臺灣博碩士論文加值系統

轉酮酶 (TK) 存在于五碳糖代謝路徑中可雙向催化二碳單元在磷酸化的酮糖與醛糖上之轉移，其催化機制已被研究且被認為相似雜環碳烯卡賓催化的苯偶姻縮合反應，其中碳¬-碳鍵的斷裂與生成是透過其反應中心親電性與親核性來回變化。在此研究計畫中，我們將探討在Pichia stipits CBS6054菌株中所發現的轉酮酶 (TKps) 是否完全適用于當前之電子對反應機制，此菌株為一個高效率五碳糖代謝反應之工業用菌株。根據目前的實驗結果發現，無論受質存在於否，其中當作輔酶的硫胺素二磷酸鹽上 (ThDP) 的噻唑環會自發性的產生彎曲效應。在此我們提出此彎曲效應是由於形成非凱庫勒雙自由基反應，且不同於被發現在丙酮酸硫鐵蛋白氧化酶 (PFOR) 中基於受質氧化的單電子反應，非凱庫勒雙自由基的形成有利於其二碳單元轉移之進行。為了證實推測，我們使用X光晶體繞射之方法，對轉酮酶中酵素活性區域內的噻唑-二碳單元之各種配體結構進行繞射分析。藉由原子級解析度的晶體結構，我們可觀察並確認各種可能異構物上的電子配置，並發現推測中的中間異構物構型是否存在。配合生物物理、生物化學實驗方法與電腦模擬計算的結果，在此我們發現一個從未有過先例的雙向單電子的反應機制。

The enzyme transketolase (TK) reversibly transfers a two-carbon unit between ketosugar donors and aldosugar acceptors in the pentose metabolism (the PPP pathway). The catalytic mechanism of TK has long been analogized with the N-heterocyclic carbene-catalyzed benzoin condensation, whereby the polarity of the reaction center alters back and forth as an electrophile or a nucleophile during the C-C bond association/disassociation. In this study, we come to question whether the currently accepted electron-pair model is applicable to the TK in Pichia stipits CBS6054 (TKps), an industrial strain known for its high efficient pentose metabolism. The fact is that our experiment data revealed that the thiazole ring of the thiamine diphosphate (ThDP) coenzyme with/without presence of substrates bends in a dynamic manner. We thus proposed that this ring bending is due to formation of non-Kekulé diradicals, differing from single electron relay upon substrate oxidation exemplified as pyruvate ferredoxin oxidoreductase (PFOR). The formation of non-Kekulé diradicals is chemically favorable while executing the two-carbon unit transfer reaction. To consolidate this reasoning, we take advantage of X-ray crystallography in a bid to snapshot all possible postures that thiazole/thiazolium -ligand adapts while a two-carbon unit transferring in the active site of TKps. We are pursuing atomic-level resolutions of crystal complexes, because it would allow us to visualize/confirm the true electron configurations of all possible conformers. In conjunction with other biophysical, biochemical and computational techniques/experiments, an unprecedented reversible single-electron mechanism is expected to be revealed here.

中文摘要...................................................i
Abstract..................................................ii
Contents.................................................iii
List of Figures...........................................iv
List of Tables............................................iv
Chapter 1. Introduction....................................1
1.1. A review of TK.....................................1
1.1.1. Distribution of TK............................1
1.1.1. The structure of TK...........................1
1.1.3. Substrate specificity of TK...................1
1.2. Transketolase in Pichia stipitis...................2
1.3. Catalytic mechanism of TK..........................3
Chapter 2. Materials and Methods...........................5
2.1. Cloning, expression and purification...............5
2.2. Crystallization and data collection................5
2.3. Structure determination and refinement.............6
2.4. LC-MS analysis.....................................6
2.5. Stability assay of ThDP............................7
2.6. Deuterium exchange assay...........................8
2.7. NMR analysis.......................................8
2.8. DFT calculations...................................9
2.9. Electron paramagnetic resonance spectroscopy.......9
Chapter 3. Results and Discussion.........................10
3.1. Thiazole ring bending................................10
3.2. Stability of ThDP in TKps.........................12
3.3. Relative energy state.............................14
3.4. Crystal complexes of TKps.........................15
3.5. Radical model for TKps............................16
3.6. Model validation..................................18
Chapter 4. Conclusion.....................................21
Reference..............................................46-49
Appendixes................................................50
List of Figures
Figure 1. Close-up views of thiazolium of ThDP in holo-TKps crystal complexes.........................................24
Figure 2. Mass/NMR spectra and LC traces of ThDP in time-course experiment.........................................26
Figure 3. Modified ThDP in the active site of TKps........28
Figure 4. Reaction coordinate.............................29
Figure 5. Ligands in the active site of TKps..............31
Figure 6. Mass, NMR and cwEPR spectra...................................................35
List of Tables
Table 1. Data collection and refinement statistics for TKps......................................................36
Table 2. Selected bond lengths (Å) of ThDP and intermediates in the TKps complexes.......................40
Table 3. Selected bond lengths (Å) of ThDP and intermediates in the TKps complexes.......................41
List of schemes
Scheme 1. TK-catalyzed reactions, chemical structures, and isomerism of ThDP.........................................43
Scheme 2. Current catalytic model of cofactor ThDP in the TK-catalyzed reaction.....................................44
Scheme 3. Proposed single-electron transfer mechanism.....45

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