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 acetyl-CoA) 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.
Highlights
Melatonin (5-methoxy-N-acetyltryptamine) is a pineal hormone that modulates a variety of endocrinological, neurophysiological, and behavioral functions in vertebrates [1]
Partial purification was obtained by glutathione affinity chromatography, and a 47-kDa band identified by SDSPAGE (Fig. 1, lane 4) was shown to correspond to GSThAANAT contaminated with a bacterial chaperone. hAANAT was cleaved by thrombin (Fig. 1, lane 5) to give both a 21-kDa hAANAT (Fig. 1, lane 7) and a 26-kDa GST band (Fig. 1, lane 6) after mixed affinity chromatography on benzamidine/glutathione-agarose
Western blot analysis of pure and cleaved GSThAANAT with anti-GST antibodies revealed a GST band at 47 kDa corresponding to GST-hAANAT and a 26-kDa band corresponding to GST. hAANAT was not adsorbed on the mixed affinity column and neither was the chaperone (Fig. 1, lane 7), whereas both thrombin and GST were retained (Fig. 1, lane 6)
Summary
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 conversion of N-acetyl-5-hydroxytryptamine to melatonin is catalyzed by hydroxyindole-O-methyltransferase This transferase is expressed at relatively constant levels, and the rate of this step is regulated by the availability of N-acetyl5-hydroxytryptamine. Two techniques are available for the measurement of AANAT activity, the highly sensitive, extraction-based assay of Deguchi [27] and the chromatographic assay of Thomas et al [36] The latter has been developed for and is limited to fluorescent compounds [37], whereas the former is difficult to automatize. Synthetic substrates of AANAT and new inhibitors are discovered and described
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
