Bm-iAANAT3: Expression and characterization of a novel arylalkylamine N-acyltransferase from Bombyx mori

• Published in: Archives of biochemistry and biophysics 2019-01, Vol.661, p.107-116
• Main Authors: Battistini, Matthew R., O'Flynn, Brian G., Shoji, Christopher, Suarez, Gabriela, Galloway, Lamar C., Merkler, David J.
• Format: Article
• Language: English
• Subjects: AANAT; Acetylation; acyl coenzyme A; amines; amino acids; Animals; Arylalkylamine N-Acetyltransferase - biosynthesis; Arylalkylamine N-Acetyltransferase - chemistry; Arylalkylamine N-Acetyltransferase - genetics; Bombyx - enzymology; Bombyx - genetics; Bombyx mori; carbon; catalytic activity; Chemical mechanism; excretion; Gene Expression; insect pests; Insect Proteins - biosynthesis; Insect Proteins - chemistry; Insect Proteins - genetics; Kinetic mechanism; Kinetics; melanosis; N-acylarylakylamine; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; sclerotization; site-directed mutagenesis; Substrate Specificity; thioesters; transferases
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2018.11.015

The arylalkylamine N-acyltransferases (AANATs) are enzymes that catalyze the acyl-CoA-dependent formation of N-acylarylalkylamides: acyl-CoA + arylalkylamine → N-acylarylalkylamides + CoA-SH. Herein, we describe our study of a previously uncharacterized AANAT from Bombyx mori: Bm-iAANAT3. Bm-iAANAT3 catalyzes the direct formation of N-acylarylalkylamides and accepts a broad range of short-chain acyl-CoA thioesters and amines as substrates. Acyl-CoA thioesters possessing an acyl chain length >10 carbon atoms are not substrates for Bm-iAANAT3. We report that Bm-iAANAT3 is a “versatile generalist”, most likely, functioning in amine acetylation – a reaction in amine inactivation/excretion, cuticle sclerotization, and melanism. We propose a kinetic and chemical mechanism for Bm-iAANAT3 that is consistent with our steady-state kinetic analysis, dead-end inhibition studies, determination of the pH-rate profiles, and site-directed mutagenesis of a catalytically important amino acid in Bm-iAANAT3. These mechanistic studies of Bm-iAANAT3 will foster the development of novel compounds targeted against this enzyme and other insect AANATs for the control of insect pests. [Display omitted] •Bm-iAANAT3 exhibits promiscuous substrate specificity for acyl-CoA and amine substrates.•Acetylation occurs via ordered sequential mechanism, with acetyl-CoA binding first and acetylated amine released last.•pH-activity profiles for Bm-iAANAT3 suggest a chemical mechanism with Glu-27 functioning as a base during catalysis.