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.

Identification and characterization of two arylalkylamine N-acetyltransferases in the yellow fever mosquito, Aedes aegypti.

In this study we provide a molecular and biochemical identification of two arylalkylamine N-acetyltransferases (aaNAT) from Aedes aegypti mosquitoes. N-acetyldopamine, the enzyme product of aaNAT, was detected in Ae. aegypti, indicating the presence of an aaNAT in this mosquito. A BLAST search of the Ae. aegypti genome, using sequence information from an activity-verified Drosophila aaNAT, identified thirteen putative aaNAT sequences sharing 13-48% sequence identity with the Drosophila enzyme. Eight of the thirteen putative aaNAT proteins were expressed using a bacterial expression system. Screening of purified recombinant proteins against 5-hydroxytryptamine, dopamine, methoxytryptamine, norepinephrine, octopamine, tryptamine, and tyramine substrates, established that two of the putative aaNATs are active to the tested arylalkylamines. We therefore named them aaNAT1 and 2, respectively. Analysis of the transcriptional profiles of the two aaNAT genes from Ae. aegypti revealed that aaNAT1 is more abundant in the whole body of larvae and pupae, and aaNAT2 is more abundant in the head of adult mosquitoes. Based on their substrate and transcriptional profiles, together with previous reports from other insects, we suggest that the two aaNATs play diverse roles in Ae. aegypti, with aaNAT1 primarily involved in sclerotization and aaNAT2 mainly in neurotransmitter inactivation. Our data provide a beginning to a more comprehensive understanding of the biochemistry and physiology of aaNATs from the Ae. aegypti and serve as a reference for studying the aaNAT family of proteins from other insect species.

Cloning and early expression pattern of two melatonin biosynthesis enzymes in the turbot (Scophthalmus maximus).

Melatonin biosynthesis from serotonin involves the sequential activation of the arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT). Photoperiod synchronizes a daily rhythm in pineal and retinal melatonin secretion through controlling AANAT activity. Teleost fish possess two Aanat, one expressed in the retina (AANAT1) and the other expressed in the pineal gland (AANAT2). We report here the full-length cloning of Aanat1, Aanat2, SmHiomt and Otx5 (orthodenticle homeobox homolog 5) in the turbot (Scophthalmus maximus, Sm), a flatfish belonging to an evolutionary recent group of Teleost. The temporal expression pattern of the genes investigated is consistent with the idea that OTX5 is needed for photoreceptor specification, and that the pineal gland differentiates before the retina. SmAanat2 expression remained pineal specific during the period of time investigated, whereas SmOtx5 and SmHiomt expressions were seen in both the retina and pineal gland. Our results do not support the existence of a second SmHiomt, as is the case for SmAanat. Neither SmAanat2 nor SmHiomt mRNAs displayed cyclic accumulation in the pineal organ of embryos and larvae maintained under a light-dark cycle from fertilization onward. This is in marked contrast with the situation observed with zebrafish Aanat2, indicating that the molecular mechanisms controlling the development of the pineal melatonin system have been modified during the evolution of Teleost.

Purification of the recombinant human serotonin N-acetyltransferase (EC 2.3.1.87): further characterization of and comparison with AANAT from other species.

Melatonin is synthesized by a series of enzymes, the penultimate one, serotonin N-acetyltransferase, catalyzing the limiting reaction. In the present study, we compared the recombinant serotonin N-acetyltransferases from rat, ovine, and human. The human protein is particularly difficult to purify because it interacts strongly with a putative chaperone protein from bacteria whereas the rat and sheep enzymes, which interact less strongly with this protein, have been purified close to homogeneity. We identified the contaminating protein as GroEL, the bacterial equivalent of Hsp60. We present numerous catalytic activities (substrate and cosubstrate specificities as well as inhibitor specificities) measured on the three species enzymes from which we deduced that the presence of the chaperone might partly explain the differences between the various species enzyme characteristics, beside the inter-species ones resulting from sequence differences. Despite several trials reported in the literature, a purification to homogeneity of the human (recombinant) enzyme has never been described. We present a new purification method, by using an original denaturation/renaturation process in which the enzyme is immobilized on an affinity chromatography column. The enzyme is then eluted in an active and pure form (i.e., absence of chaperone). The up-scaled system permitted us to perform the necessary experiments for the measurement of more accurate affinities of human serotonin N-acetyltransferase towards its main natural substrates, showing that only the activity of the enzyme towards phenylethylamine was modified.