Characterization of succinate dehydrogenase flavoprotein from Staphylococcus aureus ATCC 12600

Abstract

Background: Staphylococcus aureus possesses complete tricarboxylic acid (TCA) cycle. Succinate dehydrogenase (SDH) links TCA cycle with electron transport chain and could therefore be an ideal target in the development of new antimicrobials. Hence, present study is aimed at characterization of SDH flavoprotein from Staphylococcus aureus ATCC 12600. Methods: Staphylococcus aureus ATCC 12600 was grown in brain heart infusion (BHI) broth and from the membrane fraction SDH was isolated and purified using diethyl aminoethyl (DEAE) cellulose. The kinetic parameters Michaelis constant (KM), maximal velocity (Vmax) and rate constant (kcat) for both native (SDH) and recombinant succinate dehydrogenase (rSDH) enzyme flavoproteins were determined through Hanes-Woolf plot. The SDH flavoprotein gene was amplified using polymerase chain reaction (PCR) and was cloned in pQE30 vector and expressed in DH5 strain of Escherichia coli. The molecular weights of native and rSDH flavoproteins were determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Results: SDH flavoprotein was purified from the membrane fraction of S.aureus and the present methodology gave 40 folds purification with molecular weight of 66kDa. The Vmax, KM and kcat of SDH flavoprotein are 199±1 μM/mg/min, 143.5±0.1 μM and 995±4/min. The pure recombinant SDH flavoprotein exhibited similar properties with that of native SDH flavoprotein. Conclusions: In multidrug-resistant strains of Staphylococcus aureus up regulation of SDH was observed favouring increased biofilm formation which is one of the key pathogenic factor and in view of the differences observed in the kinetics of Staphylococcus aureus SDH flavoprotein with other bacteria this enzyme is probably regarded as an ideal drug target in the development of new antimicrobials.

Keywords

• Succinate dehydrogenase flavoprotein
• KM
• Vmax
• Staphylococcus aureus