Research Of Bacterial Sir2 Proteins

Sir2 (Silent information regulator 2) are NAD~+ dependent deacetylase whichcatalyze a unique protein deacetylation reaction. It requires the coenzyme NAD~+ andproduces nicotinamide and o-acetyl-ADP-ribose (OAADPr). Sir2 are conservedenzymes originated from bacterium to human; these proteins are involved in thecontrol of gene silencing, metabolic regulation, apoptosis and senescence. Most ofresearch of Sir2 concentrated on the eukaryotic cells, only few reports aboutprokaryotic sir2 could be seen.In this study, the 840 bp cobB gene was cloned from the E. coli k12 genome and714 bp of sir2 was amplified from Mycobacterium tuberculosis H37Rv genomerespectively. Expression vectors such as pET32a-sir2 and pET32a-cobB wereconstructed. Recombinant plasmids were transformed into E. coli AD494 forexpression of interest protein.Purification and characterization tests revealed that these two enzymes haveNAD~+ dependent deacetylase and weak ADP-ribosyltransferase activity, while Sir2has much higher ADP-ribosyltransferase activity than CobB. Their optimal pH is9.0±1.0 and the optimal temperature is 25℃. Inhibition tests showed that NAM, NA,POA, HN can inhibit the NAD~+ dependent deacetylase activity of CobB under acidicpH condition (pH 6.0), whereas only NAM and HN are effective under basic pHcondition (pH 8.0). Similar results could be seen in Sir2 tests, but PZA has weakinhibition effect to Sir2 under acidic pH condition. Furthermore, theΔcobB mutant ismore sensitive to acid stress and heat shock treatment, and more resistant to starvationstress and pyrazinamide treatment. And over-expression of CobB in vivo caused aprominent morphological change.This study of bacterial Sir2 proteins is helpful to elucidate the drugs resistant mechanism and physiological function of Sir2, and give some hints for the PZA drug-treatment mechanism of M. tuberculosis.