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

摘要

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°C, pH 7.5. Heat-inactivation studies showed that EcSTH retains 50% activity after 5h at 50°C. The enzyme was stable at 4°C for 25 days. The apparent K_m values of EcSTH were 68.29μM for NADPH and 133.2μM 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 2mM adenine nucleotide and inhibited by divalent metal ions: Mn~(2+), Co~(2+), Zn~(2+), Ni~(2+) and Cu~(2+). However, after preincubation for 30min, most divalent metal ions had little effect on EcSTH activity, except Zn~(2+), Ni~(2+) and Cu~(2+). The enzymatic analysis could provide the important basic knowledge for EcSTH utilizations.