Duality of serotonin-N-acetyltransferase in the gilthead seabream (Sparus aurata): molecular cloning and characterization of recombinant enzymes

Aug 11, 2004General and comparative endocrinology

Two forms of the serotonin-acetylating enzyme in gilthead seabream: cloning and study of their properties

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Abstract

The presence of two distinct AANAT enzymes in the gilthead seabream (Sparus aurata) suggests specialized roles in melatonin production.

sbAANAT1 is exclusively expressed in the retina, while sbAANAT2 is found only in the pineal gland.
Recombinant retinal sbAANAT1 has high substrate affinity and low activity, inhibited by high concentrations of substrates and products.
Recombinant pineal sbAANAT2 shows low substrate affinity but high activity, remaining unaffected by substrate or product concentrations.
Retinal sbAANAT1 preferentially acetylates various arylalkylamines, whereas pineal sbAANAT2 mainly acetylates indoleethylamines, particularly serotonin.
These findings support the notion that teleosts have evolved two AANAT enzymes with distinct functions due to gene duplication.

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Serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase, AANAT) is the key enzyme in the biosynthesis of melatonin in the pineal gland and retinal photoreceptors. Rhythmic AANAT activity drives rhythmic melatonin production in these tissues. The presence of two AANATs, AANAT1 and AANAT2, has been previously demonstrated in three fresh water teleosts. This duality, the result of early gene duplication, is unique to teleost species. In this study, the cDNAs encoding for AANAT1 and AANAT2 were cloned from a marine fish, the gilthead seabream (sb, Sparus aurata). Northern blot hybridization analysis indicates that sbAANAT1 and sbAANAT2 are exclusively expressed in the retina and pineal gland, respectively. Bacterially expressed recombinant sbAANATs exhibit differential enzyme kinetics. Recombinant retinal sbAANAT1 has relatively high substrate affinity and low activity rate; it is inhibited by high substrate and product concentrations. In contrast, recombinant pineal sbAANAT2 exhibits low substrate affinity and high activity rate and is not inhibited by substrates or products. The two recombinant enzymes also exhibit differential substrate preference. Retinal sbAANAT1 acetylates a range of arylalkylamines while pineal sbAANAT2 preferentially acetylates indoleethylamines, especially serotonin. The different spatial expression patterns, enzyme kinetics, and substrate preferences of the two sbAANATs support the hypothesis that, as a consequence of gene duplication, teleosts have acquired two AANATs with different functions. Pineal AANAT2 specializes in the production of large amounts of melatonin that is released into the circulation and exerts an endocrine role. Retinal AANAT1, on the other hand, is involved in producing low levels of melatonin that execute a paracrine function. In addition, retinal AANAT1 may carry out an as yet unknown function that involves acetylation of arylalkylamines other than serotonin.

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Duality of serotonin-N-acetyltransferase in the gilthead seabream (Sparus aurata): molecular cloning and characterization of recombinant enzymes

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