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 sequence similarity 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 fungal 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.

Fig 1. GNAT fold is the acetylation scaffold in the acetyltransferase superfamily.

The fold positions the two substrates in such a way that the acetyl group of Ac-CoA approaches the N-terminus of the protein acceptor in the middle of the V-shaped splay between β4 and β5 strands–marked with the red circle. Four structural motifs have been identified in the GNAT fold: motif A consists of the β4 strand and α3 helix, motif B is the β5 strand and α4 helix, motif C includes the β1 strand and α1 helix, and motif D consists in the β2 and β3 strands [2].

Fig 2. Simplified view of the resulting sequence similarity network.

Each node represents one cluster from the original network. Edges connect two nodes in the simplified network if there is at least one edge between any nodes of the corresponding clusters in the full network. Node colors correspond to their degree, i.e. the number of connections to the neighboring nodes. Each node in the network has a unique number assigned by clusterONE [72]. The numbers serve as cluster names in cases where the cluster is uncharacterized. All nodes circled in red are known and experimentally confirmed N-terminal acetyltransferases (10 –NAA10, 8 –NAA20, 2 –NAA30, 20 –NAA40, 9 –NAA50, 24 –NAA60, 97 –NAA70 and 63 –NAA80). Of importance is also cluster 135, which contains the catalytically inactive yeast NAA50. The network shows four NAT groups. Group 1 consists of two subgroups–Group 1a which contains NAA10 and NAA20 and Group 1b which contains NAA30.

Fig 3. Characteristic sequence motif fingerprints of NAT Groups 1 to 4.

Sequence motifs were calculated as described in the Material and Methods section and using sequences from the SSN clusters. Each position in the motif is represented by a colored bar and a one-letter code for the amino acid frequently found at that position in the GNAT fold. The height of colored bar is proportional to the frequency of the corresponding amino acid. The colors correspond to the type of amino acid (blue: R, H, K; red: D, E; green: C, S, G, Y, T; black: P, F, V, L, I, A; orange: M, W; purple: N, Q). Group 5 is not shown as there is no structure of NAA70 available.

Fig 4. DALI dendrogram for structural similarity between acetyltransferases.

The dendrogram is the result of hierarchical clustering of structures. The known NATs are closer to one another than to the rest of the superfamily. Note the non-NAT acetyltransferases located close to known NATs.

Fig 5. Unrooted phylogenetic tree of the acetyltransferase superfamily.

The tree contains only those sequences for which we could find significant relationships in the SSN. A gray background is used to highlight the branches on the tree that are populated exclusively by uncharacterized sequences.

Fig 6. Variations of sequence motifs in key positions on the GNAT fold suggest novel NATs with different ligand specificities.

When we compare the sequence motifs of NAA50 (cluster 9) and NAA60 (cluster 24) to the corresponding motifs of their surrounding clusters, we notice a number of small but meaningful differences (A). These differences occur on key positions of the GNAT fold and are illustrated here on the X-ray structure of NAA50 (PDB 3TFY) (B) The sequence differences located on the α1-α2 loop, β4 and β5 strands and β6-β7 loop residues are likely to result in altered specificity. The structure superimposition between human NAA50 from cluster 9 (orange, PDB 3TFY) and yeast NAA50 from cluster 135 (green, PDB 4XNH) highlights the small differences between residues involved in substrate binding in these two proteins with reportedly different specificities [88] (C).

Fig 7. Purification and DTNB-based activity assays of the putative NATs N1Q410 and A0A194XTA9.

Putative NAT activities were tested by DTNB-based assays. 3μM of purified N1Q410 (A) and A0A194XTA9 (B) were incubated with a selection of 24 amino acids-long synthetic peptides (300 μM), and Ac-CoA (300 μM) for 1 hour at 37°C. The formation of Nt-acetylated product was spectrophotometrically determined. Shown is the mean ± SD (n = 3).