Limits...
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.

Show MeSH
Dopamine inhibits tryptamine acetylation by AANAT1a and AANAT2. Dopamine inhibition of tryptamine acetylation was analyzed using the radiochemical assay. The concentrations of tryptamine selected approximated the Km values of each enzyme (0.15 and 1.5 mM for AANAT1a and AANAT2, respectively), in the presence of increasing concentrations of dopamine (0, 0.1, 1, 5 mM or 0, 1, 10, 50 mM for AANAT1a and AANAT2, respectively). Other conditions were as previously determined for wild type AANAT1a and AANAT2 [22]. Values are given as mean ± SE.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-marinedrugs-09-00906: Dopamine inhibits tryptamine acetylation by AANAT1a and AANAT2. Dopamine inhibition of tryptamine acetylation was analyzed using the radiochemical assay. The concentrations of tryptamine selected approximated the Km values of each enzyme (0.15 and 1.5 mM for AANAT1a and AANAT2, respectively), in the presence of increasing concentrations of dopamine (0, 0.1, 1, 5 mM or 0, 1, 10, 50 mM for AANAT1a and AANAT2, respectively). Other conditions were as previously determined for wild type AANAT1a and AANAT2 [22]. Values are given as mean ± SE.

Mentions: Two reasons may account for the low ability of AANAT2 to acetylate phenylethylamines: (1) phenylethylamines do not bind to the catalytic pocket of AANAT2; or, (2) phenylethylamines do bind to the catalytic pocket of AANAT2 but are not acetylated by the enzyme. In the latter case, an inhibition of indolethylamines acetylation would be expected in the presence of phenylethylamines. To discriminate between the two options, acetylation of subsaturating tryptamine concentrations, at about the Km value of each enzyme (0.15 mM for AANAT1a and 1.5 mM for AANAT2) was measured in the absence or presence of increasing dopamine concentrations: 0.1 mM or 1mM for AANAT1a and AANAT2, respectively (∼1×); 1 mM or 10 mM for AANAT1a and AANAT2, respectively (∼6×); and 5 mM or 50 mM for AANAT1a and AANAT2, respectively (∼30×) the tryptamine concentrations (Figure 3). Similar inhibition patterns (Pearson correlation, R = 0.87, p = 0.024) were found for both enzymes, with minimal inhibition at the 1:1 dopamine:tryptamine ratio, ∼30% inhibition at the 6:1 dopamine:tryptamine ratio, and ∼ 60% inhibition at the 30:1 ratio (Figure 3). These results suggest that dopamine binds to the catalytic pockets of AANAT1a and AANAT2 at similar affinities, which are lower than those of tryptamine.


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

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

Dopamine inhibits tryptamine acetylation by AANAT1a and AANAT2. Dopamine inhibition of tryptamine acetylation was analyzed using the radiochemical assay. The concentrations of tryptamine selected approximated the Km values of each enzyme (0.15 and 1.5 mM for AANAT1a and AANAT2, respectively), in the presence of increasing concentrations of dopamine (0, 0.1, 1, 5 mM or 0, 1, 10, 50 mM for AANAT1a and AANAT2, respectively). Other conditions were as previously determined for wild type AANAT1a and AANAT2 [22]. Values are given as mean ± SE.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-marinedrugs-09-00906: Dopamine inhibits tryptamine acetylation by AANAT1a and AANAT2. Dopamine inhibition of tryptamine acetylation was analyzed using the radiochemical assay. The concentrations of tryptamine selected approximated the Km values of each enzyme (0.15 and 1.5 mM for AANAT1a and AANAT2, respectively), in the presence of increasing concentrations of dopamine (0, 0.1, 1, 5 mM or 0, 1, 10, 50 mM for AANAT1a and AANAT2, respectively). Other conditions were as previously determined for wild type AANAT1a and AANAT2 [22]. Values are given as mean ± SE.
Mentions: Two reasons may account for the low ability of AANAT2 to acetylate phenylethylamines: (1) phenylethylamines do not bind to the catalytic pocket of AANAT2; or, (2) phenylethylamines do bind to the catalytic pocket of AANAT2 but are not acetylated by the enzyme. In the latter case, an inhibition of indolethylamines acetylation would be expected in the presence of phenylethylamines. To discriminate between the two options, acetylation of subsaturating tryptamine concentrations, at about the Km value of each enzyme (0.15 mM for AANAT1a and 1.5 mM for AANAT2) was measured in the absence or presence of increasing dopamine concentrations: 0.1 mM or 1mM for AANAT1a and AANAT2, respectively (∼1×); 1 mM or 10 mM for AANAT1a and AANAT2, respectively (∼6×); and 5 mM or 50 mM for AANAT1a and AANAT2, respectively (∼30×) the tryptamine concentrations (Figure 3). Similar inhibition patterns (Pearson correlation, R = 0.87, p = 0.024) were found for both enzymes, with minimal inhibition at the 1:1 dopamine:tryptamine ratio, ∼30% inhibition at the 6:1 dopamine:tryptamine ratio, and ∼ 60% inhibition at the 30:1 ratio (Figure 3). These results suggest that dopamine binds to the catalytic pockets of AANAT1a and AANAT2 at similar affinities, which are lower than those of tryptamine.

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