Overexpression and biochemical characterization of soluble pyridine nucleotide transhydrogenase from Escherichia coli

The soluble pyridine nucleotide transhydrogenase (STH) is an energy-independent flavoprotein that directly catalyzes hydride transfer between NAD(H) and NADP(H) to maintain homeostasis of these two redox cofactors. The sth gene in Escherichia coli was cloned and expressed as a fused protein (EcSTH). The purified EcSTH displayed maximal activity at 35 degrees C, pH 7.5. Heat-inactivation studies showed that EcSTH retains 50% activity after 5 h at 50 degrees C. The enzyme was stable at 4 degrees C for 25 days. The apparent K(m) values of EcSTH were 68.29 mu M for NADPH and 133.2 mu M for thio-NAD+. The k(cat)/K(m) ratios showed that EcSTH had a 1.25-fold preference for NADPH over thio-NAD+. Product inhibition studies showed that EcSTH activity was strongly inhibited by excess NADPH, but not by thio-NAD+. EcSTH activity was enhanced by 2 mM adenine nucleotide and inhibited by divalent metal ions: Mn2+, Co2+, Zn2+, Ni2+ and Cu2+. However, after preincubation for 30 min, most divalent metal ions had little effect on EcSTH activity, except Zn2+, Ni2+ and Cu2+. The enzymatic analysis could provide the important basic knowledge for EcSTH utilizations.