Molecular Evolution of Multiple Arylalkylamine N-Acetyltransferase (AANAT) in Fish

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.

Subfunctionalization of arylalkylamine N‐acetyltransferases in the sea bass Dicentrarchus labrax: two‐ones for one two

“…Aanat enzymes of vertebrates usually display a significantly higher catalytic efficiency for indolethylamines versus phenylethylamines . We bring here evidence that Aanat1 enzymes acetylated both indolethylamines and phenylethylamines in accordance with our previous findings in other species . In addition, Aanat1a and Aanat1b behaved similarly with regard to temperature; their optimum activity was reached at 37°C.…”

Unique arylalkylamine N-acetyltransferase-2 polymorphism in Salmonids and profound variations in thermal stability and catalytic efficiency conferred by two residues

“…The four enzymes exhibited the classical AANAT2 pattern of substrate preferences: they acetylated indolethylamines much better than phenylethylamines (Benyassi et al, 2000;Cazaméa-Catalan et al, 2012;Coon et al, 1999;Gothilf et al, 1999;Zilberman-Peled et al, 2004;Zilberman-Peled et al, 2011). The kinetic constants were within the range of those already reported for other AANAT2, except for saAANAT2_VV, which had a catalytic efficiency for indolethylamines at least twofold higher than that of the other enzymes.…”

“…Position 160 has already been suggested to be an important position for substrate selectivity in AANATs. In a recent study, the naturally occurring Ile160 in seabream (Sparus aurata) AANAT2 was mutated to methionine (Zilberman-Peled et al, 2011). The authors chose this mutation because of the natural occurrence of methionine at this position in seabream AANAT1 and ovine AANAT.…”

“…Most interestingly, the saAANAT2_IA and saAANAT2_VV variants displayed a high degree of conservation with the trout AANAT2, from which they differ by only one or two amino acids at the same positions. This provided a unique opportunity to study the impact of two residues that occupy key positions in the enzyme structure, and are important (at least position 160) for substrate selectivity (Zilberman-Peled et al, 2011). In order to further explore the role these residues play, we constructed a hybrid enzyme presenting a Val114 and an Ala160; this _VA version complemented the natural saAANAT2_IA, saAANAT2_VV and omAANAT2_IV combinations.…”

Drastic neofunctionalization associated with evolution of the timezyme AANAT 500 Mya

“…The encoded proteins (206 and 207 aa) exhibited the two defining features of the GCN5 N-acetyltransferase superfamily, motifs A and B (SI Appendix, Fig. S1) (9,(22)(23)(24)(25)(26)(27)(28)(29). In addition, the sequences exhibited defining characteristics of the VT-AANATs of bony vertebrates, including motifs C and D, PKA sites nested in 14-3-3 binding domains, a Cys-ProLeu peptide sequence in a floppy loop within the binding region, and closely positioned histidine residues in the active site.…”