胃幽門螺旋桿菌之菸鹼醯胺腺嘌呤雙核苷酸-啶黃素氧化還原脢功能的研究

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研究生：	楊瑞榮 Ray-Rong Yang
論文名稱：	胃幽門螺旋桿菌之菸鹼醯胺腺嘌呤雙核苷酸-啶黃素氧化還原脢功能的研究 Molecular Cloning and Functional Characterization Of NAD(P)H:Flavin oxidoreductase From Helicobacter Pylori
指導教授：	黃海美 Haimei Huang
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2006
畢業學年度：	94
語文別：	中文
論文頁數：	83
中文關鍵詞：	氧化'C1棜孻Q 、氮還原脢、双硫鍵還原脢、胃幽門螺旋桿菌
外文關鍵詞：	oxidoreductase, nitroreductase, disuphide reductase, H. pylori
相關次數：	點閱：2 下載：0
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HP0642在胃幽門螺旋桿菌中被預測為NAD(P)H:Flavin
oxidoreductase，為分子量26.2 kDa的單套體，主要利用NADH或者NADPH 當作電子捐贈者，還原氧化態的黃素。純化的HP0642為暗黃色並且具有黃素蛋白吸收光譜。液像薄膜色層分析法分析重組HP0642含有FMN輔因子。利用螢光檢測法檢測1莫耳的重組HP0642蛋白結合56.7 nmole非共價鍵結FMN和20 nmole的FAD輔因子。
重組HP0642在pH 8.5，有傘高的NADH 氧化脢酵素活性。NADH氧化脢的Km 和 Kcat分別為73.6 ± 0.3 μM 和 64.0 ± 1.6 S-1。 N-ethylmaleimide，ZnCl2 或者HgCl2 在1mM的濃度時，可以抑制超過70% (96、 70 and 78%) 的酵素活性。重組HP0642的NADH:Flavin oxidoreductase 酵素活性在 0-20 μM FMN和 100 μM NADH時測得。 NADH:Flavin oxidoreductase酵素活性對FMN的Km 和 Kcat分別為0.8±0.2 μM 和119.9 ± 6.1。最適合重組HP0642酵素活性溫度在10 到 40 ℃。重組HP0642的NADH:Flavin oxidoreductase 酵素活性在AMP和FAD存在下會被抑制。重組HP0642也具有NADPH氧化脢酵素活性並且隨者受質NADPH增加而活性增加在0-50 μM NADPH 濃度間。重組HP0642酵素活性在超過50μM NADPH濃度下呈現受質增加而活性下降的現象。外加FMN和FAD(0-500 μM)會輕微改變NADPH 氧化脢活性。
重組HP0642除了NAD(P)H:Flavin oxidoreductase活性之外，還具有双硫鍵還原脢活性能夠還原双硫鍵物質。双硫鍵還原脢對DTNB受質的Km 和 Kcat分別為8.2±0.4 μM 和 51.3±4.4 S-1。當沒有NADPH或著有NADH存在時，是沒有活性的。重組HP0642在pH 11，有最高的双硫鍵還原脢活性。
功能分析的結果指出重組HP0642在pH7.5，37 ℃含有5 mM MgCl2的緩衝溶液中，能催化ATPase活性。重組HP0642 ATPase的Km和Vmax為1.66 mM和0.2741。
而且，氮還原脢活性也能當作氮還原脢還原硝基化合物。分光光度計分析指出重組HP0642蛋白屬於氧不敏感氮還原脢(第一型)。在受質nitrofurazone(0-20 μM)和100 μM NADPH的濃度下，對nitrofurazone受質的Km和Vmax分別為6.15±2.3 μM and 1648±9.2 S-1。重組HP0642的氮還原脢酵素活性只能利用NADPH當作還原劑而還原nitrofurazine。初速度的研究顯示出氮還原脢進行順序催化機制。5 mM 濃度的二價銅離子可以增加1.3倍的氮還原脢酵素活性。在相同的濃度下，氮還原脢酵素活性會被二價鎂離子(56.7%)、二價錳離子(28.3%)、二價鈣離子(54.3%)、二價鈷離子(12.97%)、二價鐵離子和二價鋅離子所抑制。 Acetate 和nicontic acid會抑制重組HP0642蛋白的氮還原脢活性。當外加的FMN和FAD存在時，重組HP0642存在微弱但是可測得的氮還原脢活性。因此， HP0642是屬於多功能的酵素。

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

謝誌
Chinese abstract..........................................I
Abstract.................................................II
Contents................................................III
Introduction..............................................1
  I.  Helicobacter pylori in peptic ulcer disease.........1
  II. Functional and structural genomics studies
      on H.pylori.........................................1
  III.Bioinformatics, structural and functional
      information of HP0642...............................2
  IV. The physiological importance of NAD(P)H: Flavin
      oxidoreductase (HP0642).............................3
  V.  The background of NAD(P)H: Flavin oxidoreductase
      (HP0642)............................................3
  VI. The background for riboflavin, FAD, FMN.............4
Motives...................................................6
Materials and methods.....................................8
  I.  Bacterial strains, Plasmids, Enzymes, Mediums and
      Reagents............................................8
  II. Polyclonal Antibody Productio......................14

  III.Western Blot Analysis..............................14
  IV. H. pylori culture conditions for assays............15
  V.  The properties for recHP0642—to determine the
      cofactor...........................................16
  VI. Functional Assays for recHP0642....................18
Results..................................................22
  I.  Bioinformatics for HP0642..........................22
  II. HP0642 gene construction and protein purification..23
  III.Preparation of polyclonal anti-HP0642 anti-body....24
  IV. Expression of HP0642 protien in H. pylori at
      different growth stages or differernt pH...........24
  V.  Physical properties of rec-HP0642 protein..........24
  VI. Enzyme properties of recHP0642 protein.............24
Discussion...............................................35
  I.  Multiple enzyme activities for recHP0642 protein    ..35
  II. Stable NADH:Flavin oxidoreductase activity for two
      month storage rec-HP0642 protein but not yet known to
      other enzyme activities............................35
  III.Is anti-serum against rec-HP0642 protein specific in
      usage for assay HP0642 protein expression in vivo?.35
  IV. The properties of the recHP0642....................36
  V.  Enzyme properties..................................37
  VI. The effect on inhibitor for recHP0642 enzyme
      activity...........................................44
References...............................................46
Tables...................................................49
Figures..................................................54
Appendix.................................................82

                                

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