Article

Analysis of serotonin N-acetyltransferase regulation in vitro and in live cells using protein semisynthesis

Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Biochemistry (Impact Factor: 3.38). 10/2008; 47(39):10407-19. DOI: 10.1021/bi801189d
Source: PubMed

ABSTRACT Serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT)] is a key circadian rhythm enzyme that drives the nocturnal production of melatonin in the pineal. Prior studies have suggested that its light and diurnal regulation involves phosphorylation on key AANAT Ser and Thr residues which results in 14-3-3zeta recruitment and changes in catalytic activity and protein stability. Here we use protein semisynthesis by expressed protein ligation to systematically explore the effects of single and dual phosphorylation of AANAT on acetyltransferase activity and relative affinity for 14-3-3zeta. AANAT Thr31 phosphorylation on its own can enhance catalytic efficiency up to 7-fold through an interaction with 14-3-3zeta that lowers the substrate K m. This augmented catalytic profile is largely abolished by double phosphorylation at Thr31 and Ser205. A possible basis for this difference is the dual anchoring of doubly phosphorylated AANAT via one 14-3-3zeta heterodimer. We have developed a novel solution phase assay for accurate K D measurements of 14-3-3zeta-AANAT interaction using 14-3-3zeta fluorescently labeled with rhodamine by expressed protein ligation. We have also generated a doubly fluorescently labeled AANAT which can be used to assess the stability of this protein in a live cell, real-time assay by fluorescence resonance energy transfer measured by microscopic imaging. These studies offer new insights into the molecular basis of melatonin regulation and 14-3-3zeta interaction.

0 Bookmarks
 · 
81 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of melatonin and other N-acetylarylalkylamides from the corresponding arylalkylamine and acetyl-CoA. The N-acetylation of arylalkylamines is a critical step in Drosophila melanogaster for the inactivation of the bioactive amines and the sclerotization of the cuticle. Two AANAT variants (AANATA and AANATB) have been identified in D. melanogaster, in which AANATA differs from AANATB by the truncation of 35 amino acids from the N-terminus. We have expressed and purified both D. melanogaster AANAT variants (AANATA and AANATB) in Escherichia coli and used the purified enzymes to demonstrate that this N-terminal truncation does not affect the activity of the enzyme. Subsequent characterization of the kinetic and chemical mechanism of AANATA identified an ordered sequential mechanism, with acetyl-CoA binding first, followed by tyramine. We used a combination of pH-activity profiling and site-directed mutagenesis to study prospective residues believed to function in AANATA catalysis. These data led to an assignment of Glu-47 as the general base in catalysis with an apparent pKa of 7.0. Using the data generated for the kinetic mechanism, structure-function relationships, pH-rate profiles, and site-directed mutagenesis, we propose a chemical mechanism for AANATA.
    Biochemistry 12/2014; 53(49). DOI:10.1021/bi5006078 · 3.38 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Circadian Rhythmicity is present in the sleeping and breeding patterns of animals, including human beings and also related with brain wave activity, hormone production, cell regeneration and other biological activities. Melatonin is thought to play important roles in regulating circadian rhytmicity of the animals. Arylalkylamine-N-acetyltransferase (AANAT) is an enzyme which is responsible for the melatonin metabolism. In this study AANAT enzyme is targeted for the control of sleeping sickness and other irregular circadian rhythmicity by regulating the melatonin formation. AANAT protein 3D-structure was modeled, followed by loop modeling, refinement through energy minimization processes by molecular dynamics simulation and validation. Analysis of the Ramachandran plot shows 90.9% amino acids falls in the allowed region. The modeled protein was docked with N-Acetyl Serotonin. Combinatorial library was generated by using N-Acetyl Serotonin as a reference molecule and molecules having 80% similarity to N-Acetyl Serotonin was selected from Zinc database. These molecules were virtually screened by MOLEGRO virtual docker and top 5 molecules were selected and docked by using AutoDock. The AutoDock result shows that the ZINC01587152 molecule is having best interactions with the receptor protein. On the basis of this study we can suggest that the ZINC01587152 molecule is the best ligand against AANAT enzyme. It may be further synthesized and tested for sleep related disorders.
    Bioinformation 08/2013; 9(15):771-6. DOI:10.6026/97320630009771 · 0.50 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Die Untersuchung biologischer Fragestellungen mit chemischen Methoden wird meist als “Chemische Biologie” bezeichnet und setzt voraus, dass biologisch relevante Makromoleküle, wie Peptide und Proteine, chemisch zugänglich sind. Auf der Festphasensynthese von Peptiden aufbauend wurden viele chemoselektive Ligations- und Modifikationstechniken zur Verknüpfung synthetischer Peptide oder funktionaler Einheiten zu größeren synthetischen, biologisch relevanten Makromolekülen entwickelt. Dieser Aufsatz fasst die aktuellen Entwicklungen auf dem Gebiet der chemoselektiven Ligations- und Modifikationsstrategien zusammen und illustriert ihre Anwendbarkeit an Beispielen aus der chemischen Totalsynthese von Proteinen bis hin zur Semisynthese natürlicher modifizierter Proteine.
    Angewandte Chemie 01/2008; 120(52):10182-10228. DOI:10.1002/ange.200801313

Preview

Download
1 Download
Available from