Characterization of Arylalkylamine N-Acyltransferase from Tribolium castaneum: An Investigation into a Potential Next-Generation Insecticide Target.

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.

Cloning and characterization of a serotonin N-acetyltransferase from a gymnosperm, loblolly pine (Pinus taeda).

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis in both animals and plants. SNAT catalyzes serotonin into N-acetylserotonin, an immediate precursor for melatonin biosynthesis by N-acetylserotonin methyltransferase (ASMT). We cloned the SNAT gene from a gymnosperm loblolly pine (Pinus teada). The loblolly pine SNAT (PtSNAT) gene encodes 255 amino acids harboring a transit sequence with 67 amino acids and shows 67% amino acid identity with rice SNAT when comparing the mature polypeptide regions. Purified recombinant PtSNAT showed peak activity at 55°C with the K(m) (428 µM) and Vmax (3.9 nmol/min/mg protein) values. As predicted, PtSNAT localized to chloroplasts. The SNAT mRNA was constitutively expressed in all tissues, including leaf, bud, flower, and pinecone, whereas the corresponding protein was detected only in leaf. In accordance with the exclusive SNAT protein expression in leaf, melatonin was detected only in leaf at 0.45 ng per gram fresh weight. Sequence and phylogenetic analysis indicated that the gymnosperm PtSNAT had high homology with SNATs from all plant phyla (even with cyanobacteria), and formed a clade separated from the angiosperm SNATs, suggestive of direct gene transfer from cyanobacteria via endosymbiosis.

Analysis of serotonin N-acetyltransferase regulation in vitro and in live cells using protein semisynthesis.

Serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT)] is a key circadian rhythm enzyme that drives the nocturnal production of melatonin in the pineal. Prior studies have suggested that its light and diurnal regulation involves phosphorylation on key AANAT Ser and Thr residues which results in 14-3-3zeta recruitment and changes in catalytic activity and protein stability. Here we use protein semisynthesis by expressed protein ligation to systematically explore the effects of single and dual phosphorylation of AANAT on acetyltransferase activity and relative affinity for 14-3-3zeta. AANAT Thr31 phosphorylation on its own can enhance catalytic efficiency up to 7-fold through an interaction with 14-3-3zeta that lowers the substrate K m. This augmented catalytic profile is largely abolished by double phosphorylation at Thr31 and Ser205. A possible basis for this difference is the dual anchoring of doubly phosphorylated AANAT via one 14-3-3zeta heterodimer. We have developed a novel solution phase assay for accurate K D measurements of 14-3-3zeta-AANAT interaction using 14-3-3zeta fluorescently labeled with rhodamine by expressed protein ligation. We have also generated a doubly fluorescently labeled AANAT which can be used to assess the stability of this protein in a live cell, real-time assay by fluorescence resonance energy transfer measured by microscopic imaging. These studies offer new insights into the molecular basis of melatonin regulation and 14-3-3zeta interaction.

Human placental trophoblasts synthesize melatonin and express its receptors.

Although the role of melatonin on fetal development has been the subject of a number of studies, little is known about the function of melatonin in the placenta. We previously showed that melatonin receptors are expressed and are functional in JEG-3 and BeWo cell lines, both in vitro models of human trophoblast. Local synthesis of melatonin in placenta has been proposed, but the human placenta's ability to synthesize melatonin de novo has never been studied. The purpose of this study was to investigate the expression [reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis] and activity (radiometric assay) of melatonin synthesizing enzymes, and characterize the expression of the melatoninergic receptors in human term villous trophoblast. The results show that arylalkylamine N-acetyltransferase and hydroxyindole O-methyltransferase melatonin synthesizing enzymes are expressed and active in villous trophoblast as well as in JEG-3 and BeWo placental choriocarcinoma cells. In addition, immunohistochemical analysis demonstrated the presence of MT1, MT2, and retinoid-related orphan nuclear receptor alpha melatonin receptor proteins in both villous cytotrophoblast and syncytiotrophoblast (STB) as well as in endothelial cells surrounding the fetal capillaries and in the villous mesenchymal core. RT-PCR and western blot analysis in primary cultures of human term trophoblast confirmed the expression of all three melatonin receptors in villous cytotrophoblast and STB cells. This study demonstrates for the first time a local synthesis of melatonin and expression of its receptors in human trophoblasts and strongly suggests a paracrine, autocrine, and/or intracrine role for this indolamine in placental function and development as well as in protection from oxidative stress.

Arylalkylamine N-acetyltransferase: "the Timezyme".

Arylalkylamine N-acetyltransferase controls daily changes in melatonin production by the pineal gland and thereby plays a unique role in biological timing in vertebrates. Arylalkylamine N-acetyltransferase is also expressed in the retina, where it may play other roles in addition to signaling, including neurotransmission and detoxification. Large changes in activity reflect cyclic 3',5'-adenosine monophosphate-dependent phosphorylation of arylalkylamine N-acetyltransferase, leading to formation of a regulatory complex with 14-3-3 proteins. This activates the enzyme and prevents proteosomal proteolysis. The conserved features of regulatory systems that control arylalkylamine N-acetyltransferase are a circadian clock and environmental lighting.

Protein kinase structure and function analysis with chemical tools.

Protein kinases are the largest enzyme superfamily involved in cell signal transduction and represent therapeutic targets for a range of diseases. There have been intensive efforts from many labs to understand their catalytic mechanisms, discover inhibitors and discern their cellular functions. In this review, we will describe two approaches developed to analyze protein kinases: bisubstrate analog inhibition and phosphonate analog utilization. Both of these methods have been used in combination with the protein semisynthesis method expressed protein ligation to advance our understanding of kinase-substrate interactions and functional elucidation of phosphorylation. Previous work on the nature of the protein kinase mechanism suggests it follows a dissociative transition state. A bisubstrate analog was designed against the insulin receptor kinase to mimic the geometry of a dissociative transition state reaction coordinate distance. This bisubstrate compound proved to be a potent inhibitor against the insulin receptor kinase and occupied both peptide and nucleotide binding sites. Bisubstrate compounds with altered hydrogen bonding potential as well as varying spacers between the adenine and the peptide demonstrate the importance of the original design features. We have also shown that related bisubstrate analogs can be used to potently block serine/threonine kinases including protein kinase A. Since many protein kinases recognize folded protein substrates for efficient phosphorylation, it was advantageous to incorporate the peptide-ATP conjugates into protein structures. Using expressed protein ligation, a Src-ATP conjugate was produced and shown to be a high affinity ligand for the Csk tyrosine kinase. Nonhydrolyzable mimics of phosphoSer/phosphoTyr can be useful in examining the functionality of phosphorylation events. Using expressed protein ligation, we have employed phosphonomethylene phenylalanine and phosphonomethylene alanine to probe the phosphorylation of Tyr and Ser, respectively. These tools have permitted an analysis of the SH2-phosphatases (SHP1 and SHP2), revealing a novel intramolecular stimulation of catalytic activity mediated by the corresponding phosphorylation events. They have also been used to characterize the cellular regulation of the melatonin rhythm enzyme by phosphorylation.

Cellular stability of serotonin N-acetyltransferase conferred by phosphonodifluoromethylene alanine (Pfa) substitution for Ser-205.

Large changes in the activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) in the pineal gland control the rhythmic production of the time-keeping hormone melatonin. The activity of AANAT reflects changes in the amount and activation state of the AANAT protein, both of which increase at night. The molecular basis of this regulation is now becoming known, and recent data indicate that this involves phosphorylation-dependent binding to the 14-3-3 protein at two sites, one of which, Ser-205, is located several residues from the C terminus. In this study, we determined whether substitution of this residue with a non-hydrolyzable the phosphoserine/phosphothreonine mimetic would promote binding to the 14-3-3 protein and enhance cellular stability. To accomplish this, a C-terminal AANAT peptide containing the phosphonodifluoromethylene alanine at Ser-205 was synthesized and fused to bacterially expressed AANAT(30-199) using expressed protein ligation. The resulting semisynthetic protein has enhanced affinity for the expressed 14-3-3 protein and exhibits greater cellular stability in microinjection experiments, as compared with the unmodified AANAT. Enhanced 14-3-3 binding was also observed using humanized ovine AANAT, which has a different C-terminal sequence (Gly-Cys) than the ovine enzyme (Asp-Arg), indicating that that characteristic is not unique to the ovine enzyme. These studies provide the first evidence that substitution of Ser-205 with the stable phosphomimetic amino acid phosphonodifluoromethylene alanine enhances binding to 14-3-3 and the cellular stability of AANAT and are consistent with the view that Ser-205 phosphorylation plays a critical role in the regulation of AANAT activity and melatonin production.

Inactivation of human arylamine N-acetyltransferase 1 by hydrogen peroxide and peroxynitrite.

Arylamine N-acetyltransferases (NAT) are xenobiotic-metabolizing enzymes responsible for the acetylation of many arylamine and heterocyclic amines. They therefore play an important role in the detoxification and activation of numerous drugs and carcinogens. Two closely related isoforms (NAT1 and NAT2) have been described in humans. NAT2 is present mainly in the liver and intestine, whereas NAT1 is found in a wide range of tissues. Interindividual variations in NAT genes have been shown to be a potential source of pharmacological and/or pathological susceptibility. Evidence now shows that redox conditions may also contribute to overall NAT activity. This chapter summarizes current knowledge on human NAT1 regulation by reactive oxygen and nitrogen species.