Abstract
The NAD(P)H:Flavin oxidoreductase from H. pylori, named HP0642, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins using NADH or NADPH as electron donor. The purified recHP0642 protein was a pale yellow color and exhibits a flavoprotein-like absorption spectrum. Thin-layer chromatography analysis showed that recHP0642 contains FMN rather than FAD. Fluorescence method determined that recHP0642 protein contains 56.7 nmole of non-covalent bound FMN and 20 nmole FAD of per mole of protein. Rec-HP0642 has highest NADH-dependent oxidase activity at pH 8.5. The Km and Kcat of its NADH-dependent oxidase is 73.6 ± 0.3 μM and 64.0 ± 1.6 S-1, respectively. N-ethylmaleimide, ZnCl2 or HgCl2 at 1 mM could inhibit more than 70% (96, 70 and 78%) enzyme activity. The NADH-flavin oxidoreductase of rec-HP0642 was measured in the presence of 0-20 μM FMN and 100 μM NADH. The Km FMN and Vmax FMN of its NADH-Flavin oxidoreductase is 0.8±0.2 μM and 119.9 ± 6.1, respectively. RecHP0642 showed optimum activities at 10 to 40 ℃. NADH-Flavin oxidoreductase of rec-HP0642 was inhibited in the presence of AMP and FAD. Rec-HP0642 also has NADPH-dependent oxidase activity in which the activity increases between 0-50 μM NADPH substrate concentration. The enzyme activity of rec-HP0642 showed dose-dependent decrease in NADPH dose higher than 50 μM. The exogenously added FMN and FAD (0-500 μM) slightly change the NADPH oxidase activity. In addition to NAD(P)H:Flavin oxidoreductase activity, rec-HP0642 protein could reduce disulphide-compound to act as disulphide reductase. The calculated Km and Kcat for the disulphide reducatse reaction is 8.2±0.4 μM and 51.3±4.4 S-1, respectively. Without NADPH or in the presence of NADH no activity was observed. The highest disulphide reductase activity of recHP0642 protein appeared in reaction buffer at pH 11. Results from functional assays showed that recHP0642 prossesed ATPase activity in reaction buffer containing 5 mM MgCl2 at pH 7.5 and 37 ℃. The kinetic parameters of the recHP0642 for the ATP ase activity have been determined to have the apparent Km value of 1.66 mM for ATP substrate and a Vmax value of 0.2741. Furthermore, it could reduce nitro-compound to act as nitroreductase. Optical spectrum of the purified recHP0642 protein indicated that be classified as oxygen-sensitive nitroreductase (type- I). The Km NFZ and Kcat NFZ for nitrofurazone substrate (0-20 μM) at 100 μM NADPH are 6.15±2.3 μM and 1648±9.2 S-1, respectively. The nitroreductase activities of recHP0642 only utilize NADPH as a source of reducing equivalents and can reduce nitrofurazone. Initial velocity studies have been performed which establish unambiguously a sequential kinetic mechanism. At 5mM of Cu2+ ion could induce the 1.3-fold of nitroreductase activity. At this concentration, the activities were inhibited by the Mg2+ (56.7%), Mn2+ (28.3%), Ca2+ (54.3%),Co2+ (12.97%), Fe2+ and Zn2+. Acetate and nicontic acid suppress nitroreductase activity of recHP0642 protein. In the presence of exogenously added FMN and FAD, recHP0642 exhibited a weak but measurable nitroreductase activity. The HP0642 was regarded as multiple function enzyme