TPPのToday’s Pin Point 平成２２年１月８日 石井　正治.

Presentation on theme: "TPPのToday’s Pin Point 平成２２年１月８日 石井　正治."— Presentation transcript:

1 TPPのToday’s Pin Point 平成２２年１月８日 石井　正治

2 雑誌会の背景 (1/5) ・アミノトランスフェラーゼ基質としてのグリオキシル酸（亀谷氏データ） ・メタボローム解析によるグリオキシル酸存在性
・C1代謝によるホルムアルデヒド生成可能性 ・ホルムアルデヒドの自己触媒反応によるC2,C3化合物生成可能性 ・糖代謝反応経由によるC2,C3化合物生成可能性

3 背景 (2/5): 代謝物の菌体中存在量 Lactate: 14700 pmol/109 cells
Glucose 6-phosphate: 3800 Succinate: Glyoxylate: Glycolate: Glycerate: 3-Phosphoglycerate:

4 背景 (3/5):H. ｔhermophilus 研究進捗状況
CO2 Oxaloacetate Citryl-CoA Citrate Succinyl-CoA Oxalosuccinate Isocitrate 2-Oxoglutarate Succinate Fumarate Malate Pyruvate Acetyl-CoA Phosphoenolpyruvate Glycerate-2P Glycerate-3P Glycerate-1,3P Glyceraldehyde-3P Glycerone-P b-D-Fructose-1,6P2 b-D-Fructose-6P a-D-Glucose-6P HTH1644 HTH (cfiAB) HTH0836 (icd) HTH1737, 0201 (ccsAB) HTH (korAB) HTH (forDABGE) HTH1421, 0983, 1908, 1512, 1863 (frdABCDE) HTH (porEDABG) HTH0756 (mdh) HTH0757, 0301 (fumAB) HTH0755 (acnA) HTH (sucCD) HTH1674 HTH0611 HTH1015 HTH0107 HTH0978 HTH0103/0183/1861 HTH0340 HTH0010/1286 Gluconeogenesis Reductive TCA cycle HTH1452, 1598 (pycAB) HTH0311 (ccl) 7412 6052 13503 11896 (kor) 13056 (for) 8931 319- 3061 3301,679 7084 4332 8863,6182 3223,2878 14459 3862 979 1874/243/1870 4637 6109 665 6658 8055 1106 赤字：酵素学的解析終了 青字：遺伝子アノテーション済み 緑字：マイクロアレイシグナル値

5 背景 (4/5)：POR推定反応機構 ？ FEBS J. in press

6 背景 (5/5) Glyoxylate carboligase(TPP酵素）はreversible?
Tartronate semialdehyde Glyoxylate Carbon dioxide

7 TPPの構造と機能 ・ Oxidative and non-oxidative decarboxylation of 2-oxoacids
ピリミジン部 チアゾール部 ・ Oxidative and non-oxidative decarboxylation of 2-oxoacids ・ Interketol transfer between sugar phosphates

8 Glyoxylate carboligase (GCL)?
Tartronate semialdehyde Glyoxylate Carbon dioxide ・ The enzyme is a homotetramer and requires FAD, ThDP and Mg2+ for catalysis. ・ The (R)-tartronate　semialdehyde produced by GCL is subsequently converted to D-glycerate by tartronate semialdehyde reductase (TSAR). ・ These reactions enable Escherichia coli and other microorganisms to　use the two-carbon compounds glycolate and glyoxylate as their　sole carbon sources for growth.

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10 GCLの構造 (residues 186–357) (residues 358–592) (residues 1–185)
NAT. CHEM. BIOL. 4 (2008)

11 Glyoxylate carboligase (Pseudomonas oxalaticus)
フラビンの反応への関与性 Glyoxylate carboligase (Pseudomonas oxalaticus) 分子量：230,000 (sucrose gradient centrifugation) 　　　　　：61,000 (SDS-PAGE) FAD含量：2モル（230,000あたり） 酵素を酸性硫安処理に供すると、分子量は115,000となり酵素は失活 失活した酵素にFAD添加すると、分子量は230,000となり、活性も回復 活性型Glyoxylate carboligaseはテトラマーで、 FADは テトラマー形成に　必要 Stopped-flow spectrophotometric studyからは、酵素反応おけるフラビンの還元性は全く示されていない。 Biochemistry 10 (1971)

12 TPPが関わる反応の基盤 プロトンリレープロセスによるC2-Hの引き抜き FEBS Journal 276 (2009) 2447–2453

13 TPP (ThDP)と隣接するVal 51 大腸菌由来GCL NAT. CHEM. BIOL. 4 (2008)

14 Val 51の位置の妥当性 大腸菌由来GCL NAT. CHEM. BIOL. 4 (2008)

15 WT並びに各種変異体の活性 NAT. CHEM. BIOL. 4 (2008)

16 C2-Hの脱プロトン化 60 s–1 380 s–1 210 s–1 kobs values
NAT. CHEM. BIOL. 4 (2008)

17 Determination of ThDP C2-H deprotonation rates
The kinetics of hydrogen deuterium exchange of the C2-H of ThDP were measured by 1H NMR as previously described. Immediately before the exchange experiments, GCL was preincubated at 25 ℃ in 0.1 M potassium phosphate buffer, pH 7.6, containing 5 mM Mg2+. The rates were measured by mixing equal volumes of enzyme (fully reconstituted with all cofactors) with 99.9% (v/v) D2O and quenching with acid in a rapid quench flow apparatus (RQF-3, KinTek) as previously described.

18 C2-H exchange と 活性との相関 ～GCLを除いて～
FEBS Journal 276 (2009) 2447–2453

19 GCLの反応機構 NAT. CHEM. BIOL. 4 (2008)

20 各ステップの活性 (1/2) NAT. CHEM. BIOL. 4 (2008)

21 各ステップの活性 (2/2) C6’-H NAT. CHEM. BIOL. 4 (2008)

22 Kinetic analysis of microscopic rate constants
The net forward rate constants for steps 1 to 4 were calculated from the integrated areas of the NMR peaks of ThDP and of the individual covalent ThDP intermediates by the method described for AHAS (acetohydroxyacid synthase). The 1H spectra of the acid quench–derived reaction intermediates were recorded on a Bruker Avance 400 MHz NMR spectrometer at 300 K.

23 TPPを取り囲む環境とC2脱プロトン化 NAT. CHEM. BIOL. 4 (2008)

24 We thus see that stabilization of the iminopyrimidine form of ThDP by interaction of a carboxylate with N1’, which is crucial for all the many other ThDP-dependent enzymes studied previously, is counterproductive for the last catalytic steps in GCL and hence unfavorable for the overall reaction

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