title: Overexpression and biochemical characterization of soluble pyridine nucleotide transhydrogenase from Escherichia coli.
abstract: 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 5 h at 50 °C. The enzyme was stable at 4 °C for 25 days. The apparent K(m) values of EcSTH were 68.29 μM for NADPH and 133.2 μM 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: Mn(2+) , Co(2+) , Zn(2+) , Ni(2+) and Cu(2+) . However, after preincubation for 30 min, 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.
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Cao
authors.forename: Ping
authors.initials: P
authors.lastname: Song
authors.forename: Qin
authors.initials: Q
authors.lastname: Xu
authors.forename: Ruirui
authors.initials: R
authors.lastname: Su
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhu
mesh_terms: D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D009250:NADP Transhydrogenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012995:Solubility
chemical_list: D029968:Escherichia coli Proteins; D009250:NADP Transhydrogenases