Substrate Specificity and Inhibition Studies of Human SerotoninN-Acetyltransferase

Abstract

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the reaction of serotonin with acetyl-CoA to form N-acetylserotonin and plays a major role in the regulation of the melatonin circadian rhythm in vertebrates. In the present study, the human cloned enzyme has been expressed in bacteria, purified, cleaved, and characterized. The specificity of the human enzyme toward substrates (natural as well as synthetic arylethylamines) and cosubstrates (essentially acyl homologs of acetylCoA) has been investigated. Peptide combinatorial libraries of tri-, tetra-, and pentapeptides with various amino acid compositions were also screened as potential sources of inhibitors. We report the findings of several peptides with low micromolar inhibitory potency. For activity measurement as well as for specificity studies, an original and rapid method of analysis was developed. The assay was based on the separation and detection of N-[ 3 H]acetylarylethylamine formed from various arylethylamines and tritiated acetyl-CoA, by means of high performance liquid chromatography with radiochemical detection. The assay proved to be robust and flexible, could accommodate the use of numerous synthetic substrates, and was successfully used throughout this study. We also screened a large number of pharmacological bioamines among which only one, tranylcypromine, behaved as a substrate. The synthesis and survey of simple arylethylamines also showed that AANAT has a large recognition pattern, including compounds as different as phenyl-, naphthyl-, benzothienyl-, or benzofuranyl-ethylamine derivatives. An extensive enzymatic study allowed us to pinpoint the amino acid residue of the pentapeptide inhibitor, S 34461, which interacts with the cosubstrate-binding site area, in agreement with an in silico study based on the available coordinates of the hAANAT crystal.Melatonin (5-methoxy-N-acetyltryptamine) is a pineal hormone that modulates a variety of endocrinological, neurophysiological, and behavioral functions in vertebrates (1). It is involved in the regulation of circadian rhythms and in the reproduction of photoperiodic species (2). The chronobiotic effects of melatonin in humans have been mainly studied in circadian rhythm sleep disorders (3). Moreover, alterations of the melatonin profiles have been reported in other biological rhythm disorders (3). Melatonin exerts its effects through at least three targets: 2 receptor subtypes, mt 1 and MT 2 , and a binding site, MT 3 (4). The two first ones have been cloned (5-6) and their pharmacological effects largely studied, and several specific and potent ligands (7-9) discovered. The MT 3 subtype is still a putative binding site under intensive research from purification attempts to pharmacological characterizations (10 -11). Since melatonin is implicated in several types of mild to severe pathologies, including mood disorders (3, 12), it is considered a valuable therapeutic target. Beside the classical search for agonists and antagonists of the melatonin receptors, a series of programs was launched t...

Identification of the Melatonin-binding SiteMT 3 as the Quinone Reductase 2

“…The oxidoreductive properties of the QR 2 open the way for a novel enzymatic investigation of the highly debated antioxidant properties of melatonin (for review, see Ref 27). Hence, in addition to the G protein-coupled receptors of melatonin (mt 1 and MT 2 ) and the transferase arylalkylamine N-acetyltransferase which controls the limiting step of melatonin biosynthesis (28), MT 3 /QR 2 appears as a fourth molecular target to explore the multiple facets of melatonin action.…”

Molecular evidence that melatonin is enzymatically oxidized in a different manner than tryptophan: investigations with both indoleamine 2,3-dioxygenase and myeloperoxidase

“…Grohmann et al [17]). Our laboratory group has been involved for several years in understanding the mechanisms of action of melatonin at the molecular level, including the enzymology of its biosynthesis [18][19][20][21] and its interaction with melatonin receptors [22][23][24][25]. In order to explore further the catabolism of melatonin, which to date is based on only a few reports, we expressed recombinant human IndoDO in bacteria, leading to expression of a protein tagged with a hexahistidine moiety [12].…”

New substrate analogues of human serotonin N‐acetyltransferase produce in situ specific and potent inhibitors

“…Expression, production and purification of hAANAT have been described by Ferry et al [7]. In brief, the human arylalkylamine N-acetyltransferase cDNA coding region (kindly provided by D. C. Klein and S. L. Coon 5 , NIH, Bethesda, USA) was inserted into the bacterial expression vector pGEX-4T (Pharmacia, Les Ulis, France), leading to the expression of a protein fused to glutathione-S-transferase.…”

“…Five classes of hAANAT inhibitors have been reported in the literature to date: loose structural analogues of the indoleamine hormone [9,10], tetrapeptides issued from the deconvolution of combinatorial libraries [7], heterocyclic compounds from a large screen of natural plant extracts and synthetic molecules [11], bisubstrate analogues (i.e. Coenzyme A derivatives) [12] and the N-bromoacetamido derivatives (precursors of potent inhibitors) [13].…”

“…The synthesis of melatonin from serotonin is directed by a series of enzymes, of which arylalkylamine (serotonin) N-acetyltransferase (AANAT; EC 2.3.1.87) catalyses the penultimate and rate-limiting step. The human AANAT enzyme has been cloned [6] and purified, and the specificity of its substrates and cosubstrates have been described [7]. A large program of chemistry and screening was launched in order to discover potent inhibitors of this particular enzyme and to investigate the effects of the reduction (or total inhibition) of nocturnal melatonin production.…”