Isolation and characterization of an arylalkylamine <i>N</i>‐acetyltransferase from <i>Drosophila melanogaster</i>

Hintermann, Edith; Jenö, Paul; Meyer, Urs

doi:10.1016/0014-5793(95)01198-n

Cited by 48 publications

(34 citation statements)

References 25 publications

(23 reference statements)

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“…2, lane B*), consistent with the presence of related genes. During our purification of aaNAT1 based on tryptamine acetylation activity (20), fractions with biphasic enzyme kinetics, separation of two activity peaks on hydroxylapatite columns, and evidence for two additional protein spots after two-dimensional SDS/ PAGE also suggested the presence of additional aaNAT enzymes (data not shown).…”

Section: Resultsmentioning

confidence: 88%

“…With the primer combination sp2O/apG, a PCR product was isolated that was 298-bp long and contained the sequences coding for all four internal peptides identified in aaNAT1 (Fig. IA) (20). Surprisingly, this DNA fragment contained no poly(A) tail, presumably due to amplification by only one primer (26).…”

Section: Resultsmentioning

confidence: 99%

“…Extracts were prepared by homogenization either of whole flies (1 g per 5 ml) or of heads only (25 heads per 500 ,ul), and aaNAT activity was measured as described (20). Melatonin concentration was determined by RIA (Nichols Institute, Geneva).…”

Section: Methodsmentioning

confidence: 99%

“…aaNAT activity has been described in several insect species (14)(15)(16)(17)(18), including D. melanogaster (2,19), but none of these enzymes could be purified to derive antibodies or sufficient internal sequence information for cloning. We have purified an aaNAT from D. melanogaster using tryptamine acetylation as a functional enzyme assay (20). In the present study, we document the isolation of the corresponding cDNA (aaNAT1) encoding an enzymatically active aaNAT, and discuss the presence of several aaNAT enzymes in D. melanogaster.…”

mentioning

confidence: 94%

“…Three days after transfection, the monolayers were rinsed with PBS (137 mM NaCl/8 mM Na2HPO412 H20/2.7 mM KCl/1.47 mM KH2PO4, pH 7.2) and harvested in 400 ,lI ice-cold homogenization buffer (20). Cells were sonicated on ice and centrifuged at 15,000 g for 5 min, and enzyme activity was measured (20) Northern blot analysis was performed with the radiolabeled 757-bp probe using 20 ,ug total RNA from whole flies to assess transcript length and with 10 ,g total RNA from heads only for the day/night experiment. The same high stringency conditions as described for Southern blot analysis were used.…”

mentioning

confidence: 99%

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Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut.

Hintermann

Grieder

Amherd

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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In insects, neurotransmitter catabolism, melatonin precursor formation, and sclerotization involve arylalkylamine N-acetyltransferase (aaNAT, EC 2.3.1.87) activity. It is not known if one or multiple aaNAT enzymes are responsible for these activities. We recently have purified an aaNAT from Drosophila melanogaster. Here, we report the cloning of the corresponding aaNAT cDNA (aaNAT1) that upon COS cell expression acetylates dopamine, tryptamine, and the immediate melatonin precursor serotonin. aaNAT1 represents a novel gene family unrelated to known acetyltransferases, except in two weakly conserved amino acid motifs. In situ hybridization studies of aaNAT1 mRNA in embryos reveal hybridization signals in the brain, the ventral cord, the gut, and probably in oenocytes, indicating a broad tissue distribution of aaNAT1 transcripts. Moreover, in day/ night studies we demonstrate a diurnal rhythm of melatonin concentration without a clear-cut change in aaNATI mRNA levels. The data suggest that tissue-specific regulation of aaNAT1 may be associated with different enzymatic functions and do not exclude the possibility of additional aaNAT genes.Amine acetylation in insects by the enzyme arylalkylamine N-acetyltransferase (aaNAT, EC 2.3.1.87) is involved in at least three different physiological functions: (i) It appears to be the major route of neurotransmitter catabolism (1-4), inactivating biogenic amines including tryptamine, tyramine, octopamine, serotonin, and norepinephrine. (ii) An acetylated amine, acetyldopamine, is subsequently oxidized to the respective quinone, which cross-links different proteins and/or chitin, resulting in stabilization and hardening (sclerotization) of the insect cuticle (5). (iii) Acetylation of serotonin plays an important role in the regulation of photoperiodically influenced physiological and behavioral processes (6). The major regulatory hormone is melatonin (7, 8), which is synthesized by the methylation of acetylserotonin. In vertebrates, melatonin is secreted periodically with high concentration at night, caused by a 10-to 100-fold nocturnal increase in aaNAT activity (8). The final steps of aaNAT activation are still unclear and presumably involve new RNA and protein synthesis (9-12). All living organisms are influenced by environmental factors such as light and dark and thus need mechanisms to coordinate their physiological processes in response to seasonal changes. Serotonin, aaNAT activity, and melatonin have indeed been found in several organs (eye, optic lobe, and brain) of various invertebrate species in which melatonin concentration also shows a rhythmic behavior (13). However, it is not known if aaNAT activity and melatonin are subject to diurnal variation in Drosophila melanogaster. aaNAT activity has been described in several insect species (14-18), including D. melanogaster (2, 19), but none of these enzymes could be purified to derive antibodies or sufficient internal sequence information for cloning. We have purified an aaNAT from D. melanogaster using tryptam...

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Section: Resultsmentioning

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 94%

mentioning

confidence: 99%

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Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut.

Hintermann

Grieder

Amherd

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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Arylalkalamine N‐acetyltransferase‐1 acts on a secondary amine in the yellow fever mosquito, Aedes aegypti

et al. 2022

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Arylalkylamine N-acetyltransferase (aaNAT) in Aedes aegypti is primarily involved in cuticle pigmentation and formation. The reported arylalkylamine substrates are all primary amines. In this study, we report a novel substrate, a secondary amine, of Ae. aegypti aaNAT1. The recombinant aaNAT1 protein exhibited high activity to a secondary amine, epinephrine, which has not been reported for any aaNATs previously. Structure-activity relationship study demonstrated that aaNAT1 has an epinephrine-binding site, and molecular docking and dynamic simulation showed that epinephrine is quite stable in the active cavity. Further functional studies demonstrated that epinephrine affected mosquito fecundity, egg hatching and development. The new biochemical function of aaNAT1 in metabolizing epinephrine could reduce some negative effects of the compound in the mosquito.

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Parasitism-Linked Block of Host Plasma Melanization

Shelby

Adeyeye

Okot-Kotber

et al. 2000

Journal of Invertebrate Pathology

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Isolation and characterization of an arylalkylamine N‐acetyltransferase from Drosophila melanogaster

Cited by 48 publications

References 25 publications

Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut.

Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut.

Arylalkalamine N‐acetyltransferase‐1 acts on a secondary amine in the yellow fever mosquito, Aedes aegypti

Parasitism-Linked Block of Host Plasma Melanization

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