interestingly, an animal homolog of arylalkylamine N-acetyltransferase ( AANAT ) is present in the genome of C. reinhardtii , but not in other green algae or higher plants [ 3 , 5 , 6 ].…”

Identification of a serotonin N-acetyltransferase from Staphylococcus pseudintermedius ED99

“…Frontiers in Microbiology 07 frontiersin.org different catalytic residues (De Angelis et al, 1998;Dutnall et al, 1998;Hickman et al, 1999;Dyda et al, 2000;Liao et al, 2021). This variation could be behind the enzyme's wide substrate range and its involvement in alternative pathways in different species (Tan et al, 2016).…”