Graduation Year


Document Type




Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department


Major Professor

David J. Merkler, Ph.D.

Committee Member

Bill J. Baker, Ph.D.

Committee Member

Ioannis Gelis, Ph.D.

Committee Member

Yu Chen, Ph.D.


AANAT, Acylation, Fatty Acid Amide, Insecticide, Tribolium castaneum


Arylalkylamine N-acyltransferases (AANATs) have in recent years been suggested as potential new insecticide targets. These promiscuous enzymes are involved in the N-acylation of biogenic amines to form N-acylamides. Mammalian AANAT is predominantly associated with circadian rhythm regulation, as it catalyzes the formation of N-acetylserotonin, the precursor of melatonin, from serotonin. In insects, this process is a key step in melanism, as well as hardening of the cuticle, removal of biogenic amines, and in the biosynthesis of fatty acid amides. The unique nature of each insect AANAT (iAANAT) isoform characterized indicates that while catalyzing similar reactions, each insect accommodates an assembly of iAANATs relatively exclusive to that organism. This implies a high potential for selectivity in insecticide design, while also maintaining polypharmacology, and would make iAANATs a valuable target to combat insecticide resistance. As highlighted by the World Health Organization, however, to develop an effective resistance management plan (RMP), intrinsic biological knowledge of existing and potential targets is vital to understand the development of resistance.

This dissertation is dedicated to the consolidation and expansion of our knowledge of iAANATs both mechanistically and structurally. Significant efforts have thus far been put in to characterizing this group of enzymes and their products in several different insects. These efforts are highlighted and are built upon with the identification and characterization of three novel iAANATs; TcAANAT1b and TcAANAT0 from Tribolium castaneum, and Bm-iAANAT3 from Bombyx mori. TcAANAT1b is a truncated form of the putative dopamine N-acetyltransferase of T. castaneum: TcAANAT1. This shortened ‘splicoform’ was shown to be a significantly more active form of the enzyme, indicating a catalytic importance of the N-terminus of iAANATs. TcAANAT0 was shown to be a much more promiscuous enzyme, catalyzing the formation of short chain N-acylarylalkylamines via ordered sequential mechanism, with short-chain acyl-CoAs (C2-C10) functioning in the role of acyl-donor. The first crystal structure was also obtained for TcAANAT0 bound to acetyl-CoA, revealing valuable information about its active site. Bm-iAANAT3 was found to be a “versatile generalist”, functioning via a random kinetic mechanism. pH-rate profiles generated for both enzymes indicate key catalytic residues which were used to solve the chemical mechanism of both TcAANAT0 and Bm-iAANAT3. Both these enzymes were also found to catalyze the formation of succinylated and malonated amides respectively, indicating a potential panel of biologically occurring N-dicarboxylated amines awaiting discovery. None of the above enzymes were found to catalyze the formation of long-chain fatty acid amides, however. The previous characterization of two enzymes, Bm-iAANAT and DmAANATL2, which do perform this role in vitro indicates that some evolutionary divergence took place to separate short and long-chain acylators. This divergence, which led to the specialization of the arylalkylamine N-acetyltransferases, was found to likely be a result of organization of the amine binding pocket, resulting in an active site less accommodating to long-chain acyl-CoAs.

The combination of kinetic analysis and crystallography, alongside phylogenetic analysis shines light on the heterogeneity of insect AANATs and garners further discussion into their core differences and some approaches possible to utilize these enzymes in insecticide design.

Included in

Biochemistry Commons