Accepted_test

Nα-acetyltransferases (Nα-ATs) acetylate the α-amino group of a wide range of substrates (amino acids, proteins, serotonin, antibiotics, etc.), thereby modulating their activity and metabolism. Nα-ATs are used in biotechnology to targeted N-terminal protein and peptides acetylation. Nα-ATs belong to GNAT super-family acetyltransferase (AT), characterized by a conserved three-dimensional structure but a low percentage of sequence identity, which makes it difficult to correctly annotate them in genomes and predict their substrate specificity. In this study we used bioinformatics methods to conduct comparative study at the primary, secondary and tertiary levels of organization of all annotated “putative/predicted/probable” acetyltransferases in the genome of the bacterium T. thermophilus HB8. This allowed us to define the specific criteria for the Nα-AT prediction. Six “probable” Nα-ATs were selected from more than 10 AT enzymes in T. thermophilus (Tth-Nα-AT). The substrate specificity of the Tth-Nα-ATs were predicted based on their comparative study with orthologous from other bacteria. Several Tth-Nα-ATs were cloned, purified and specific acetyltransferase activity was tested in vitro with a model protein. The results of the work made it possible to identify specific criteria for searching, predicting and selecting Nα-ATs among the sets of GNAT-ATs. The data obtained can subsequently form the basis for creating an algorithm for identifying Nα-ATs in the genomes of other organisms, which is currently, undoubtedly, very important for the correct annotation of enzymes of the Nα-AT family and prediction of their substrate specificity.