Classification and phylogeny for the annotation of novel eukaryotic GNAT acetyltransferases

Abstract

The enzymes of the GCN5-related N-acetyltransferase (GNAT) superfamily count more than 870 000 members through all kingdoms of life and share the same structural fold. GNAT enzymes transfer an acyl moiety from acyl coenzyme A to a wide range of substrates including aminoglycosides, serotonin, glucosamine-6-phosphate, protein N-termini and lysine residues of histones and other proteins. The GNAT subtype of protein N-terminal acetyltransferases (NATs) alone targets a majority of all eukaryotic proteins stressing the omnipresence of the GNAT enzymes. Despite the highly conserved GNAT fold, sequence similarity is quite low between members of this superfamily even when substrates are similar. Furthermore, this superfamily is phylogenetically not well characterized. Thus functional annotation based on homology is unreliable and strongly hampered for thousands of GNAT members that remain biochemically uncharacterized. Here we used sequence similarity networks to map the sequence space and propose a new classification for eukaryotic GNAT acetyltransferases. Using the new classification, we built a phylogenetic tree, representing the entire GNAT acetyltransferase superfamily. Our results show that protein NATs have evolved more than once on the GNAT acetylation scaffold. We use our classification to predict the function of uncharacterized sequences and verify by in vitro protein assays that two fungi genes encode NAT enzymes targeting specific protein N-terminal sequences, showing that even slight changes on the GNAT fold can lead to change in substrate specificity. In addition to providing a new map of the relationship between eukaryotic acetyltransferases the classification proposed constitutes a tool to improve functional annotation of GNAT acetyltransferases. Author SummaryEnzymes of the GCN5-related N-acetyltransferase (GNAT) superfamily transfer an acetyl group from one molecule to another. This reaction is called acetylation and is one of the most common reactions inside the cell. The GNAT superfamily counts more than 870 000 members through all kingdoms of life. Despite sharing the same fold the GNAT superfamily is very diverse in terms of amino acid sequence and substrates. The eight N-terminal acetyltransferases (NatA, NatB, etc.. to NatH) are a GNAT subtype which acetylates the free amine group of polypeptide chains. This modification is called N-terminal acetylation and is one of the most abundant protein modifications in eukaryotic cells. This subtype is also characterized by a high sequence diversity even though they share the same substrate. In addition the phylogeny of the superfamily is not characterized. This hampers functional annotation based on homology, and discovery of novel NATs. In this work we set out to solve the problem of the classification of eukaryotic GCN5-related acetyltransferases and report the first classification framework of the superfamily. This framework can be used as a tool for annotation of all GCN5-related acetyltransferases. As an example of what can be achieved we report in this paper the computational prediction and in vitro verification of the function of two previously uncharacterized N-terminal acetyltransferases. We also report the first acetyltransferase phylogenetic tree of the GCN5 superfamily. It indicates that N-terminal acetyltransferases do not constitute one homogeneous protein family, but that the ability to bind and acetylate protein N-termini had evolved more than once on the same acetylation scaffold. We also show that even small changes in key positions can lead to altered enzyme specificity.