| dc.description.abstract | Helicobacter pylori is a gram-negative epsilon proteobacterium infecting half of the world population. H. pylori is a naturally competent bacterium with a huge repertoire of Restriction-Modification (RM) systems. The bacterium lacks a mismatch DNA repair system, lexA gene responsible for SOS response, and starvation/stress-responding alternative sigma factor. These factors make it essential to understand the physiology of bacteria to manage H. pylori-related diseases. In recent years, protein acetylation stands out as a vital regulatory system of cellular processes such as virulence, acid stress survival, transcription, motility, and metabolic pathways. In this study, Western blotting-based acetylome analysis of different strains of H. pylori suggest a prominent and significantly different acetylation profile in H. pylori, strain. Mass spectrometry-based acetylome analysis found 384 acetylation sites on 236. HP0935, a possible protein N-acetyltransferase belonging to GNAT superfamily was identified. Unlike most GNAT superfamily acetyltransferases HP0935 remains as a monomer in the solution. Biochemical analysis suggests that HP0935 acetylates the respective N-α amino group of lysine, arginine, methionine, and serine. In addition, HP0935 acetylates the N-ε amino group of lysine. Crystals of HP0935 were grown by the sitting drop vapor diffusion method, and the structure was solved to 1.93 Å resolution. The crystal structure of HP0935 showed a proper GNAT fold, which further validates that the protein belongs to the GNAT superfamily. The co-crystal structure of HP0935 and acetyl-CoA complex suggests that Glu77, His115, and Tyr127 and a conserved water molecule could play essential roles in the catalysis. In general, glutamate and histidine in most GNATs act as a general base that deprotonate the amino group of substrates. In other GNATs, conserved water molecules could perform a similar activity. Tyrosine in the structure acts as a general acid that protonates the leaving thiolate anion during catalysis. Point mutations of HP0935 E77Q, H115A, and Y127F were created by site-directed mutagenesis to understand the catalytic mechanism. Acetylation activity of these mutants against lysine showed that mutant E77Q and H115A have comparable activity to the wild type. In comparison, mutant Y127F completely lost its activity. These results suggest that HP0935 contains catalytic activity where a conserved water molecule probably performs the deprotonation of the amino group of substrates and Tyr127 acts as a general acid. In search of the protein substrates for HP0935, HPDprA, DNA processing protein A which plays an essential role in natural transformation found to be acetylated by HP0935. SPR analysis suggests that HPDprA interacts with HP0935. Enzymatically acetylated K133 residue and non-enzymatically acetylated K127 residue were found to play a regulatory role in the DNA binding activity of HPDprA. Next, acetylation of HPDprA inhibits its known stimulatory effects on the activity of HPyAVI DNA methyltransferase. Two other proteins, HPyAVI, an N6 adenine methyltransferase, and HPUvrD helicase were found to be substrates of HP0935. It was observed that enzymatic acetylation stimulates the DNA methyltransferase activity of HPyAVI. Biochemical analysis of acetylated HPUvrD suggests that both enzymatic and non-enzymatic acetylation stimulate its ATPase activity by ~30%. Acetylated HPUvrD also showed high helicase activity against 3’overhang DNA substrate in comparison to wild-type HPUvrD. These results demonstrate a role for acetylation in Natural Transformation and DNA repair pathways of H. pylori. | en_US |
