Role of arylalkylamine N-acetyltransferase 7 in reproduction and limb pigmentation of Aedes aegypti.

Arylalkylamine N-acetyltransferase (aaNAT) is a crucial enzyme that catalyses the transfer of acetyl groups from acetyl coenzyme A to arylalkylamines and arylamines. Evolutionary studies have identified a distinct class of aaNATs specific to mosquitoes, yet their functions remain elusive. This study focuses on Ae-aaNAT7, a mosquito-unique gene in Aedes aegypti (Diptera:Culicidae), to explore its functionality. Temporal and spatial expression analysis of Ae-aaNAT7 mRNA revealed high expression during embryonic development and in first-instar larvae, with notable expression in the limbs of adult mosquitoes based on tissue expression profiling. By further employing CRISPR/Cas9 technology for loss-of-function studies, our investigation revealed a reduction in the area of white spotting in the limbs of Ae-aaNAT7 mutant adult mosquitoes. Further investigation revealed a significant decrease in the fecundity and hatchability of the mutants. Dissection of the ovaries from Ae-aaNAT7 heterozygous mutants showed a noticeable reduction in the oocyte area compared with wild type. Dissection of the exochorion of the eggs from Ae-aaNAT7 homozygous mutants consistently revealed a striking absence of mature embryos. In addition, RNA interference experiments targeting Ae-aaNAT7 in males resulted in a reduction in fecundity, but no effect on hatchability was observed. These collective insights underscore the substantial impact of Ae-aaNAT7 on reproduction and its pivotal contribution to adult limb pigmentation in Ae. aegypti. These revelations offer insights pivotal for the strategic design of future insecticide targets.

Synthesis, Quantification, and Characterization of Fatty Acid Amides from In Vitro and In Vivo Sources.

Fatty acid amides are a diverse family of underappreciated, biologically occurring lipids. Herein, the methods for the chemical synthesis and subsequent characterization of specific members of the fatty acid amide family are described. The synthetically prepared fatty acid amides and those obtained commercially are used as standards for the characterization and quantification of the fatty acid amides produced by biological systems, a fatty acid amidome. The fatty acid amidomes from mouse N18TG2 cells, sheep choroid plexus cells, Drosophila melanogaster, Bombyx mori, Apis mellifera, and Tribolium castaneum are presented.

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

The assumption that structural or sequential homology between enzymes implies functional homology is a common misconception. Through in-depth structural and kinetic analysis, we are now beginning to understand the minute differences in primary structure that can alter the function of an enzyme completely. Alternative splicing is one method for which the activity of an enzyme can be controlled, simply by altering its length. Arylalkylamine N-acetyltransferase A (AANATA) in D. melanogaster, which catalyzes the N-acetylation of biogenic amines, has multiple splicoforms - alternatively spliced enzyme isoforms - with differing tissue distribution. As demonstrated here, AANAT1 from Tribolium castaneum is another such enzyme with multiple splicoforms. A screening assay was developed and utilized to determine that, despite only a 35 amino acid truncation, the shortened form of TcAANAT1 is a more active form of the enzyme. This implies regulation of enzyme metabolic activity via alternative splicing.

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

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.

Knockdown of arylalkylamine N-acetyltransferase-like 2 in Drosophila melanogaster.

Drosophila melanogaster produces fatty acid amides, and thus, provides a model to unravel the pathways for their biosynthesis. We previously demonstrated that arylalkylamine N-acetyltransferase-like 2 (AANATL2) from D. melanogaster will catalyze the formation of long-chain N-acylserotonins and N-acyldopamines in vitro. Generating silencing RNA via the UAS/GAL4 bipartite approach for targeted gene expression effectively decreased the endogenous levels of the AANATL2 transcripts in D. melanogaster, as shown by reverse transcription quantitative polymerase chain reaction. Consistent with these data, western blot analysis of the offspring of the AANATL2 knockdown flies using an anti-AANATL2 antibody revealed a significant reduction in the expression of the AANATL2 protein. Reduced expression of AANATL2 decreased the cellular levels of N-palmitoyldopamine (PALDA), providing strong evidence that AANATL2 is responsible for the biosynthesis of PALDA in vivo. This is the first time that the expression of an AANAT has been reduced in D. melanogaster to link one of these enzymes to the in vivo production of an N-acylarylalkylamide.

Primary fatty amides in plasma associated with brain amyloid burden, hippocampal volume, and memory in the European Medical Information Framework for Alzheimer's Disease biomarker discovery cohort.

INTRODUCTION: A critical and as-yet unmet need in Alzheimer's disease (AD) is the discovery of peripheral small molecule biomarkers. Given that brain pathology precedes clinical symptom onset, we set out to test whether metabolites in blood associated with pathology as indexed by cerebrospinal fluid (CSF) AD biomarkers. METHODS: This study analyzed 593 plasma samples selected from the European Medical Information Framework for Alzheimer's Disease Multimodal Biomarker Discovery study, of individuals who were cognitively healthy (n = 242), had mild cognitive impairment (n = 236), or had AD-type dementia (n = 115). Logistic regressions were carried out between plasma metabolites (n = 883) and CSF markers, magnetic resonance imaging, cognition, and clinical diagnosis. RESULTS: Eight metabolites were associated with amyloid ß and one with t-tau in CSF, these were primary fatty acid amides (PFAMs), lipokines, and amino acids. From these, PFAMs, glutamate, and aspartate also associated with hippocampal volume and memory. DISCUSSION: PFAMs have been found increased and associated with amyloid ß burden in CSF and clinical measures.

Glycine N-acyltransferase-like 3 is responsible for long-chain N-acylglycine formation in N18TG2 cells.

Long-chain fatty acid amides are signaling lipids found in mammals and other organisms; however, details of the metabolic pathways for the N-acylglycines and primary fatty acid amides (PFAMs) have remained elusive. Heavy-labeled precursor and subtraction lipidomic experiments in mouse neuroblastoma N18TG2 cells, a model cell line for the study of fatty acid amide metabolism, establish the biosynthetic pathways for the N-acylglycines and the PFAMs. We provide evidence that the N-acylglycines are formed by a long-chain specific glycine-conjugating enzyme, glycine N-acyltransferase-like 3 (GLYATL3). siRNA knockdown of GLYATL3 in the N18TG2 cells resulted in a decrease in the levels of the N-acylglycines and the PFAMs. This is the first report of an enzyme responsible for long-chain N-acylglycine production in cellula. The production of the PFAMs in N18TG2 cells was reported to occur by the oxidative cleavage of the N-acylglycines, as catalyzed by peptidylglycine α-amidating monooxygenase (PAM). siRNA knockdown of PAM resulted in an accumulation of [(13)C18]N-oleoylglycine and decreased levels of [(13)C18]oleamide when the N18TG2 cells were grown in the presence of [(13)C18]oleic acid. The addition of [1-(13)C]palmitate to the N18TG2 cell growth media led to the production of a family of [1-(13)C]palmitoylated fatty acid amides, consistent with the biosynthetic pathways detailed herein.

Mechanistic binding insights for 1-deoxy-D-Xylulose-5-Phosphate synthase, the enzyme catalyzing the first reaction of isoprenoid biosynthesis in the malaria-causing protists, Plasmodium falciparum and Plasmodium vivax.

We have successfully truncated and recombinantly-expressed 1-deoxy-D-xylulose-5-phosphate synthase (DXS) from both Plasmodium vivax and Plasmodium falciparum. We elucidated the order of substrate binding for both of these ThDP-dependent enzymes using steady-state kinetic analyses, dead-end inhibition, and intrinsic tryptophan fluorescence titrations. Both enzymes adhere to a random sequential mechanism with respect to binding of both substrates: pyruvate and D-glyceraldehyde-3-phosphate. These findings are in contrast to other ThDP-dependent enzymes, which exhibit classical ordered and/or ping-pong kinetic mechanisms. A better understanding of the kinetic mechanism for these two Plasmodial enzymes could aid in the development of novel DXS-specific inhibitors that might prove useful in treatment of malaria.

Inactivation of peptidylglycine α-hydroxylating monooxygenase by cinnamic acid analogs.

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the final reaction in the maturation of α-amidated peptide hormones. Peptidylglycine α-hydroxylating monooxygenase (PHM) is the PAM domain responsible for the copper-, ascorbate- and O2-dependent hydroxylation of a glycine-extended peptide. Peptidylamidoglycolate lyase is the PAM domain responsible for the Zn(II)-dependent dealkylation of the α-hydroxyglycine-containing precursor to the final α-amidated peptide. We report herein that cinnamic acid and cinnamic acid analogs are inhibitors or inactivators of PHM. The inactivation chemistry exhibited by the cinnamates exhibits all the attributes of a suicide-substrate. However, we find no evidence for the formation of an irreversible linkage between cinnamate and PHM in the inactivated enzyme. Our data support the reversible formation of a Michael adduct between an active site nucleophile and cinnamate that leads to inactive enzyme. Our data are of significance given that cinnamates are found in foods, perfumes, cosmetics and pharmaceuticals.

Mechanistic and Structural Analysis of a Drosophila melanogaster Enzyme, Arylalkylamine N-Acetyltransferase Like 7, an Enzyme That Catalyzes the Formation of N-Acetylarylalkylamides and N-Acetylhistamine.

Arylalkylamine N-acetyltransferase like 7 (AANATL7) catalyzes the formation of N-acetylarylalkylamides and N-acetylhistamine from acetyl-CoA and the corresponding amine substrate. AANATL7 is a member of the GNAT superfamily of >10000 GCN5-related N-acetyltransferases, many members being linked to important roles in both human metabolism and disease. Drosophila melanogaster utilizes the N-acetylation of biogenic amines for the inactivation of neurotransmitters, the biosynthesis of melatonin, and the sclerotization of the cuticle. We have expressed and purified D. melanogaster AANATL7 in Escherichia coli and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Information about the substrate specificity provides insight into the potential contribution made by AANATL7 to fatty acid amide biosynthesis because D. melanogaster has emerged as an important model system contributing to our understanding of fatty acid amide metabolism. Characterization of the kinetic mechanism of AANATL7 identified an ordered sequential mechanism, with acetyl-CoA binding first followed by histamine to generate an AANATL7·acetyl-CoA·histamine ternary complex prior to catalysis. Successive pH-activity profiling and site-directed mutagenesis experiments identified two ionizable groups: one with a pKa of 7.1 that is assigned to Glu-26 as a general base and a second pKa of 9.5 that is assigned to the protonation of the thiolate of the coenzyme A product. Using the data generated herein, we propose a chemical mechanism for AANATL7 and define functions for other important amino acid residues involved in substrate binding and regulation of catalysis.

Mechanistic and structural analysis of Drosophila melanogaster arylalkylamine N-acetyltransferases.

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of melatonin and other N-acetylarylalkylamides from the corresponding arylalkylamine and acetyl-CoA. The N-acetylation of arylalkylamines is a critical step in Drosophila melanogaster for the inactivation of the bioactive amines and the sclerotization of the cuticle. Two AANAT variants (AANATA and AANATB) have been identified in D. melanogaster, in which AANATA differs from AANATB by the truncation of 35 amino acids from the N-terminus. We have expressed and purified both D. melanogaster AANAT variants (AANATA and AANATB) in Escherichia coli and used the purified enzymes to demonstrate that this N-terminal truncation does not affect the activity of the enzyme. Subsequent characterization of the kinetic and chemical mechanism of AANATA identified an ordered sequential mechanism, with acetyl-CoA binding first, followed by tyramine. We used a combination of pH-activity profiling and site-directed mutagenesis to study prospective residues believed to function in AANATA catalysis. These data led to an assignment of Glu-47 as the general base in catalysis with an apparent pKa of 7.0. Using the data generated for the kinetic mechanism, structure-function relationships, pH-rate profiles, and site-directed mutagenesis, we propose a chemical mechanism for AANATA.

Expression, purification, and characterization of mouse glycine N-acyltransferase in Escherichia coli.

Glycine N-acyltransferase (GLYAT) is a phase II metabolic detoxification enzyme for exogenous (xenobiotic) and endogenous carboxylic acids; consisting of fatty acids, benzoic acid, and salicylic acid. GLYAT catalyzes the formation of hippurate (N-benzoylglycine) from the corresponding glycine and benzoyl-CoA. Herein, we report the successful expression, purification, and characterization of recombinant mouse GLYAT (mGLYAT). A 34kDa mGLYAT protein was expressed in Escherichia coli and purified to homogeneity by nickel affinity chromatography to a final yield of 2.5mg/L culture. Characterization for both amino donors and amino acceptors were completed, with glycine serving as the best amino donor substrate, (kcat/Km)app=(5.2±0.20)×10(2)M(-1)s(-1), and benzoyl-CoA serving as the best the amino acceptor substrate, (kcat/Km)app=(4.5±0.27)×10(5)M(-1)s(-1). Our data demonstrate that mGLYAT will catalyzed the chain length specific (C2-C6) formation of N-acylglycines. The steady-state kinetic constants determined for recombinant mGLYAT for the substrates benzoyl-CoA and glycine, were shown to be consistent with other reported species (rat, human, bovine, ovine, and rhesus monkey). The successful recombinant expression and purification of mGLYAT can lead to solve unanswered questions associated with this enzyme, consisting of what is the chemical mechanism and what catalytic residues are essential for the how this phase II metabolic detoxification enzyme conjugates glycine to xenobiotic and endogenous carboxylic acids.

Function, structure, evolution, regulation of a potent drug target, arylalkylamine N-acetyltransferase.

Arylalkylamine N-acetyltransferase (aaNAT) catalyzes the transacetylation of acetyl coenzyme A to arylamines and arylalkylamines. Based on three-dimensional structural information, aaNAT belongs to the GCN5-related N-acetyltransferases superfamily with a conserved acetyl-CoA binding domain (Dyda et al., 2000). By comparison of sequence similarity, aaNAT is usually divided into vertebrate aaNAT (VT-aaNAT) and non-vertebrate aaNAT (NV-aaNAT) (Cazaméa-Catalan et al., 2014). Insects have evolved multiple aaNATs in comparison to mammals, thus more diverse functions are also reflected in insects. This chapter will summarize previous studies on the function, regulation, structure and evolution of aaNAT, and provide insight into future pest management.

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

Arylalkylamine N-acetyltransferase (aaNAT), considered a potential new insecticide target, catalyzes the acetylation of arylalkylamine substrates such as serotonin and dopamine and, hence, mediates diverse functions in insects. However, the origin of insect aaNATs (iaaNATs) and the evolutionary process that generates multiple aaNATs in mosquitoes remain largely unknown. Here, we have analyzed the genomes of 33 species to explore and expand our understanding of the molecular evolution of this gene family in detail. We show that aaNAT orthologs are present in Bacteria, Cephalochordata, Chondrichthyes, Cnidaria, Crustacea, Mammalia, Placozoa, and Teleoste, as well as those from a number of insects, but are absent in some species of Annelida, Echinozoa, and Mollusca as well as Arachnida. Particularly, more than 10 aaNATs were detected in the Culicinae subfamily of mosquitoes. Molecular evolutionary analysis of aaNAT/aaNAT-like genes in mosquitoes reveals that tandem duplication events led to gene expansion in the Culicinae subfamily of mosquitoes more than 190 million years ago. Further selection analysis demonstrates that mosquito aaNATs evolved under strongly positive pressures that generated functional diversity following gene duplication events. Overall, this study may provide novel insights into the molecular evolution of the aaNAT family in mosquitoes.

A mass spectrometry-based method to screen for α-amidated peptides.

Amidation is a post-translational modification found at the C-terminus of ~50% of all neuropeptide hormones. Cleavage of the C(α)-N bond of a C-terminal glycine yields the α-amidated peptide in a reaction catalyzed by peptidylglycine α-amidating monooxygenase (PAM). The mass of an α-amidated peptide decreases by 58 Da relative to its precursor. The amino acid sequences of an α-amidated peptide and its precursor differ only by the C-terminal glycine meaning that the peptides exhibit similar RP-HPLC properties and tandem mass spectral (MS/MS) fragmentation patterns. Growth of cultured cells in the presence of a PAM inhibitor ensured the coexistence of α-amidated peptides and their precursors. A strategy was developed for precursor and α-amidated peptide pairing (PAPP): LC-MS/MS data of peptide extracts were scanned for peptide pairs that differed by 58 Da in mass, but had similar RP-HPLC retention times. The resulting peptide pairs were validated by checking for similar fragmentation patterns in their MS/MS data prior to identification by database searching or manual interpretation. This approach significantly reduced the number of spectra requiring interpretation, decreasing the computing time required for database searching and enabling manual interpretation of unidentified spectra. Reported here are the α-amidated peptides identified from AtT-20 cells using the PAPP method.

Primary fatty acid amide metabolism: conversion of fatty acids and an ethanolamine in N18TG2 and SCP cells.

Primary fatty acid amides (PFAM) are important signaling molecules in the mammalian nervous system, binding to many drug receptors and demonstrating control over sleep, locomotion, angiogenesis, and many other processes. Oleamide is the best-studied of the primary fatty acid amides, whereas the other known PFAMs are significantly less studied. Herein, quantitative assays were used to examine the endogenous amounts of a panel of PFAMs, as well as the amounts produced after incubation of mouse neuroblastoma N(18)TG(2) and sheep choroid plexus (SCP) cells with the corresponding fatty acids or N-tridecanoylethanolamine. Although five endogenous primary amides were discovered in the N(18)TG(2) and SCP cells, a different pattern of relative amounts were found between the two cell lines. Higher amounts of primary amides were found in SCP cells, and the conversion of N-tridecanoylethanolamine to tridecanamide was observed in the two cell lines. The data reported here show that the N(18)TG(2) and SCP cells are excellent model systems for the study of PFAM metabolism. Furthermore, the data support a role for the N-acylethanolamines as precursors for the PFAMs and provide valuable new kinetic results useful in modeling the metabolic flux through the pathways for PFAM biosynthesis and degradation.

Production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli.

Most mammalian bioactive peptides possess a C-terminal amino acid amide moiety. The presence of the C-terminal amide is a significant impediment to the recombinant production of α-amidated peptides. α-Amidated peptides are produced in vivo by the enzymatic cleavage of a precursor with a C-terminal glycine residue. Peptidylglycine α-hydroxylating monooxygenase catalyzes the key step in the oxidation of the glycine-extended precursors to the α-amidated peptide. Herein, we detail the production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli possessing a N-terminal fusion to thioredoxin (Trx). Trx was fused to hPHMcc to enhance the yield of the resulting 52 kDa protein as a soluble and catalytically active enzyme. The Trx-hPHMcc-His(6) fusion was purified to homogeneity and exhibited steady-state kinetic parameters that were similar to purified rat PHMcc. The bacterial production of recombinant hPHMcc will foster efforts to generate α-amidated peptides by the co-expression of hPHMcc and the α-amidated peptide precursors in E. coli or the in vitro amidation of recombinantly expressed α-amidated peptide precursors.

Peptidylglycine α-amidating monooxygenase as a therapeutic target or biomarker for human diseases.

Peptides play a key role in controlling many physiological and neurobiological pathways. Many bioactive peptides require a C-terminal α-amide for full activity. The bifunctional enzyme catalysing α-amidation, peptidylglycine α-amidating monooxygenase (PAM), is the sole enzyme responsible for amidated peptide biosynthesis, from Chlamydomonas reinhardtii to Homo sapiens. Many neuronal and endocrine functions are dependent upon amidated peptides; additional amidated peptides are growth promoters in tumours. The amidation reaction occurs in two steps, glycine α-hydroxylation followed by dealkylation to generate the α-amide product. Currently, most potentially useful inhibitors target the first reaction, which is rate-limiting. PAM is a membrane-bound enzyme that visits the cell surface during peptide secretion. PAM is then used again in the biosynthetic pathway, meaning that cell-impermeable inhibitors or inactivators could have therapeutic value for the treatment of cancer or psychiatric abnormalities. To date, inhibitor design has not fully exploited the structures and mechanistic details of PAM.

N-acylethanolamines as novel alcohol dehydrogenase 3 substrates.

N-acylethanolamines (NAEs) are members of the fatty acid amide family. The NAEs have been proposed to serve as metabolic precursors to N-acylglycines (NAGs). The sequential oxidation of the NAEs by an alcohol dehydrogenase and an aldehyde dehydrogenase would yield the N-acylglycinals and/or the NAGs. Alcohol dehydrogenase 3 (ADH3) is one enzyme that might catalyze this reaction. To define a potential role for ADH3 in NAE catabolism, we synthesized a set of NAEs and evaluated these as ADH3 substrates. NAEs were oxidized by ADH3, yielding the N-acylglycinals as the product. The (V/K)(app) values for the NAEs included here were low relative to cinnamyl alcohol. Our data show that the NAEs can serve as alcohol dehydrogenase substrates.

The Biosynthesis and Metabolism of the N-Acylated Aromatic Amino Acids: N-Acylphenylalanine, N-Acyltyrosine, N-Acyltryptophan, and N-Acylhistidine.

The fatty acid amides are a family of lipids composed of two chemical moieties, a fatty acid and a biogenic amine linked together in an amide bond. This lipid family is structurally related to the endocannabinoid anandamide (N-arachidonoylethanolamine) and, thus, is frequently referred to as a family of endocannabinoid-related lipids. The fatty acid amide family is divided into different classes based on the conjugate amine; anandamide being a member of the N-acylethanolamine class (NAE). Another class within the fatty acid amide family is the N-acyl amino acids (NA-AAs). The focus of this review is a sub-class of the NA-AAs, the N-acyl aromatic amino acids (NA-ArAAs). The NA-ArAAs are not broadly recognized, even by those interested in the endocannabinoids and endocannabinoid-related lipids. Herein, the NA-ArAAs that have been identified from a biological source will be highlighted and pathways for their biosynthesis, degradation, enzymatic modification, and transport will be presented. Also, information about the cellular functions of the NA-ArAAs will be placed in context with the data regarding the identification and metabolism of these N-acylated amino acids. A review of the current state-of-knowledge about the NA-ArAAs is to stimulate future research about this underappreciated sub-class of the fatty acid amide family.