臺灣博碩士論文加值系統

天然二級代謝物的結構類型多樣且複雜豐富，具有廣泛的生物活性，成為新穎藥物研發的重要來源。藉由研究生合成酵素的功能，可以促進生物催化劑的分子設計研究及產生多樣性衍生物。第三型聚酮合成酶DpgA及輔因子非依存性氧化酶DpgC為參與醣胜肽類抗生素萬古黴素(vancomycin)以及teicoplanin中不尋常胺基酸3, 5-dihydroxyphenylglycine (DPG)生合成之關鍵酵素。DpgA是催化聚酮肽鏈的延長以及環化反應進而形成dihydroxyphenylacetyl-CoA (DPA-CoA)，然而其分子催化機轉依然不清楚。因此，我們想利用合成二種DPA-CoA中間產物di- and tri-ketidyl-CoA (DK-CoA and TK-CoA)進行其催化作用機制探討。每個中間產物進一步的與穩定同位素碳-13 (13C)標定的malonyl-CoA (MA-CoA)在DpgA、B及D催化下可以得到各種碳-13標定DPA-CoA。我們並以一次、二次、三次質譜及核磁共振解析碳-13原子在DPA-CoA上的分佈位置，藉以窺知DpgA其反應機制，特別的是polyketidyl-CoA可以當作起始單元分子以及延長單元分子進行縮合反應。然而若缺乏末端羧基的polyketone-CoA則只能當作延長單元分子。由此得知此末端羧基為環化反應中扮演重要的角色，且環化反應可能不需要酵素的參與。
另一方面，DpgC的作用是利用消耗一分子氧氣使DPA-CoA生成3,5-dihydroxyphenylglyoxylate (DPGx)，而罕見的DpgC不需任何的輔因子卻具有催化四個電子氧化反應的能力，然而其分子作用機轉大部分仍不清楚，包括如何催化位置上碳-氫鍵氧化反應與硫酯键水解。在此篇研究中，我們利用合成一系列基質類似物、使用氧-18 (18O)穩定同位素標定及X光蛋白結晶學，解析出DpgC的反應作用機制及蛋白複合體結構，有助於從分子層面上完善其催化機轉。DPA-CoA與氧氣首先形成一籠形複合體，然後於a碳位置上重組形成過氧陰離子(peroxide anion)。此過氧陰離子接著攻擊硫酯键，形成一個四元環分子二氧雜環丁烷，並伴隨著不均勻斷裂(hetrolytic cleavage)最後產生DPGx。期待此一研究成果，可以拓展這些酵素功能上的運用，將有助於創造更多獨特的小分子化合物。

Secondary metabolites are rich in molecular complexity and diversity in addition to varied biological activities. The study of enzymes involved in the synthesis of secondary metabolites could facilitate biocatalyst design and thus expand the scope of secondary metabolites. The type III polyketide synthase DpgA and the cofactor-free oxygenase DpgC catalyze two key steps in the biosynthesis of the essential building block 3,5-dihydroxyphenylglycine (DPG), a non-proteinogenic amino-acid building unit in the vancomycin and teicoplanin family of antibiotics. The reactions of DpgA include polyketone chain-length elongation and subsequent ring cyclization reactions to form dihydroxyphenylacetyl-CoA (DPA-CoA), while the detailed mechanisms behind these reactions remain elusive. To better understand the catalytic details, two labile DPA-CoA intermediates, di- and tri-ketidyl-CoA (DK-CoA and TK-CoA), were designed and chemically synthesized. Each intermediate along with [13C3]-malonyl-CoA (MA-CoA) in the presence of DpgABD was able to form 13C-enriched DPA-CoA. The product was subjected to NMR and MSn analyses, by　which the positions of 13C atoms in the partially 13C-enriched DPA-CoA were determined, thus shedding new light on how polyketide chain length elongates and cyclizes in the DpgA-dependent enzyme reaction. In contrast to the conventional mechanism, polyketidyl-CoA can act as the starter or extender, whereas polyketone-CoA without the terminal carboxyl group can only serve as an extender. As a result, the terminal carboxyl group is crucial for ring cyclization that likely occurs on CoA instead of the enzyme.
DpgC was known to catalyze conversion of 3,5-dihydroxyphenylglyoxylate (DPGx) from DPA-CoA at the expense of one molecule of dioxygen. Oddly, DpgC is an enzyme that contains no cofactor but capable of catalyzing a 4-electron oxidation reaction. Despite this interesting fact, the catalytic mechanism that virtually governs both  C-H oxidation and thioester hydrolysis remains largely unknown. To answer this question, we came to probe this catalytic issue by utilizing an array of synthetic analogues as well as the 18O stable isotope labelling technique. We also took advantage of X-ray crystallography to obtain crystal structures of DpgC in complex with ligands in a hope to visualize the chemical transformation. In conjunction with chemical examinations, our analysis have detailed the catalytic mechanism at the molecular level, where dioxygen and DPA-CoA first form a caged complex and then recombine to form a peroxide anion species at a-C. This peroxide anion then attacks thioester carbon to form a four-membered dioxetane species that subsequently undergoes hetrolytic cleavage to form DPGx. We believe the molecular detailing of the given enzymes has paved a strong foundation for future biocatalyst design, which should accelerate development of unnatural natural products, for example, in tackling difficult-to-treat diseases.

Contents
中文摘要........i
Abstract........iii
Contents........v
List of Figures........vii
List of Tables........ix
Chapter 1. Introduction........1-4
1.1 The emergence of drug resistant pathogens........1
1.2 Nonproteinogenic amino acid 3,5-dihydroxyphenylglycine........1
1.3 DpgA........2
1.4 DpgC........4
Chapter 2. DpgA........5-20
2.1. Materials and Methods........5
2.1.1. Cloning, expression and purification........5
2.1.2. Site-directed mutagenesis........6
2.1.3. Enzymatic assay........7
2.1.4. Preparation of [13C3]malonyl-CoA and [13C4]acetoacetyl-CoA........7
2.1.5. Synthesis of Diketide anhydride........7
2.1.6. Synthesis of Triketide anhydride........9
2.1.7. Enzyme assays with di- and tri-ketidyl-CoA........11
2.1.8. Compound characterization........11
2.2. Results and Discussion........12
2.2.1. Synthesis of polyketidyl-CoA intermediates........12
2.2.2. Synthesis of polyketidyl-CoA intermediates: Determination of the true reaction route........13
2.2.3. Distribution of 13C atoms in DPA-CoA........14
2.2.4. Unexpected starter and extender........15
2.2.5. Where does chain cyclization occur?........17
2.2.6. KS/NRPS-related type III PKSs might be evolutionarily related........18
2.3. Conclusions........20
Chapter 3. DpgC........21-34
3.1. Materials and Methods........21
3.1.1. Cloning, expression and purification........21
3.1.2. Analytical ultracentrifuge analysis........21
3.1.3. Enzymatic assay........22
3.1.4. Incubations with 18O2........23
3.1.5. Crystallization and data collection........23
3.1.6. Structure determination and refinement........24
3.1.7. Synthesis of phenylacetyl-NAC analogues........24
3.1.8 Synthesis of 2-fluoro-2-phenylacetyl-NAC analogues........25
3.1.9. Compound characterization........26
3.2. Results and Discussion........27
3.2.1. Enzyme specificity........27
3.2.2. Stereoselectivity of DpgC........29
3.2.3. Stable isotope labelling analysis........30
3.2.4. Overall structure description........32
3.2.5. Active Site of DpgC........32
3.3. Summary........34
Reference........77
Appendix........85

List of Figures
Figure 1. Biosynthesis of DPG and possible mechanisms of DpgA........35
Figure 2. ClustalW alignment of CHSs........36
Figure 3. Synthetic protocol and assay strategy........37
Figure 4. Mass spectra for diketidyl-CoA and triketidyl-CoA........38
Figure 5. Preparation of [13C3]-malonyl- and [13C4]-acetoacetyl-CoA using succinyl-CoA transferase........40
Figure 6. LC traces for DpgABD enzymatic reactions........41
Figure 7. NMR, MS and MSn spectra for DPA-CoA........43
Figure 8. Distribution of 13C atoms (●) in the presence of two [13C3]MA-CoA and one DK-CoA for the formation of DPA-CoA........44
Figure 9. Catalytic mechanism of DpgA........45
Figure 10. Polyketone chain cyclization in the formation of DPA-CoA........46
Figure 11. Homology modeling of DpgA........48
Figure 12. Phylogenetic tree of 29 peptides........49
Figure 13. SDS-PAGE analysis, gel filtration chromatography and analytical ultracentrifugation analysis for DpgC........50
Figure 14. Structure and mass spectra of chemically synthesized aliphatic ring-NAC derivatives........51
Figure 15. Structure and mass spectra of chemically synthesized N-acetyl derivatives....52
Figure 16. HPLC traces and mass spectra for the DpgC products obtained in N-acetyl derivatives reactions........53
Figure 17. HPLC traces for the DpgC products obtained in N-acetyl derivatives reactions........54
Figure 18. Structure and mass spectra of chemically synthesized substitution on C-NAC derivatives........55
Figure 19. HPLC traces for the DpgC products obtained in substitution on C-CoA derivatives reactions and mass spectra of standard........56
Figure 20. Mass spectra for the DpgC products obtained in substitution on C-CoA derivatives reactions........57
Figure 21. TIC traces for the DpgC products obtained in substitution on C-CoA derivatives reactions and mass spectra of standard........58
Figure 22. Structure and mass spectra of chemically synthesized substitution on benzene-NAC derivatives........59
Figure 23. HPLC traces for the DpgC products obtained in substitution on benzene-CoA derivatives reactions........60
Figure 24. Mass spectra for the DpgC products obtained in substitution on benzene-CoA derivatives reactions........61
Figure 25. Chiral resolution for the DpgC products........62
Figure 26. Schematic of the assay analyzing oxygen incorporation into DPGx........63
Figure 27. MS and MS2 spectra for DPGx........64
Figure 28. MS spectra for phenylglyoxlate........65
Figure 29. Two proposed catalytic mechanism carried out in DpgC........66
Figure 30. Overall structure of DpgC........67
Figure 31. The CoA substrate binding site of DpgC........68
Figure 32. The active site of DpgC/CoA complex structure........69
Figure 33. The active site of DpgC/p-tolylglyoxlate complex structure........70
Figure 34. The active site of DpgC/phenylglyoxlate complex structure........71
Figure 35. Superimposition of DpgC·p-tolylglyoxlate, DpgC·phenylglyoxlate and DPA-NH-CoA bound DpgC structures........72

List of Tables
Table 1. Structures of aliphatic ring-CoA and N-acetyl derivatives and their availability in enzymatic reactions........73
Table 2. Structures of substitution on C-CoA derivatives and their availability in enzymatic reactions........74
Table 3. Structures of substitution on benzene-CoA derivatives and their availability in enzymatic reactions........75
Table 4. Data collection and refinement statistics for DpgC structures........76

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