Abstract
Recombinant HP0642 protein was obtained from gene cloning and expression in E. coli SG13009 system by Mr. Yang, Ray-Rong in 2006. Preliminary data indicated that this protein performed majority function as (1) NAD(P)H-flavin oxidoreductase activity and (2) NADPH-dependent nitroreductase activity according to various substrates. Further in vitro and in vivo characterization and kinetics determination of HP0642 protein are presented in this study. Purified rec-HP0642 protein from Ni-NTA super-flow column has a molecular weight of 26.7 kDa at the yield of 10-12 mg from per liter of E. coli culture. Analytical ultracentrifugation data indicated the purified protein forms a dimer. Purified protein exhibits a flavoprotein-like absorption spectrum. Thin layer chromatography results showed that the HP0642 protein contains FMN co-factor, but not FAD. Molar ratio of FMN and protein is 0.5 by fluorescence measurement along with separated FMN and FAD standard. The sera against rec-HP0642 protein had been produced in rabbits. The endogenous HP0642 of H. pylori was detected by western blotting with 1:5000 dilution of the 5th boost anti-serum. No difference in HP0642 protein expression from samples harvested from 3-24 h after re-plated cultures. However, HP0642 expression was about 2-fold up regulation on acidic (pH 5.5) Brucella agar plates or Brucella broth for 2 h. NAD(P)H oxidase and NAD(P)H:flavin oxidoreductase activity was confirmed by the consumption of NAD(P)H after incubating 10 □g HP0642 protein at 25℃ for 3 min in buffer containing 50 mM Tris-HCl (pH 8.5). The Km(NADPH) is 6.4 ± 0.7 □M and Vmax(NADPH) is 591.4 ± 27.2 □M•min-1•mg-1 for its NADPH oxidase activity. And, the Km(NADH) is 37.5 ± 3.8 □M and Vmax(NADH) is 318.0 ± 4.4 □M•min-1•mg-1 for its NADH oxidase activity. Extra addition of flavin (FMN, FAD or riboflavin) would significantly increase the rec-HP0642 enzyme activity in the presence of either NADPH or NADH substrate. NADPH preference to NADH was found as the substrate of the rec-HP0642 oxidase in spite of flavin addition or not. An intercepting pattern appeared in several Lineweaver-Burk plots in which were generated from enzyme reaction including 2-16 □M FMN and 2-16 □M NADPH substrates. This indicated that HP0642 NADPH:flavin oxidoreductase activity follows a sequential mechanism. Nitroreductase activity was confirmed by the consumption of nitrofurazone after incubating 0.5 □g HP0642 protein and NADPH, but not NADH at 25℃ for 3 min in 50 mM Tris-HCl (pH 8.5). The Km(NFZ) is 3.4 ± 0.1 □M and Vmax(NFZ) is 3181.3 ± 64.0 □M•min-1•mg-1 for its enzyme activity in the presence of 100 □M of NADPH. The Km(NADPH) is 29.5 ± 2.4 □M and Vmax(NADPH) is 2468.7 ± 20.3 □M•min-1•mg-1 for its enzyme activity in the presence of 10 □M of nitrofurazone. FMN or FAD addition would not increase the enzyme activity. A parallel pattern double-reciprocal plots showed in enzyme reactions including 1-3 □M Nitrofurazone and 25-200 □M NADPH substrates. This indicated that HP0642 nitroreductase activity follows a ping-pong mechanism. Either NADP+ or Lumichrome inhibited the NADPH oxidase activity. There is no divalent cation cofactor involve in the NADPH oxidase activity. ptimal pH range from 7 to 9 was observed either for HP0642 NADPH oxidase or nitroreductase activity. The IC50 of the thermal stability for either NADPH oxidase or nitroreductase activity is about 55℃. This temperature matched the Tm of the HP0642 protein from circular dichroism data.