An insight into the role of the N-terminal domain of Salmonella CobB in oligomerization and Zn 2+ mediated inhibition of the deacetylase activity.

Prokaryotic deacetylases are classified into nicotinamide adenine dinucleotide (NAD+ )-dependent sirtuins and Zn2+ -dependent deacetylases. NAD+ is a coenzyme for redox reactions, thus serving as an essential component for energy metabolism. The NAD+ -dependent deacetylase domain is quite conserved and well characterized across bacterial species like CobB in Escherichia coli and Salmonella , Rv1151c in Mycobacterium , and SirtN in Bacillus subtilis . E. coli CobB is the only bacterial deacetylase with a known crystal structure (PDB ID: 1S5P), which has 91% sequence similarity with Salmonella CobB (SeCobB). Salmonella encodes two CobB isoforms, SeCobBS and SeCobBL , with a difference of 37 amino acids in its N-terminal domain (NTD). The hydrophobic nature of NTD leads to the stable oligomerization of SeCobBL . The homology modeling-based predicted structure of SeCobB showed the presence of a zinc-binding motif of unknown function. Tryptophan fluorescence quenching induced by ZnCl2 showed that Zn2+ has a weak interaction with SeCobBS but higher binding affinity toward SeCobBL , which clearly demonstrated the crucial role of NTD in Zn2+ binding. In the presence of Zn2+ , both isoforms had significantly reduced thermal stability, and a greater effect was observed on SeCobBL . Dynamic light scattering (DLS) studies reflected a ninefold increase in the scattering intensity of SeCobBL upon ZnCl2 addition in contrast to an ∼onefold change in the case of SeCobBS , indicating that the Zn2+ interaction leads to the formation of large particles of SeCobBL . An in vitro lysine deacetylase assay showed that SeCobB deacetylated mammalian histones, which can be inhibited in the presence of 0.25-1.00 mM ZnCl2 . Taken together, our data conclusively showed that Zn2+ strongly binds to SeCobBL through the NTD that drastically alters its stability, oligomeric status, and enzymatic activity in vitro .