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.
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