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David C Klein - One of the best experts on this subject based on the ideXlab platform.
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LECTURE The 2004 Aschoff/Pittendrigh Lecture: Theory of the Origin of the Pineal Gland— A Tale of Conflict and Resolution
2016Co-Authors: David C KleinAbstract:Abstract A theory is presented that explains the evolution of the pinealocyte from the common ancestral photoreceptor of both the pinealocyte and retinal photoreceptor. Central to the hypothesis is the previously unrecognized conflict between the two chemistries that define these cells—melatonin synthesis and retinoid recycling. At the core of the conflict is the formation of adducts com-posed of two molecules of retinaldehyde and one molecule of serotonin, analo-gous to formation in the retina of the toxic bis-retinyl ethanolamine (A2E). The hypothesis argues that early in chordate evolution, at a point before the genes required for melatonin synthesis were acquired, retinaldehyde—which is essen-tial for photon capture—was depleted by reacting with naturally occurring Arylalkylamines (tyramine, serotonin, tryptamine, phenylethylamine) and xenobiotic Arylalkylamines. This generated toxic bis-retinyl Arylalkylamines (A2AAs). The acquisition of Arylalkylamine N-acetyltransferase (AANAT) pre-vented this by N-acetylating the Arylalkylamines. Hydroxyindole-O-methyltransferase enhanced detoxification in the primitive photoreceptor b
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© 2000 International Society for Neurochemistry Chick Pineal Melatonin Synthesis: Light and Cyclic AMP Control Abundance of Serotonin N-Acetyltransferase Protein
2013Co-Authors: Martin Zatz, Jonathan A Gastel, James R. Heath, David C KleinAbstract:Abstract: Melatonin production in the pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N-acetyltransferase [Arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87], the penultimate enzyme in melatonin synthesis. The rhythm is generated by an endogenous circadian clock. In the chick, a clock is located in the pinealocyte, which also contains two phototransduction systems. One controls melatonin production by adjusting the clock and the other acts distal to the clock, via cyclic AMP mechanisms, to switch melatonin synthesis on and off. Unlike the clock in these cells, cyclic AMP does not appear to regulate activity by altering AA-NAT mRNA levels. The major changes in AA-NAT mRNA levels induced by the clock seemed likely (but not certain) to generate comparable changes in AA
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molecular evolution of multiple Arylalkylamine n acetyltransferase aanat in fish
Marine Drugs, 2011Co-Authors: Bina Zilbermanpeled, David C Klein, Sharron Bransburgzabary, Yoav GothilfAbstract:Arylalkylamine N-acetyltransferase (AANAT) catalyzes the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to Arylalkylamines, including indolethylamines and phenylethylamines. Multiple aanats are present in teleost fish as a result of whole genome and gene duplications. Fish aanat1a and aanat2 paralogs display different patterns of tissue expression and encode proteins with different substrate preference: AANAT1a is expressed in the retina, and acetylates both indolethylamines and phenylethylamines; while AANAT2 is expressed in the pineal gland, and preferentially acetylates indolethylamines. The two enzymes are therefore thought to serve different roles. Here, the molecular changes that led to their specialization were studied by investigating the structure-function relationships of AANATs in the gilthead seabream (sb, Sperus aurata). Acetylation activity of reciprocal mutated enzymes pointed to specific residues that contribute to substrate specificity of the enzymes. Inhibition tests followed by complementary analyses of the predicted three-dimensional models of the enzymes, suggested that both phenylethylamines and indolethylamines bind to the catalytic pocket of both enzymes. These results suggest that substrate selectivity of AANAT1a and AANAT2 is determined by the positioning of the substrate within the catalytic pocket, and its accessibility to catalysis. This illustrates the evolutionary process by which enzymes encoded by duplicated genes acquire different activities and play different biological roles.
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evidence that proline focuses movement of the floppy loop of Arylalkylamine n acetyltransferase ec 2 3 1 87
Journal of Biological Chemistry, 2008Co-Authors: Jiri Pavlicek, Steven L Coon, Joan L Weller, Surajit Ganguly, Sergio A Hassan, Dan L Sackett, David C KleinAbstract:Arylalkylamine N-acetyltransferase (AANAT) catalyzes the N-acetylation of serotonin, the penultimate step in the synthesis of melatonin. Pineal AANAT activity increases at night in all vertebrates, resulting in increased melatonin production. This increases circulating levels of melatonin, thereby providing a hormonal signal of darkness. Kinetic and structural analysis of AANAT has determined that one element is floppy. This element, termed Loop 1, is one of three loops that comprise the Arylalkylamine binding pocket. During the course of chordate evolution, Loop 1 acquired the tripeptide CPL, and the enzyme became highly active. Here we focused on the functional importance of the CPL tripeptide and found that activity was markedly reduced when it was absent. Moreover, increasing the local flexibility of this tripeptide region by P64G and P64A mutations had the counterintuitive effect of reducing activity and reducing the overall movement of Loop 1, as estimated from Langevin dynamics simulations. Binding studies indicate that these mutations increased the off-rate constant of a model substrate without altering the dissociation constant. The structural kink and local rigidity imposed by Pro-64 may enhance activity by favoring configurations of Loop 1 that facilitate catalysis and do not become immobilized by intramolecular interactions.
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Arylalkylamine n acetyltransferase the timezyme
Journal of Biological Chemistry, 2007Co-Authors: David C KleinAbstract:Arylalkylamine N-acetyltransferase controls daily changes in melatonin production by the pineal gland and thereby plays a unique role in biological timing in vertebrates. Arylalkylamine N-acetyltransferase is also expressed in the retina, where it may play other roles in addition to signaling, including neurotransmission and detoxification. Large changes in activity reflect cyclic 3',5'-adenosine monophosphate-dependent phosphorylation of Arylalkylamine N-acetyltransferase, leading to formation of a regulatory complex with 14-3-3 proteins. This activates the enzyme and prevents proteosomal proteolysis. The conserved features of regulatory systems that control Arylalkylamine N-acetyltransferase are a circadian clock and environmental lighting.
Steven L Coon - One of the best experts on this subject based on the ideXlab platform.
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evolution of aanat expansion of the gene family in the cephalochordate amphioxus
BMC Evolutionary Biology, 2010Co-Authors: Jiri Pavlicek, Sandrine Sauzet, Laurence Besseau, Steven L Coon, Joan L Weller, Gilles Boeuf, Pascaline Gaildrat, Marina V Omelchenko, Eugene V Koonin, Jack FalconAbstract:The Arylalkylamine N-acetyltransferase (AANAT) family is divided into structurally distinct vertebrate and non-vertebrate groups. Expression of vertebrate AANATs is limited primarily to the pineal gland and retina, where it plays a role in controlling the circadian rhythm in melatonin synthesis. Based on the role melatonin plays in biological timing, AANAT has been given the moniker "the Timezyme". Non-vertebrate AANATs, which occur in fungi and protists, are thought to play a role in detoxification and are not known to be associated with a specific tissue. We have found that the amphioxus genome contains seven AANATs, all having non-vertebrate type features. This and the absence of AANATs from the genomes of Hemichordates and Urochordates support the view that a major transition in the evolution of the AANATs may have occurred at the onset of vertebrate evolution. Analysis of the expression pattern of the two most structurally divergent AANATs in Branchiostoma lanceolatum (bl) revealed that they are expressed early in development and also in the adult at low levels throughout the body, possibly associated with the neural tube. Expression is clearly not exclusively associated with the proposed analogs of the pineal gland and retina. blAANAT activity is influenced by environmental lighting, but light/dark differences do not persist under constant light or constant dark conditions, indicating they are not circadian in nature. bfAANATα and bfAANATδ' have unusually alkaline (> 9.0) optimal pH, more than two pH units higher than that of vertebrate AANATs. The substrate selectivity profiles of bfAANATα and δ' are relatively broad, including alkylamines, Arylalkylamines and diamines, in contrast to vertebrate forms, which selectively acetylate serotonin and other Arylalkylamines. Based on these features, it appears that amphioxus AANATs could play several roles, including detoxification and biogenic amine inactivation. The presence of seven AANATs in amphioxus genome supports the view that Arylalkylamine and polyamine acetylation is important to the biology of this organism and that these genes evolved in response to specific pressures related to requirements for amine acetylation.
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evidence that proline focuses movement of the floppy loop of Arylalkylamine n acetyltransferase ec 2 3 1 87
Journal of Biological Chemistry, 2008Co-Authors: Jiri Pavlicek, Steven L Coon, Joan L Weller, Surajit Ganguly, Sergio A Hassan, Dan L Sackett, David C KleinAbstract:Arylalkylamine N-acetyltransferase (AANAT) catalyzes the N-acetylation of serotonin, the penultimate step in the synthesis of melatonin. Pineal AANAT activity increases at night in all vertebrates, resulting in increased melatonin production. This increases circulating levels of melatonin, thereby providing a hormonal signal of darkness. Kinetic and structural analysis of AANAT has determined that one element is floppy. This element, termed Loop 1, is one of three loops that comprise the Arylalkylamine binding pocket. During the course of chordate evolution, Loop 1 acquired the tripeptide CPL, and the enzyme became highly active. Here we focused on the functional importance of the CPL tripeptide and found that activity was markedly reduced when it was absent. Moreover, increasing the local flexibility of this tripeptide region by P64G and P64A mutations had the counterintuitive effect of reducing activity and reducing the overall movement of Loop 1, as estimated from Langevin dynamics simulations. Binding studies indicate that these mutations increased the off-rate constant of a model substrate without altering the dissociation constant. The structural kink and local rigidity imposed by Pro-64 may enhance activity by favoring configurations of Loop 1 that facilitate catalysis and do not become immobilized by intramolecular interactions.
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evolution of Arylalkylamine n acetyltransferase emergence and divergence
Molecular and Cellular Endocrinology, 2006Co-Authors: Steven L Coon, David C KleinAbstract:The melatonin rhythm-generating enzyme, Arylalkylamine N-acetyltransferase (AANAT) is known to have recognizable ancient homologs in bacteria and fungi, but not in other eukaryotes. Analysis of new cDNA and genomic sequences has identified several additional homologs in other groupings. First, an AANAT homolog has been found in the genome of the cephalochordate amphioxus, representing the oldest homolog in chordates. Second, two AANAT homologs have been identified in unicellular green algae. The homologs in amphioxus, unicellular green algae, fungi and bacteria are similarly primitive in that they lack sequences found in vertebrate AANATs that are involved in regulation and that facilitate binding and catalysis. In addition, all these sequences are intronless. These features are consistent with horizontal transfer of the AANAT ancestor from bacteria to green algae, fungi and chordates. Lastly, a third AANAT gene has been found in teleost fish, suggesting that AANAT genes serve multiple functions in addition to melatonin synthesis.
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characterization of the saccharomyces cerevisiae homolog of the melatonin rhythm enzyme Arylalkylamine n acetyltransferase ec 2 3 1 87
Journal of Biological Chemistry, 2001Co-Authors: Surajit Ganguly, David C Klein, Padmaja Mummaneni, Peter J Steinbach, Steven L CoonAbstract:Abstract Arylalkylamine N-acetyltransferase (AANAT, serotonin N-acetyltransferase, EC 2.3.1.87) plays a unique transduction role in vertebrate physiology by converting information about day and night into a hormonal signal: melatonin. Only vertebrate members of the AANAT family have been functionally characterized. Here a putative AANAT from Saccharomyces cerevisiae (scAANAT) was studied to determine whether it possessed the catalytic activity of the vertebrate enzyme. scAANAT is 47% similar to ovine AANAT, but lacks the regulatory N- and C-terminal flanking regions conserved in all vertebrate AANATs. It was found to have enzyme activity generally typical for AANAT family members, although the substrate preference pattern was somewhat broader, the specific activity was lower, and the pH optimum was higher. Deletion of scAANAT reduced Arylalkylamine acetylation by S. cerevisiaeextracts, indicating that scAANAT contributes significantly to this process. The scAANAT sequence conformed to the three-dimensional structure of ovine AANAT catalytic core; however, an important structural element (loop 1) was found to be shorter and to lack a proline involved in substrate binding. These differences could explain the lower specific activity of scAANAT, because of the importance of loop 1 in catalysis. Data base analysis revealed the presence of putative AANATs in other fungi but not in the nearly complete genomes of Drosophila melanogaster or Caenorhabditis elegans. These studies indicate that the catalytic and kinetic characteristics of fungal and vertebrate enzymes can be considered to be generally similar, although some differences exist that appear to be linked to changes in one structural element. Perhaps the most striking difference is that fungal AANATs lack the regulatory domains of the vertebrate enzyme, which appear to be essential for the regulatory role the enzyme plays in photochemical transduction.
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role of a pineal camp operated Arylalkylamine n acetyltransferase 14 3 3 binding switch in melatonin synthesis
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Surajit Ganguly, Mark D Rollag, M A A Namboodiri, Steven L Coon, Joan L Weller, Jonathan A Gastel, Christian Schwartz, Howard Jaffe, Alison B Hickman, Tomas ObsilAbstract:The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, Arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC 2.3.1.87). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN → RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the Km for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.
Joan L Weller - One of the best experts on this subject based on the ideXlab platform.
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evolution of aanat expansion of the gene family in the cephalochordate amphioxus
BMC Evolutionary Biology, 2010Co-Authors: Jiri Pavlicek, Sandrine Sauzet, Laurence Besseau, Steven L Coon, Joan L Weller, Gilles Boeuf, Pascaline Gaildrat, Marina V Omelchenko, Eugene V Koonin, Jack FalconAbstract:The Arylalkylamine N-acetyltransferase (AANAT) family is divided into structurally distinct vertebrate and non-vertebrate groups. Expression of vertebrate AANATs is limited primarily to the pineal gland and retina, where it plays a role in controlling the circadian rhythm in melatonin synthesis. Based on the role melatonin plays in biological timing, AANAT has been given the moniker "the Timezyme". Non-vertebrate AANATs, which occur in fungi and protists, are thought to play a role in detoxification and are not known to be associated with a specific tissue. We have found that the amphioxus genome contains seven AANATs, all having non-vertebrate type features. This and the absence of AANATs from the genomes of Hemichordates and Urochordates support the view that a major transition in the evolution of the AANATs may have occurred at the onset of vertebrate evolution. Analysis of the expression pattern of the two most structurally divergent AANATs in Branchiostoma lanceolatum (bl) revealed that they are expressed early in development and also in the adult at low levels throughout the body, possibly associated with the neural tube. Expression is clearly not exclusively associated with the proposed analogs of the pineal gland and retina. blAANAT activity is influenced by environmental lighting, but light/dark differences do not persist under constant light or constant dark conditions, indicating they are not circadian in nature. bfAANATα and bfAANATδ' have unusually alkaline (> 9.0) optimal pH, more than two pH units higher than that of vertebrate AANATs. The substrate selectivity profiles of bfAANATα and δ' are relatively broad, including alkylamines, Arylalkylamines and diamines, in contrast to vertebrate forms, which selectively acetylate serotonin and other Arylalkylamines. Based on these features, it appears that amphioxus AANATs could play several roles, including detoxification and biogenic amine inactivation. The presence of seven AANATs in amphioxus genome supports the view that Arylalkylamine and polyamine acetylation is important to the biology of this organism and that these genes evolved in response to specific pressures related to requirements for amine acetylation.
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evidence that proline focuses movement of the floppy loop of Arylalkylamine n acetyltransferase ec 2 3 1 87
Journal of Biological Chemistry, 2008Co-Authors: Jiri Pavlicek, Steven L Coon, Joan L Weller, Surajit Ganguly, Sergio A Hassan, Dan L Sackett, David C KleinAbstract:Arylalkylamine N-acetyltransferase (AANAT) catalyzes the N-acetylation of serotonin, the penultimate step in the synthesis of melatonin. Pineal AANAT activity increases at night in all vertebrates, resulting in increased melatonin production. This increases circulating levels of melatonin, thereby providing a hormonal signal of darkness. Kinetic and structural analysis of AANAT has determined that one element is floppy. This element, termed Loop 1, is one of three loops that comprise the Arylalkylamine binding pocket. During the course of chordate evolution, Loop 1 acquired the tripeptide CPL, and the enzyme became highly active. Here we focused on the functional importance of the CPL tripeptide and found that activity was markedly reduced when it was absent. Moreover, increasing the local flexibility of this tripeptide region by P64G and P64A mutations had the counterintuitive effect of reducing activity and reducing the overall movement of Loop 1, as estimated from Langevin dynamics simulations. Binding studies indicate that these mutations increased the off-rate constant of a model substrate without altering the dissociation constant. The structural kink and local rigidity imposed by Pro-64 may enhance activity by favoring configurations of Loop 1 that facilitate catalysis and do not become immobilized by intramolecular interactions.
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melatonin synthesis 14 3 3 dependent activation and inhibition of Arylalkylamine n acetyltransferase mediated by phosphoserine 205
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Surajit Ganguly, Joan L Weller, Anthony D Ho, P Chemineau, Benoit Malpaux, David C KleinAbstract:The nocturnal increase in circulating melatonin in vertebrates is regulated by the activity of Arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the melatonin pathway (serotonin → N-acetylserotonin → melatonin). Large changes in activity are linked to cyclic AMP-dependent protein kinase-mediated phosphorylation of AANAT T31. Phosphorylation of T31 promotes binding of AANAT to the dimeric 14-3-3 protein, which activates AANAT by increasing Arylalkylamine affinity. In the current study, a putative second AANAT cyclic AMP-dependent protein kinase phosphorylation site, S205, was found to be ≈55% phosphorylated at night, when T31 is ≈40% phosphorylated. These findings indicate that ovine AANAT is dual-phosphorylated. Moreover, light exposure at night decreases T31 and S205 phosphorylation, consistent with a regulatory role of both sites. AANAT peptides containing either T31 or S205 associate with 14-3-3ζ in a phosphorylation-dependent manner; binding through phosphorylated (p)T31 is stronger than that through pS205, consistent with the location of only pT31 in a mode I binding motif, one of two recognized high-affinity 14-3-3-binding motifs AANAT protein binds to 14-3-3ζ through pT31 or pS205. Two-site binding lowers the Km for Arylalkylamine substrate to ≈30 μM. In contrast, single-site pS205 binding increases the Km to ≈1,200 μM. Accordingly, the switch from dual to single pS205 binding of AANAT to 14-3-3 changes the Km for substrates by ≈40-fold. pS205 seems to be part of a previously unrecognized 14-3-3-binding motif-pS/pT (X1–2)-COOH, referred to here as mode III.
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role of a pineal camp operated Arylalkylamine n acetyltransferase 14 3 3 binding switch in melatonin synthesis
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Surajit Ganguly, Mark D Rollag, M A A Namboodiri, Steven L Coon, Joan L Weller, Jonathan A Gastel, Christian Schwartz, Howard Jaffe, Alison B Hickman, Tomas ObsilAbstract:The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, Arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC 2.3.1.87). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN → RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the Km for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.
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regulation of Arylalkylamine n acetyltransferase 2 aanat2 ec 2 3 1 87 in the fish pineal organ evidence for a role of proteasomal proteolysis
Endocrinology, 2001Co-Authors: Steven L Coon, Joan L Weller, Jack Falcon, Kristina M Galarneau, Benny Ron, Galit Chen, David C KleinAbstract:In fish, individual photoreceptor cells in the pineal organ and retina contain complete melatonin rhythm generating systems. In the pike and seabream, this includes a photodetector, circadian clock, and melatonin synthesis machinery; the trout lacks a functional clock. The melatonin rhythm is due in part to a nocturnal increase in the activity of the Arylalkylamine N-acetyltransferase (AANAT) which is inhibited by light. Two AANATs have been identified in fish: AANAT1, more closely related to AANATs found in higher vertebrates, is specifically expressed in the retina; AANAT2 is specifically expressed in the pineal organ. We show that there is a physiological day/night rhythm in pineal AANAT2 protein in the pike, and that light exposure at midnight decreases the abundance of AANAT2 protein and activity. In culture, this decrease is blocked by inhibitors of the proteasomal degradation pathway. If glands are maintained under light at night, treatment with these inhibitors increases AANAT2 activity and protei...
Surajit Ganguly - One of the best experts on this subject based on the ideXlab platform.
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evidence that proline focuses movement of the floppy loop of Arylalkylamine n acetyltransferase ec 2 3 1 87
Journal of Biological Chemistry, 2008Co-Authors: Jiri Pavlicek, Steven L Coon, Joan L Weller, Surajit Ganguly, Sergio A Hassan, Dan L Sackett, David C KleinAbstract:Arylalkylamine N-acetyltransferase (AANAT) catalyzes the N-acetylation of serotonin, the penultimate step in the synthesis of melatonin. Pineal AANAT activity increases at night in all vertebrates, resulting in increased melatonin production. This increases circulating levels of melatonin, thereby providing a hormonal signal of darkness. Kinetic and structural analysis of AANAT has determined that one element is floppy. This element, termed Loop 1, is one of three loops that comprise the Arylalkylamine binding pocket. During the course of chordate evolution, Loop 1 acquired the tripeptide CPL, and the enzyme became highly active. Here we focused on the functional importance of the CPL tripeptide and found that activity was markedly reduced when it was absent. Moreover, increasing the local flexibility of this tripeptide region by P64G and P64A mutations had the counterintuitive effect of reducing activity and reducing the overall movement of Loop 1, as estimated from Langevin dynamics simulations. Binding studies indicate that these mutations increased the off-rate constant of a model substrate without altering the dissociation constant. The structural kink and local rigidity imposed by Pro-64 may enhance activity by favoring configurations of Loop 1 that facilitate catalysis and do not become immobilized by intramolecular interactions.
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melatonin synthesis 14 3 3 dependent activation and inhibition of Arylalkylamine n acetyltransferase mediated by phosphoserine 205
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Surajit Ganguly, Joan L Weller, Anthony D Ho, P Chemineau, Benoit Malpaux, David C KleinAbstract:The nocturnal increase in circulating melatonin in vertebrates is regulated by the activity of Arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the melatonin pathway (serotonin → N-acetylserotonin → melatonin). Large changes in activity are linked to cyclic AMP-dependent protein kinase-mediated phosphorylation of AANAT T31. Phosphorylation of T31 promotes binding of AANAT to the dimeric 14-3-3 protein, which activates AANAT by increasing Arylalkylamine affinity. In the current study, a putative second AANAT cyclic AMP-dependent protein kinase phosphorylation site, S205, was found to be ≈55% phosphorylated at night, when T31 is ≈40% phosphorylated. These findings indicate that ovine AANAT is dual-phosphorylated. Moreover, light exposure at night decreases T31 and S205 phosphorylation, consistent with a regulatory role of both sites. AANAT peptides containing either T31 or S205 associate with 14-3-3ζ in a phosphorylation-dependent manner; binding through phosphorylated (p)T31 is stronger than that through pS205, consistent with the location of only pT31 in a mode I binding motif, one of two recognized high-affinity 14-3-3-binding motifs AANAT protein binds to 14-3-3ζ through pT31 or pS205. Two-site binding lowers the Km for Arylalkylamine substrate to ≈30 μM. In contrast, single-site pS205 binding increases the Km to ≈1,200 μM. Accordingly, the switch from dual to single pS205 binding of AANAT to 14-3-3 changes the Km for substrates by ≈40-fold. pS205 seems to be part of a previously unrecognized 14-3-3-binding motif-pS/pT (X1–2)-COOH, referred to here as mode III.
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characterization of the saccharomyces cerevisiae homolog of the melatonin rhythm enzyme Arylalkylamine n acetyltransferase ec 2 3 1 87
Journal of Biological Chemistry, 2001Co-Authors: Surajit Ganguly, David C Klein, Padmaja Mummaneni, Peter J Steinbach, Steven L CoonAbstract:Abstract Arylalkylamine N-acetyltransferase (AANAT, serotonin N-acetyltransferase, EC 2.3.1.87) plays a unique transduction role in vertebrate physiology by converting information about day and night into a hormonal signal: melatonin. Only vertebrate members of the AANAT family have been functionally characterized. Here a putative AANAT from Saccharomyces cerevisiae (scAANAT) was studied to determine whether it possessed the catalytic activity of the vertebrate enzyme. scAANAT is 47% similar to ovine AANAT, but lacks the regulatory N- and C-terminal flanking regions conserved in all vertebrate AANATs. It was found to have enzyme activity generally typical for AANAT family members, although the substrate preference pattern was somewhat broader, the specific activity was lower, and the pH optimum was higher. Deletion of scAANAT reduced Arylalkylamine acetylation by S. cerevisiaeextracts, indicating that scAANAT contributes significantly to this process. The scAANAT sequence conformed to the three-dimensional structure of ovine AANAT catalytic core; however, an important structural element (loop 1) was found to be shorter and to lack a proline involved in substrate binding. These differences could explain the lower specific activity of scAANAT, because of the importance of loop 1 in catalysis. Data base analysis revealed the presence of putative AANATs in other fungi but not in the nearly complete genomes of Drosophila melanogaster or Caenorhabditis elegans. These studies indicate that the catalytic and kinetic characteristics of fungal and vertebrate enzymes can be considered to be generally similar, although some differences exist that appear to be linked to changes in one structural element. Perhaps the most striking difference is that fungal AANATs lack the regulatory domains of the vertebrate enzyme, which appear to be essential for the regulatory role the enzyme plays in photochemical transduction.
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role of a pineal camp operated Arylalkylamine n acetyltransferase 14 3 3 binding switch in melatonin synthesis
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Surajit Ganguly, Mark D Rollag, M A A Namboodiri, Steven L Coon, Joan L Weller, Jonathan A Gastel, Christian Schwartz, Howard Jaffe, Alison B Hickman, Tomas ObsilAbstract:The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, Arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC 2.3.1.87). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN → RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the Km for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.
David J. Merkler - One of the best experts on this subject based on the ideXlab platform.
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identification of catalytically distinct Arylalkylamine n acetyltransferase splicoforms from tribolium castaneum
Protein Expression and Purification, 2020Co-Authors: Brian G Oflynn, Gabriela Suarez, Karin Claire Prins, Britney A Shepherd, Victoria E Forbrich, David J. MerklerAbstract:The assumption that structural or sequential homology between enzymes implies functional homology is a common misconception. Through in-depth structural and kinetic analysis, we are now beginning to understand the minute differences in primary structure that can alter the function of an enzyme completely. Alternative splicing is one method for which the activity of an enzyme can be controlled, simply by altering its length. Arylalkylamine N-acetyltransferase A (AANATA) in D. melanogaster, which catalyzes the N-acetylation of biogenic amines, has multiple splicoforms - alternatively spliced enzyme isoforms - with differing tissue distribution. As demonstrated here, AANAT1 from Tribolium castaneum is another such enzyme with multiple splicoforms. A screening assay was developed and utilized to determine that, despite only a 35 amino acid truncation, the shortened form of TcAANAT1 is a more active form of the enzyme. This implies regulation of enzyme metabolic activity via alternative splicing.
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knockdown of Arylalkylamine n acetyltransferase like 2 in drosophila melanogaster
Archives of Insect Biochemistry and Physiology, 2019Co-Authors: Ryan L Anderson, Dylan J Wallis, Alexander Aguirre, Dean Holliday, David J. MerklerAbstract:Drosophila melanogaster produces fatty acid amides, and thus, provides a model to unravel the pathways for their biosynthesis. We previously demonstrated that Arylalkylamine N-acetyltransferase-like 2 (AANATL2) from D. melanogaster will catalyze the formation of long-chain N-acylserotonins and N-acyldopamines in vitro. Generating silencing RNA via the UAS/GAL4 bipartite approach for targeted gene expression effectively decreased the endogenous levels of the AANATL2 transcripts in D. melanogaster, as shown by reverse transcription quantitative polymerase chain reaction. Consistent with these data, western blot analysis of the offspring of the AANATL2 knockdown flies using an anti-AANATL2 antibody revealed a significant reduction in the expression of the AANATL2 protein. Reduced expression of AANATL2 decreased the cellular levels of N-palmitoyldopamine (PALDA), providing strong evidence that AANATL2 is responsible for the biosynthesis of PALDA in vivo. This is the first time that the expression of an AANAT has been reduced in D. melanogaster to link one of these enzymes to the in vivo production of an N-acylarylalkylamide.
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bm iaanat3 expression and characterization of a novel Arylalkylamine n acyltransferase from bombyx mori
Archives of Biochemistry and Biophysics, 2019Co-Authors: Matthew R Battistini, Gabriela Suarez, Brian G Oflynn, Christopher Shoji, Lamar Galloway, David J. MerklerAbstract:Abstract The Arylalkylamine N-acyltransferases (AANATs) are enzymes that catalyze the acyl-CoA-dependent formation of N-acylarylalkylamides: acyl-CoA + Arylalkylamine → N-acylarylalkylamides + CoA-SH. Herein, we describe our study of a previously uncharacterized AANAT from Bombyx mori: Bm-iAANAT3. Bm-iAANAT3 catalyzes the direct formation of N-acylarylalkylamides and accepts a broad range of short-chain acyl-CoA thioesters and amines as substrates. Acyl-CoA thioesters possessing an acyl chain length >10 carbon atoms are not substrates for Bm-iAANAT3. We report that Bm-iAANAT3 is a “versatile generalist”, most likely, functioning in amine acetylation – a reaction in amine inactivation/excretion, cuticle sclerotization, and melanism. We propose a kinetic and chemical mechanism for Bm-iAANAT3 that is consistent with our steady-state kinetic analysis, dead-end inhibition studies, determination of the pH-rate profiles, and site-directed mutagenesis of a catalytically important amino acid in Bm-iAANAT3. These mechanistic studies of Bm-iAANAT3 will foster the development of novel compounds targeted against this enzyme and other insect AANATs for the control of insect pests.
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Insect Arylalkylamine N-Acyltransferases: Mechanism and Role in Fatty Acid Amide Biosynthesis
Frontiers Media S.A., 2018Co-Authors: Brian G. O'flynn, Gabriela Suarez, Aidan J. Hawley, David J. MerklerAbstract:Arylalkylamine N-acyltransferases (AANATs) catalyze the formation of an N-acylamide from an acyl-CoA thioester and an amine. One well known example is the production of N-acetylserotonin from acetyl-CoA and serotonin, a reaction in the melatonin biosynthetic pathway from tryptophan. AANATs have been identified from a variety of vertebrates and invertebrates. Considerable efforts have been devoted to the mammalian AANAT because a cell-permeable inhibitor specifically targeted against this enzyme could prove useful to treat diseases related to dysfunction in melatonin production. Insects are an interesting model for the study of AANATs because more than one isoform is typically expressed by a specific insect and the different insect AANATs (iAANATs) serve different roles in the insect cell. In contrast, mammals express only one AANAT. The major role of iAANATs seem to be in the production of N-acetyldopamine, a reaction important in the tanning and sclerotization of the cuticle. Metabolites identified in insects including N-acetylserotonin and long-chain N-fatty acyl derivatives of dopamine, histidine, phenylalanine, serotonin, tyrosine, and tryptophan are likely produced by an iAANAT. In vitro studies of specific iAANATs are consistent with this hypothesis. In this review, we highlight the current metabolomic knowledge of the N-acylated aromatic amino acids and N-acylated derivatives of the aromatic amino acids, the current mechanistic understanding of the iAANATs, and explore the possibility that iAANATs serve as insect “rhymezymes” regulating photoperiodism and other rhythmic processes in insects
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insect Arylalkylamine n acetyltransferases as potential targets for novel insecticide design
Biochemistry & Molecular Biology Journal, 2018Co-Authors: Brian G Oflynn, Aidan J. Hawley, David J. MerklerAbstract:Crop protection against destructive pests has been at the forefront of recent agricultural advancements. Rapid adaptive evolution has led to insects becoming immune to the chemicals employed to quell their damage. Insecticide resistance is a serious problem that negatively impacts food production, food storage, human health, and the environment. To make matters more complicated are the strict regulations in place on insecticide development, driven by rising public concern relating to the harmful effects these chemicals have on the environment and on society. A key component to solving the problem of insect resistance, while keeping public welfare in mind, is the identification of novel insectspecific protein targets. One unexplored target for the development of new targeted insecticides are the insect Arylalkylamine N-acetyltransferases (iAANATs). This group of enzymes, shown to be intrinsic in the development of the insect cuticle, is an untapped well of potential for target-specific inhibition, while offering enough variety to ensure protection for non-target enzymes. In this review, we highlight kinetic, genetic and bioinformatic data showing that the iAANATs are intriguing insecticide targets that should be specific only for particular insect pests. Such a pest-specific insecticide would minimize environmental harm by eliminating such non-discriminate attacks which have made insecticides such a highly regulated industry, and would have negligible toxicity to humans and other mammals.
