An insight into the role of the N-terminal domain of Salmonella CobB in oligomerization and Zn2+ mediated inhibition of the deacetylase activity

Abstract

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, SeCobB_S and SeCobB_L, with a difference of 37 amino acids in its N-terminal domain (NTD). The hydrophobic nature of NTD leads to the stable oligomerization of SeCobB_L. 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 SeCobB_S but higher binding affinity toward SeCobB_L, 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 SeCobB_L. Dynamic light scattering (DLS) studies reflected a ninefold increase in the scattering intensity of SeCobB_L upon ZnCl₂ addition in contrast to an ∼onefold change in the case of SeCobB_S, indicating that the Zn²⁺ interaction leads to the formation of large particles of SeCobB_L. 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 SeCobB_L through the NTD that drastically alters its stability, oligomeric status, and enzymatic activity in vitro.

Keywords: CobB homology modeling; CobB oligomerization; CobB thermal stability; Salmonella nicotinamide adenine dinucleotide-dependent deacetylase (CobB); Zn2+–CobB interaction; inhibition of CobB deacetylase activity.