Functional and structural characterisation of RimL from Bacillus cereus, a new Nα-acetyltransferase of ribosomal proteins that was wrongly assigned as an aminoglycosyltransferase
International journal of biological macromolecules(2024)
Abstract
Enzymes of the GNAT (GCN5-relate N-acetyltransferases) superfamily are important regulators of cell growth and development. They are functionally diverse and share low amino acid sequence identity, making functional annotation difficult. In this study, we report the function and structure of a new ribosomal enzyme, N-alpha-acetyl transferase from Bacillus cereus (RimL(BC)), a protein that was previously wrongly annotated as an aminoglycosyltransferase. Firstly, extensive comparative amino acid sequence analyses suggested RimL(BC) belongs to a cluster of proteins mediating acetylation of the ribosomal protein L7/L12. To assess if this was the case, several well established substrates of aminoglycosyltransferases were screened. The results of these studies did not support an aminoglycoside acetylating function for RimL(BC). To gain further insight into RimL(BC) biological role, a series of studies that included MALDI-TOF, isothermal titration calorimetry, NMR, X-ray protein crystallography, and site-directed mutagenesis confirmed RimL(BC) affinity for Acetyl-CoA and that the ribosomal protein L7/L12 is a substrate of RimL(BC). Last, we advance a mechanistic model of RimL(BC) mode of recognition of its protein substrates. Taken together, our studies confirmed RimL(BC) as a new ribosomal N-alpha-acetyltransferase and provide structural and functional insights into substrate recognition by N-alpha-acetyltransferases and protein acetylation in bacteria.
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Key words
N-alpha -acetyltransferase,GNAT family,Acetyl-CoA,RimL,L7/L12 ribosomal protein,Acetylation,Protein substrate recognition
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