春日黴素 (KSM) 是一種氨基糖苷類抗生素，主要由三種化學基團：D型肌醇、春日胺基與甘氨酸亞胺來組成其化學結構。儘管春日黴素早在 50 多年前就已人類所發現，但參與其生物合成途徑之酵素仍未被詳細探討。因此在此論文研究中，我們利用結構解析與酵素活性測試證實表異構酶 KasQ 在KSM 生物合成途徑中扮演了關鍵腳色，而非先前相關研究中所提出之酵素 KasF/H。我們推測KasF/H應為乙酰轉移酶，能乙酰化KSM 使其失去其抗菌活性。在進一步針對KasQ進行生化和生物結構分析，其結果證實KasQ能將 UDP-GlcNAc 轉化為 UDP-ManNAc 作為KSM生物合成途徑的第一步。而同位素研究中也進一步印證並非尿苷二磷酸葡萄糖 (UDP-glucose)，而是帶有13C,15N之氨基尿苷二磷葡萄糖 (UDP-GlcNH2)為形成 KSM 的重要中間體。同時我們也分別證實 KasF 和 KasH，具有將 UDP-GlcNH2 和 KSM 乙酰化為 UDP-GlcNAc 和 2-N'-乙酰基 KSM的作用。實驗結果中發現到KasF 並非催化KSM生合成反應的第一步； 其功能與KasH 相同，均為春日黴素之乙酰化修飾蛋白酶，且後者KasH在抗性活性方面比前者更具特異性和反應性佳。此篇論文所完成的相關研究奠定了未來繪製完整春日黴素(KSM) 之生物合成途徑的重要基礎。

Kasugamycin (KSM), an aminoglycoside antibiotic, is composed of three chemical moieties: D-chiro-inositol, kasugamine and glycine imine. Despite being discovered more than 50 years ago, the biosynthetic pathway of KSM remains an unresolved puzzle. Here we report a structural and functional analysis for an epimerase, KasQ, that primes KSM biosynthesis rather than the previously proposed KasF/H, which instead acts as an acetyltransferase, inactivating KSM. Our biochemical and biophysical analysis determined that KasQ converts UDP-GlcNAc to UDP-ManNAc as the initial step in the biosynthetic pathway. The isotope-feeding study further confirmed that 13C,15N-glucosamine/UDP-GlcNH2 rather than glucose/UDP-Glc serves as the direct precursor for the formation of KSM. Both KasF and KasH were proposed, respectively, converting UDP-GlcNH2 and KSM to UDP-GlcNAc and 2-N’-acetyl KSM. Experimentally, KasF is unable catalyze the first step of reaction; both KasF and KasH are instead KSM-modifying enzymes, while the latter is more specific and reactive than the former in terms of the extent of resistance. The information gained here lays the foundation for mapping out the complete KSM biosynthetic pathway.

Acknowledgements i
摘 要 ii
Abstract iii
Table of contents iv
Abbreviations x
Chapter 1 1-5
Introduction 1
1.1. Kasugamycin (KSM) 1
1.2. KSM mode of action 1
1.3. Antibacterial, antifungal and antiviral activity of KSM 3
1.4. KSM proposed biosynthesis pathway and its resistance 4
Chapter 2 6-20
Materials and methods 6
2.1. Chemicals, reagents and bacterial strains 6
2.2. Cloning, expression and purification 6
2.3. Crystallization and Data Collection 8
2.4. Structure Determination and Refinement 9
2.5. Site-Directed Mutagenesis 10
2.6. Isothermal Titration Calorimetry (ITC) 10
2.7. Synthesis of 2-Acetamidoglucal (AAG) 11
2.7.1. General Information 11
2.7.2. Procedure for the synthesis of 2-acetamidoglucal (AAG) 12
2.8. Construction of pMKBAC08-KAS and pWZC07-PrpsJ-kasT 13
2.9. In Vivo Isotope Incorporation Assay 14
2.10. Disc Diffusion Assay 16
2.11. HPLC Activity Assay 16
2.12. Thin Layer Chromatography (TLC) Analysis 18
2.13. HPLC Kinetic Analysis 18
2.14. NMR Activity Assay 18
2.15. Deuterium Incorporation Assay 19
2.16. Computational Analysis 20
Chapter 3 21-76
Results and Discussion 21
3.1. Bioinformatic mining of KasQ gene 21
3.2. Biochemical Investigation of an Epimerase 21
3.3. Biochemical Characterization of Acetyltransferases 34
3.4. Crystal Structure of KasQ in Complex with UDP and UDP-Glc 47
3.5. Structural Comparison 55
3.6. KasQ Substrate Specificity 59
3.7. In vivo Isotope Incorporation Analysis 73
Chapter 4 77-79
Conclusion 77
References 80
Appendixes 89
Curriculum Vitae 97






List of Tables vi
Table 1. Summary of crystal data-collection statistics 9
Table 2. The sequences of primers used in this study to create KasQ mutants 10
Table 3. The sequences of primers used to construct pMKBAC08-KAS and pWZC07-PrpsJ-kasT 14
Table 4. Conversion of substrate to product and intermediates at different time points. 27
Table 5. Thermodynamic parameters of KasH with substrates 42
Table 6. Kinetics of KasQ_WT with UDP-GlcNAc and MgCl2 62
Table 7. Thermodynamic parameters of KasQ versus different substrates. 63
Table 8. Thermodynamic parameters of KasQ mutants in the binding affinity assay 69


















List of Figures vii
Figure 1.1 | Chemical structure of kasugamycin (KSM) 1
Figure 1.2 | Crystal structure of KSM complex with ribosome and its inhibition mechanisms 102
Figure 1.3 | KSM activity against different human pathogens 3
Figure 1.4 | Previous biosynthetic proposals for kasugamycin (KSM) 5
Figure 2.1 | Schematic representation has shown the method of in-vivo isotope incorporation assay 15
Figure 3.1| Expression and purification of KasQ 24
Figure 3.2 | Biochemical investigation of KasQ by HPLC-LTQ-MS 25
Figure 3.3 | Effects of different temperatures on KasQ activity 26
Figure 3.4 | Conversion percentage of substrate to product and intermediates 27
Figure 3.5 | Intermediate trapping and product formation monitored by 1H NMR spectroscopy 28
Figure 3.6 | Biochemical investigation of KasQ by NMR spectrometry 29
Figure 3.7 | Deuterium incorporation assay by LC-MS 30
Figure 3.8 | Deuterium incorporation assay by NMR spectroscopy 31
Figure 3.9 | 1H NMR spectrum 32
Figure 3.10 | 1H-STD NMR 33
Figure 3.11 | The sequence alignment of KasH and KasF 37
Figure 3.12 | Expression and purification of KasF and KasH 38
Figure 3.13 | In vivo resistance activity of KasF and KasH 39
Figure 3.14 | In vitro biochemical characterization of acetyltransferases KasF and KasH 40
Figure 3.15 | The acetylation reactions of KasF and KasH were examined 41
Figure 3.16 | Isothermal Titration Calorimetry (ITC) analysis 103
Figure 3.17 | Acetylation assay of KasF with UDP-GlcNH2 and AcCoA 44
Figure 3.18 | Deacetylation assay 45
Figure 3.19 | HPLC-LTQ-MS analysis for 2-N’-acyl substituted KSM analogs 46
Figure 3.20 | KasQ crystals 50
Figure 3.21 | Crystal structures of KasQ_wildtype (WT) 51
Figure 3.22 | Crystal structures of KasQ in complex with UDP or UDP-Glc 52
Figure 3.23 | Sequence alignment of KasQ with different UDP-GlcNAc 2-epimerases 53
Figure 3.24 | Biochemical assay of KasQ mutants E308A and E308Q 54
Figure 3.25 | Sequence and structural comparison of KasQ with 57
Figure 3.26 | The metal-ion binding site of KasQ 58
Figure 3.27 | Enzymatic reactions of KasQ with different NDP sugars 61
Figure 3.28 | The kinetic curve of KasQ for the epimerization reactions 62
Figure 3.29 | ITC analyses of KasQ with different nucleosides (mono/di/tri) phosphates 65
Figure 3.30 | ITC analyses of KasQ with different NDP sugars 67
Figure 3.31 | ITC analyses of KasQ with different monosaccharides 69
Figure 3.32 | ITC analyses of KasQ mutants with different NDP sugars 71
Figure 3.33 | 1H NMR spectra of KasQ in time course reactions 72
Figure 3.34 | Disc diffusion and in vivo isotope incorporation assay 75
Figure 3.35 | Isotope incorporation assay 76
Figure 4.1 | The biosynthetic gene cluster (BGC) sequenced and identified 78
Figure 4.2 | The proposed biosynthetic pathway of KSM from this study 79











List of Schemes ix
Scheme 1 | The synthetic step for AAG 2. 105

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