重組HP0642蛋白已被建立於大腸桿菌(E. coli) SG13009表現系統(楊瑞榮 碩士論文, 2006)。這個蛋白質主要的功能有二，一是具有菸鹼醯胺腺嘌呤雙核苷酸-啶黃素氧化還原酶(NAD(P)H:flavin oxidoreductase)的酵素活性，二是硝基還原酶(nitroreductase)的酵素活性。在這篇論文中呈現in vitro及in vivo所檢測出HP0642蛋白質的特性及酵素活性。
重組HP0642蛋白經由Ni-NTA管柱層析方法純化效率為10-12 mg/L E. coli，重組HP0642蛋白分子量為26.7 kDa。利用超高速離心法(ultracentrifugation)分析HP0642是以二聚體的方式存在。HP0642蛋白具有類似黃素蛋白(flavoprotein)的吸收光譜值。薄層色譜(Thin layer chromatography)實驗結果發現蛋白質HP0642利用FMN做為輔因子(co-factor)，而非FAD。FMN與HP0642的含量比值經由螢光檢測出為0.5。
抗HP0642的抗體血清經已由五次兔子注射實驗製備而成，利用西方點墨法(western blotting)可在1:5000稀釋倍率下測得HP0642蛋白。HP0642蛋白質表現量在3-24小時的H. pylori培養中並沒有差別；但在pH 5.5 的Brucella洋菜膠盤或Brucella broth培養兩小時，HP0642的蛋白質表現量會比在pH 7.2下提高約兩倍。
利用測定NAD(P)H的消耗量來確認NAD(P)H:flavin oxidoreductase的活性，反應條件為使用10 □g HP0642在25℃、pH 8.5 50 mM的Tris-HCl緩衝溶液中反應三分鐘。NADPH oxidase活性的Km(NADPH)為6.4 ± 0.7 □M，Vmax(NADPH)為591.4 ± 27.2 □M•min-1•mg-1。NADH oxidase活性的Km(NADH)為37.5 ± 3.8 □M及Vmax(NADH)為318.0 ± 4.4 □M•min-1•mg-1。無論是利用NADPH或NADH作受質，額外加入flavin，可明顯增加HP0642的活性。而無論flavin的添加與否，NAD(P)H oxidase活性傾向利用NADPH，而非NADPH為受質。利用2-16 □M的FMN以及2-16 □M的NADPH為受質做出的酵素動力雙倒數圖形呈現交叉圖形，顯示此酵素利用順序式機制(sequential mechanism)進行催化反應。
可經由測定nitrofurazone的消耗量來確認Nitroreductase的活性。反應條件為使用0.5 □g HP0642及NADPH在25℃ pH 8.5 50 □M Tris-HCl 緩衝溶液中反應三分鐘，固定NADPH量為100 □M，得知Km(NFZ)為3.4 ± 0.1 □M及Vmax(NFZ)為3181.3 ± 64.0 □M•min-1•mg-1；而固定nitrofurazone量為10 □M，得知Km(NADPH)為29.5 ± 2.4 □M及Vmax(NADPH)為2468.7 ± 20.3 □M•min-1•mg-1。添加FMN或FAD都不會增加酵素的活性，使用NADH為受質無法進行利用Nitroreductase的活性。1-3 □M的nitrofurazone以及25-200 □M的NADPH為受質做出的酵素動力論雙倒數圖形呈現平行線，表示此酵素活性利用乒乓機制(ping-pong mechanism)進行催化反應。
NADP+以及lumichrome都可以抑制NADPH oxidase活性。NADPH oxidase活性並沒有兩價陽離子作為co-factor參與其中。蛋白質HP0642的兩個酵素活性在pH 7-9都可具有理想的活性，溫度穩定性的IC50為55℃，此溫度也符合蛋白質HP0642由圓二光圖譜(circular dichroism)測定出的Tm值。

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.

Bruchhaus, I., S. Richter, and E. Tannich. 1998. Recombinant expression and biochemical characterization of an NADPH:flavin oxidoreductase from Entamoeba histolytica. Biochem J. 330 ( Pt 3):1217-21.
Bryant, C., and M. DeLuca. 1991. Purification and characterization of an oxygen-insensitive NAD(P)H nitroreductase from Enterobacter cloacae. J Biol Chem. 266:4119-25.
Covacci, A., J.L. Telford, G. Del Giudice, J. Parsonnet, and R. Rappuoli. 1999. Helicobacter pylori virulence and genetic geography. Science. 284:1328-33.
Deller, S., S. Sollner, R. Trenker-El-Toukhy, I. Jelesarov, G.M. Gubitz, and P. Macheroux. 2006. Characterization of a thermostable NADPH:FMN oxidoreductase from the mesophilic bacterium Bacillus subtilis. Biochemistry. 45:7083-91.
Dunn, B.E., H. Cohen, and M.J. Blaser. 1997. Helicobacter pylori. Clin Microbiol Rev. 10:720-41.
Faeder, E.J., and L.M. Siegel. 1973. A rapid micromethod for determination of FMN and FAD in mixtures. Anal Biochem. 53:332-6.
Fieschi, F., V. Niviere, C. Frier, J.L. Decout, and M. Fontecave. 1995. The mechanism and substrate specificity of the NADPH:flavin oxidoreductase from Escherichia coli. J Biol Chem. 270:30392-400.
Fontecave, M., R. Eliasson, and P. Reichard. 1987. NAD(P)H:flavin oxidoreductase of Escherichia coli. A ferric iron reductase participating in the generation of the free radical of ribonucleotide reductase. J Biol Chem. 262:12325-31.
Goodwin, A., D. Kersulyte, G. Sisson, S.J. Veldhuyzen van Zanten, D.E. Berg, and P.S. Hoffman. 1998. Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen-insensitive NADPH nitroreductase. Mol Microbiol. 28:383-93.
Inouye, S. 1994. NAD(P)H-flavin oxidoreductase from the bioluminescent bacterium, Vibrio fischeri ATCC 7744, is a flavoprotein. FEBS Lett. 347:163-8.
Jeffers, C.E., J.C. Nichols, and S.C. Tu. 2003. Complex formation between Vibrio harveyi luciferase and monomeric NADPH:FMN oxidoreductase. Biochemistry. 42:529-34.
Jeong, J.Y., A.K. Mukhopadhyay, D. Dailidiene, Y. Wang, B. Velapatino, R.H. Gilman, A.J. Parkinson, G.B. Nair, B.C. Wong, S.K. Lam, R. Mistry, I. Segal, Y. Yuan, H. Gao, T. Alarcon, M.L. Brea, Y. Ito, D. Kersulyte, H.K. Lee, Y. Gong, A. Goodwin, P.S. Hoffman, and D.E. Berg. 2000. Sequential inactivation of rdxA (HP0954) and frxA (HP0642) nitroreductase genes causes moderate and high-level metronidazole resistance in Helicobacter pylori. J Bacteriol. 182:5082-90.
Kobori, T., H. Sasaki, W.C. Lee, S. Zenno, K. Saigo, M.E. Murphy, and M. Tanokura. 2001. Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds: alteration of pyridine nucleotide binding by a single amino acid substitution. J Biol Chem. 276:2816-23.
Lacy, B.E., and J. Rosemore. 2001. Helicobacter pylori: ulcers and more: the beginning of an era. J Nutr. 131:2789S-2793S.
Lei, B., M. Liu, S. Huang, and S.C. Tu. 1994. Vibrio harveyi NADPH-flavin oxidoreductase: cloning, sequencing and overexpression of the gene and purification and characterization of the cloned enzyme. J Bacteriol. 176:3552-8.
Liu, M., B. Lei, Q. Ding, J.C. Lee, and S.C. Tu. 1997. Vibrio harveyi NADPH:FMN oxidoreductase: preparation and characterization of the apoenzyme and monomer-dimer equilibrium. Arch Biochem Biophys. 337:89-95.
Louie, T.M., H. Yang, P. Karnchanaphanurach, X.S. Xie, and L. Xun. 2002. FAD is a preferred substrate and an inhibitor of Escherichia coli general NAD(P)H:flavin oxidoreductase. J Biol Chem. 277:39450-5.
Malfertheiner, P., K. McColl, F. Baldi, M. Dinelli, D. Festi, F. Parente, L. Ricciardiello, E. Roda, G. Scarpulla, V. Stanghellini, and G. Torre. 1997. Update on Helicobacter pylori research. Dyspepsia. Eur J Gastroenterol Hepatol. 9:624-5.
Marshall, B.J., and J.R. Warren. 1984. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet. 1:1311-5.
Merrell, D.S., M.L. Goodrich, G. Otto, L.S. Tompkins, and S. Falkow. 2003. pH-regulated gene expression of the gastric pathogen Helicobacter pylori. Infect Immun. 71:3529-39.
Mostertz, J., C. Scharf, M. Hecker, and G. Homuth. 2004. Transcriptome and proteome analysis of Bacillus subtilis gene expression in response to superoxide and peroxide stress. Microbiology. 150:497-512.
Niviere, V., F. Fieschi, J.L. Decout, and M. Fontecave. 1999. The NAD(P)H:flavin oxidoreductase from Escherichia coli. Evidence for a new mode of binding for reduced pyridine nucleotides. J Biol Chem. 274:18252-60.
Nokhbeh, M.R., S. Boroumandi, N. Pokorny, P. Koziarz, E.S. Paterson, and I.B. Lambert. 2002. Identification and characterization of SnrA, an inducible oxygen-insensitive nitroreductase in Salmonella enterica serovar Typhimurium TA1535. Mutat Res. 508:59-70.
Parkinson, G.N., J.V. Skelly, and S. Neidle. 2000. Crystal structure of FMN-dependent nitroreductase from Escherichia coli B: a prodrug-activating enzyme. J Med Chem. 43:3624-31.
Peterson, D.R., and F.A. Carone. 1979. Renal regeneration following d-serine induced acute tubular necrosis. Anat Rec. 193:383-8.
Race, P.R., A.L. Lovering, R.M. Green, A. Ossor, S.A. White, P.F. Searle, C.J. Wrighton, and E.I. Hyde. 2005. Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone. Reversed binding orientations in different redox states of the enzyme. J Biol Chem. 280:13256-64.
Russell, T.R., and S.C. Tu. 2004. Aminobacter aminovorans NADH:flavin oxidoreductase His140: a highly conserved residue critical for NADH binding and utilization. Biochemistry. 43:12887-93.
Seo, D., K. Kamino, K. Inoue, and H. Sakurai. 2004. Purification and characterization of ferredoxin-NADP+ reductase encoded by Bacillus subtilis yumC. Arch Microbiol. 182:80-9.
Streker, K., C. Freiberg, H. Labischinski, J. Hacker, and K. Ohlsen. 2005. Staphylococcus aureus NfrA (SA0367) is a flavin mononucleotide-dependent NADPH oxidase involved in oxidative stress response. J Bacteriol. 187:2249-56.
Tanner, J.J., S.C. Tu, L.J. Barbour, C.L. Barnes, and K.L. Krause. 1999. Unusual folded conformation of nicotinamide adenine dinucleotide bound to flavin reductase P. Protein Sci. 8:1725-32.
Thomsen, L.L., J.B. Gavin, and C. Tasman-Jones. 1990. Relation of Helicobacter pylori to the human gastric mucosa in chronic gastritis of the antrum. Gut. 31:1230-6.
Tomb, J.F., O. White, A.R. Kerlavage, R.A. Clayton, G.G. Sutton, R.D. Fleischmann, K.A. Ketchum, H.P. Klenk, S. Gill, B.A. Dougherty, K. Nelson, J. Quackenbush, L. Zhou, E.F. Kirkness, S. Peterson, B. Loftus, D. Richardson, R. Dodson, H.G. Khalak, A. Glodek, K. McKenney, L.M. Fitzegerald, N. Lee, M.D. Adams, E.K. Hickey, D.E. Berg, J.D. Gocayne, T.R. Utterback, J.D. Peterson, J.M. Kelley, M.D. Cotton, J.M. Weidman, C. Fujii, C. Bowman, L. Watthey, E. Wallin, W.S. Hayes, M. Borodovsky, P.D. Karp, H.O. Smith, C.M. Fraser, and J.C. Venter. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature. 388:539-47.
Wen, Y., E.A. Marcus, U. Matrubutham, M.A. Gleeson, D.R. Scott, and G. Sachs. 2003. Acid-adaptive genes of Helicobacter pylori. Infect Immun. 71:5921-39.
Xu, Y., M.W. Mortimer, T.S. Fisher, M.L. Kahn, F.J. Brockman, and L. Xun. 1997. Cloning, sequencing, and analysis of a gene cluster from Chelatobacter heintzii ATCC 29600 encoding nitrilotriacetate monooxygenase and NADH:flavin mononucleotide oxidoreductase. J Bacteriol. 179:1112-6.
Zenno, S., H. Koike, A.N. Kumar, R. Jayaraman, M. Tanokura, and K. Saigo. 1996a. Biochemical characterization of NfsA, the Escherichia coli major nitroreductase exhibiting a high amino acid sequence homology to Frp, a Vibrio harveyi flavin oxidoreductase. J Bacteriol. 178:4508-14.
Zenno, S., H. Koike, M. Tanokura, and K. Saigo. 1996b. Conversion of NfsB, a minor Escherichia coli nitroreductase, to a flavin reductase similar in biochemical properties to FRase I, the major flavin reductase in Vibrio fischeri, by a single amino acid substitution. J Bacteriol. 178:4731-3.
Zenno, S., H. Koike, M. Tanokura, and K. Saigo. 1996c. Gene cloning, purification, and characterization of NfsB, a minor oxygen-insensitive nitroreductase from Escherichia coli, similar in biochemical properties to FRase I, the major flavin reductase in Vibrio fischeri. J Biochem (Tokyo). 120:736-44.
Zenno, S., and K. Saigo. 1994. Identification of the genes encoding NAD(P)H-flavin oxidoreductases that are similar in sequence to Escherichia coli Fre in four species of luminous bacteria: Photorhabdus luminescens, Vibrio fischeri, Vibrio harveyi, and Vibrio orientalis. J Bacteriol. 176:3544-51.
Zenno, S., K. Saigo, H. Kanoh, and S. Inouye. 1994. Identification of the gene encoding the major NAD(P)H-flavin oxidoreductase of the bioluminescent bacterium Vibrio fischeri ATCC 7744. J Bacteriol. 176:3536-43.