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Molecular evolution of multiple arylalkylamine N-acetyltransferase (AANAT) in fish.

Zilberman-Peled B, Bransburg-Zabary S, Klein DC, Gothilf Y - Mar Drugs (2011)

Bottom Line: Multiple aanats are present in teleost fish as a result of whole genome and gene duplications.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

ABSTRACT
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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Acetylation of arylalkylamines by wild type and mutant AANATs. Activities were measured in the presence of near saturating levels of AcCoA and increasing concentration of amine substrates. The graphs represent acetylation activities (mean ± SE) with serotonin and phenylethylamine. A generally similar kinetics was found with tryptamine and dopamine. The AANAT1a- α2, β4 and αa mutants did not express well and were inactive, consistent with misfolding, and therefore were not analyzed (see ‘Experimental Section’ for details). (A) AANAT1a and mutants with serotonin; (B) AANAT1a and mutants with phenylethylamine; (C) AANAT2 and mutants with serotonin; (D) AANAT2 and mutants with phenylethylamine; PEA = phenylethylamine.
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f2-marinedrugs-09-00906: Acetylation of arylalkylamines by wild type and mutant AANATs. Activities were measured in the presence of near saturating levels of AcCoA and increasing concentration of amine substrates. The graphs represent acetylation activities (mean ± SE) with serotonin and phenylethylamine. A generally similar kinetics was found with tryptamine and dopamine. The AANAT1a- α2, β4 and αa mutants did not express well and were inactive, consistent with misfolding, and therefore were not analyzed (see ‘Experimental Section’ for details). (A) AANAT1a and mutants with serotonin; (B) AANAT1a and mutants with phenylethylamine; (C) AANAT2 and mutants with serotonin; (D) AANAT2 and mutants with phenylethylamine; PEA = phenylethylamine.

Mentions: Analysis of the acetylation activity of wild type and mutant proteins was done with serotonin and tryptamine (indolethylamines) and dopamine and phenylethylamine (phenylethylamines). Figure 2 represents acetylation activity with serotonin and phenylethylamine, which are generally similar to the kinetics found with tryptamine and dopamine, respectively. Table 2 represents the catalytic efficiency in terms of Kcat/Km values. Acetylation of phenylethylamine and dopamine by AANAT2 mutants did not obey the Michaelis-Menten equation (Figure 2), and their activities are therefore expressed relative to wild type AANAT2 activity in the presence of 10 mM substrate (shaded values, Table 2); these values cannot be compared to other enzyme-substrate Kcat/Km values. The specific effect of each mutation is detailed below.


Molecular evolution of multiple arylalkylamine N-acetyltransferase (AANAT) in fish.

Zilberman-Peled B, Bransburg-Zabary S, Klein DC, Gothilf Y - Mar Drugs (2011)

Acetylation of arylalkylamines by wild type and mutant AANATs. Activities were measured in the presence of near saturating levels of AcCoA and increasing concentration of amine substrates. The graphs represent acetylation activities (mean ± SE) with serotonin and phenylethylamine. A generally similar kinetics was found with tryptamine and dopamine. The AANAT1a- α2, β4 and αa mutants did not express well and were inactive, consistent with misfolding, and therefore were not analyzed (see ‘Experimental Section’ for details). (A) AANAT1a and mutants with serotonin; (B) AANAT1a and mutants with phenylethylamine; (C) AANAT2 and mutants with serotonin; (D) AANAT2 and mutants with phenylethylamine; PEA = phenylethylamine.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3111191&req=5

f2-marinedrugs-09-00906: Acetylation of arylalkylamines by wild type and mutant AANATs. Activities were measured in the presence of near saturating levels of AcCoA and increasing concentration of amine substrates. The graphs represent acetylation activities (mean ± SE) with serotonin and phenylethylamine. A generally similar kinetics was found with tryptamine and dopamine. The AANAT1a- α2, β4 and αa mutants did not express well and were inactive, consistent with misfolding, and therefore were not analyzed (see ‘Experimental Section’ for details). (A) AANAT1a and mutants with serotonin; (B) AANAT1a and mutants with phenylethylamine; (C) AANAT2 and mutants with serotonin; (D) AANAT2 and mutants with phenylethylamine; PEA = phenylethylamine.
Mentions: Analysis of the acetylation activity of wild type and mutant proteins was done with serotonin and tryptamine (indolethylamines) and dopamine and phenylethylamine (phenylethylamines). Figure 2 represents acetylation activity with serotonin and phenylethylamine, which are generally similar to the kinetics found with tryptamine and dopamine, respectively. Table 2 represents the catalytic efficiency in terms of Kcat/Km values. Acetylation of phenylethylamine and dopamine by AANAT2 mutants did not obey the Michaelis-Menten equation (Figure 2), and their activities are therefore expressed relative to wild type AANAT2 activity in the presence of 10 mM substrate (shaded values, Table 2); these values cannot be compared to other enzyme-substrate Kcat/Km values. The specific effect of each mutation is detailed below.

Bottom Line: Multiple aanats are present in teleost fish as a result of whole genome and gene duplications.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

ABSTRACT
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.

Show MeSH