Characterization of Arylalkylamine N-Acyltransferase from Tribolium castaneum: An Investigation into a Potential Next-Generation Insecticide Target

Abstract

The growing issue of insecticide resistance has meant the identification of novel insecticide targets has never been more important. Arylalkylamine N-acyltransferases (AANATs) have been suggested as a potential new target. These promiscuous enzymes are involved in the N-acylation of biogenic amines to form N-acylamides. In insects, this process is a key step in melanism, hardening of the cuticle, removal of biogenic amines, and in the biosynthesis of fatty acid amides. The unique nature of each AANAT isoform characterized indicates each organism accommodates an assembly of discrete AANATs relatively exclusive to that organism. This implies a high potential for selectivity in insecticide design, while also maintaining polypharmacology. Presented here is a thorough kinetic and structural analysis of AANAT found in one of the most common secondary pests of all plant commodities in the world, Tribolium castaneum. The enzyme, named TcAANAT0, catalyzes the formation of short-chain N-acylarylalkylamines, with short-chain acyl-CoAs (C2–C10), benzoyl-CoA, and succinyl-CoA functioning in the role of acyl donor. Recombinant TcAANAT0 was expressed and purified from E. coli and was used to investigate the kinetic and chemical mechanism of catalysis. The kinetic mechanism is an ordered sequential mechanism with the acyl-CoA binding first. pH-rate profiles and site-directed mutagenesis studies identified amino acids critical to catalysis, providing insights about the chemical mechanism of TcAANAT0. A crystal structure was obtained for TcAANAT0 bound to acetyl-CoA, revealing valuable information about its active site. This combination of kinetic analysis and crystallography alongside mutagenesis and sequence analysis shines light on some approaches possible for targeting TcAANAT0 and other AANATs for novel insecticide design.

Identification of catalytically distinct arylalkylamine N-acetyltransferase splicoforms from Tribolium castaneum

“…As shown in Figure 2 (underlined), the catalytic site of the amine binding pocket is often located relatively close to the N-terminus in AANATs. While there is no structure yet available for TcAANAT1, several other iAANAT structures have been reported: T. castaneum (TcAANAT0) [37], D. melanogaster (DmAANATA) [40], and A. aegypti (AaNAT2, AaNAT5b and AaNAT7) [41], as well as mammalian SNAT [42,43]. SNAT has the longest N-terminus before amine binding pocket, approximately 60 residues long.…”

“…The enzyme, TcAANAT1, catalyzed the formation of acetylated amines with dopamine, tryptamine, norepinephrine, tyramine and octopamine functioning as the acceptor substrate. A BLAST search of the T. castaneum genome using TcAANAT1 as a template identified just one additional discrete TcAANAT, the recently characterized TcAANAT0 [37]. Also identified were several isoforms of TcAANAT1.…”

Evolutionary genomics analysis reveals gene expansion and functional diversity of arylalkylamine N‐acetyltransferases in the Culicinae subfamily of mosquitoes

“…As aaNAT serves as an important regulator in a variety of physiological functions, the enzyme is considered a target for the development of new insecticides (O'Flynn et al. , 2020).…”

“…These include cuticular sclerotization, the suppression of black pigmentation (Noh et al, 2016), the inactivation of biogenic amines, and the N-acylation of arylalkylamines (Tsugehara et al, 2007;Dempsey et al, 2014;O'Flynn et al, 2018). As aaNAT serves as an important regulator in a variety of physiological functions, the enzyme is considered a target for the development of new insecticides (O'Flynn et al, 2020).…”