Bm -iAANAT and its potential role in fatty acid amide biosynthesis in Bombyx mori

Abstract

The purpose of this research is to unravel the substrate specificity and kinetic properties of an insect arylalkylamine N-acyltransferase from Bombyx mori (Bm-iAANAT) and to determine if this enzyme will catalyze the formation of long chain N-acylarylalkylamides in vitro. However, the determination of substrates and products for Bm-iAANAT in vitro is no guarantee that these same molecules are substrates and products for the enzyme in the organism. Therefore, RT-PCR was performed to detect the Bm-iAANAT transcripts and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) analysis was performed on purified lipid extracts from B. mori larvae (fourth instar, Bmi4) to determine if long chain fatty acid amides are produced in B. mori. Ultimately, we found that recombinant Bm-iAANAT will utilize long-chain acyl-CoA thioesters as substrates and identified Bm-iAANAT transcripts and long-chain fatty acid amides in Bmi4. Together, these data show Bm-iAANAT will catalyze the formation of long-chain N-acylarylalkylamides in vitro and provide evidence demonstrating that Bm-iAANAT has a role in fatty acid amide biosynthesis in B. mori, as well.

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

“…In applying this strategy to identify amine substrates for Bm -iAANAT3, a relatively high concentration of each amine (20 mM or 60 mM, Table 1) was included in the pool because the K m value for glycine is reported at ≥6 mM for some of the glycine N -acyltransferases [38–40]. Pools of acyl-CoA substrates were not constructed because of the reports of long-chain acyl-CoAs acting as AANAT inhibitors [29,40,41]; therefore, we individually cross-screened amine pools against archetypal short and long-chain acyl-CoA thioesters, acetyl-CoA and oleoyl-CoA, because both have been reported as substrates for other GNAT family acyltransferases [20,21]. For Bm -iAANAT3, we found no initial velocity increase with oleoyl-CoA above the low enzyme-independent, background rate of oleoyl-CoA hydrolysis, suggesting that this enzyme does not catalyze the formation of long-chain N -acylamides.…”

“…While fatty acid amides have been recently identified in B. mori [21], Bm -iAANAT3 is probably not involved in their biosynthesis in vivo . Bm -iAANAT3 accepts a variety of amines as substrates pointing towards a cellular role for this enzyme in amine inactivation and/or excretion.…”

“…Our interest in the AANATs is derived from our work on the biosynthesis of the fatty acid amides [16,17], a family of cell signaling lipids [18]. We and others have found that AANAT-like (AANATL) enzymes will catalyze the formation of fatty acid amides from fatty acyl-CoA thioesters and the corresponding amines [17,19–21]. Database searches of insect genomes indicate that a number of iAANATs and iAANATLs could be expressed in a variety of insects [7,14,22].…”

“…In addition, we plan to extend our mechanistic work on the AANATs to focus on structural elucidation by NMR spectroscopy and X-ray crystallography. The other iAANATs we have over-expressed [20,21,29–32] have thwarted such experiments because these enzymes precipitate at high protein concentrations. Bm -iAANAT3 does not precipitate at high protein concentration enabling structural studies of this enzyme by NMR [33,34].…”

Insect Arylalkylamine N-Acyltransferases: Mechanism and Role in Fatty Acid Amide Biosynthesis

“…We have proposed that novel iAANATs exist which will catalyze the acyl-CoA-dependent formation of these N -fatty acylamides (Figure 2 ). AANATs in D. melanogaster (Dempsey et al, 2014a ) and B. mori (Anderson et al, 2018 ; Battistini, 2015 ) have been identified that will utilize long-chain fatty acyl-CoA thioesters as substrates leading to the production of these fatty acylamides. Thus, we suggest replacing the name “ N -acetyltransferase” with “ N -acyltransferase” to better reflect the most current data on this family of enzymes.…”