Characterization and kinetic analysis of Vc1509, a NAD+ dependent deacetylase from Vibrio cholerae

• Detailed enzymatic characterization of Vc1509, first NAD dependent deacetylase, CobB, of V. cholerae. • Vc1509 efficiently deacetylates histone H3K9ac and H3K18ac with high substrate affinity. • Vc1509 shows higher histone affinity than mammalian SIRT6 and E. coli CobB. • Optimization of high-density culture conditions using a bioreactor for production of Vc1509 with ∼9-fold increase in yield compared to shake flask culture. Protein lysine acetyltransferase and deacetylases play crucial role in the regulation of bacterial metabolism, stress response and virulence. Among deacetylases, sirtuin-type NAD + dependent deacetylases, CobB, play central role in bacterial homeostasis. CobB has been extensively studied in several bacterial species like E. coli, Salmonella spp. , Vibrio parahaemolyticus, Mycobacterium etc. A CobB homolog, Vc1509, has been identified among the multiple putative deacetylases present in Vibrio cholerae genome. Knockout of Vc1509 led to global hyperacetylation, but there was no further biochemical characterization of enzyme activity. Our previous study reported the involvement of Vibrio parahaemolyticus CobB in the deacetylation of mammalian histones during host-pathogen interactions. In this study, we present the first comprehensive functional and kinetic analysis of Vc1509. We established a scalable expression system for high-yield production of recombinant Vc1509 required for detailed characterization. Vc1509 is a monomeric protein and harbours conserved secondary structure features of CobB. Our data show that Vc1509 is an active NAD + dependent lysine de-acylase capable of targeting a broad range of acylated substrates, providing new insights into the enzymatic properties and substrate specificity of CobB bacterial deacetylases.