“Probable acetyltransferase” TTHA1209 Thermus thermophilus bacteria: Gene cloning, structural and functional analysis of the enzyme
Abstract
Background. In the last decade, bacterial N-acetyltransferases (ATs) have been intensively studied because they are involved in the biosynthesis/inactivation of antibiotics, form the “toxin-antitoxin” system and the acetylation status of proteins and peptides influences their virulence and pathogenicity. AT enzymes have a low percentage of amino acid sequence identity, which complicates their identification and annotation in genomes. In this regard, the aim of this work was the structural and functional analysis of a new “probable” acetyltransferase encoded by the open reading frame (ORF) TTHA1209 of the bacterium T. thermophilus.
Materials and methods. Structural analysis of the enzyme was carried out using bioinformatics methods. Genetic engineering methods were used to clone the TTHA1209 gene. Affinity chromatography was used to purify the protein. The protein activity was tested in an in vitro reaction of acetylation of the model protein parvalbumin (PA). The incorporation of an acetyl group at the N-terminus of PA was evaluated by mass spectrometry.
Results. It was found that TTHA1209 share single GNAT domain in its structure and exhibits the alternation of secondary structure elements and three-dimensional folding characteristic of N-terminal At (NAT). The similarity of the structure of the TTHA1209 enzyme with orthologous proteins from E. coli was investigated. The highest percentage identity of TTHA1209 is observed with the RimI enzyme (27%). It has been shown that the TTHA1209 enzyme acetylates parvalbumin, i.e. has a specific Nα-acetyltransferase activity.
Conclusion. The new enzyme TTHA1209 from the bacterium T. thermophilus exhibits properties characteristic of NAT at all levels of organization, has a specific activity and can be annotated in the genome as an N-terminal acetyltransferase. The results obtained create the prerequisites for further investigation of the substrate specificity and biochemical properties of the new NAT TTHA1209, which will open up prospects for its use in biotechnology for the acetylation of proteins and peptides.
EDN: PJYJXI
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References
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