指導教授：李宗璘臺灣大學：生化科學研究所王貴貞Wang, Kuei-ChenKuei-ChenWang2014-11-262018-07-062014-11-262018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261641比菲德氏菌(Bifidobacteria)為革蘭氏陽性厭氧菌，其具有獨特的lacto-N-biose I (LNB)/galacto-N-biose (GNB) 代謝路徑。比菲德氏菌利用此獨特的路徑代謝LNB及GNB，以利其共生在人類消化道中。LNB/GNB代謝路徑是由lnpABCD基因組經轉錄(transcription)、轉譯(translation)後生成。lnpA基因生成Galacto-N-biose/lacto-N-biose I磷酸化酶(galacto-N-biose/lacto-N-biose I phosphorylase, GLNBP)。 在不消耗ATP情況下，GLNBP可將LNB及GNB轉化為半乳糖-1-磷酸(galactose-1-phosphate) 及 氮-乙醯六碳糖胺 (N-acetyl-D-hexosamine)。氮-乙醯六碳糖胺包含氮-乙醯葡萄糖胺(N-acetylglucosamine, GlcNAc)及氮-乙醯半乳糖胺(N-acetylgalactosamine, GalNAc)。GLNBP有助於比菲德氏菌在厭氧環境中更有效運用ATP。接著，由lnpB基因生成的氮-乙醯六碳糖激酶 (N-acetylhexosamine 1-kinase, NahK)將氮-乙醯葡萄糖胺及氮-乙醯半乳糖胺磷酸化為氮-乙醯葡萄糖胺-1-磷酸(N-acetylglucosamine -1-phosphate, GlcNAc-1P)及氮-乙醯半乳糖胺-1-磷酸 (N-acetylgalactosamine-1-phosphate, GalNAc-1P)。NahK已被廣泛使用於酵素合成中。 雖然GLNBP及NahK已被研究多年，GLNBP及Nahk的分子催化機轉依然不清楚。因此，我們想利用X光蛋白結晶學及生物化學的方法解析出GLNBP及NahK的蛋白質結構，並進一步探討其催化作用機制。在此篇研究中，我們解析出GLNBP與LNB/GNB複合體蛋白質結構。藉由分析複合體蛋白結構，我們推測藉由無機磷酸根(inorganic phosphate)的直接攻擊，GLNBP可能是進行反轉的磷酸解反應(an inverting phosphorolytic reaction)。在NahK部分，我們解析了七個NahK與其受質(substrates)及產物(products)複合的蛋白結構。藉由這些複合體蛋白結構，我們推測NahK是進行類似SN2的磷酸化反應。NahK 是第一個具有多階段反應之複合體蛋白結構之激酶。GLNBP及NahK的蛋白複合體結構解析及催化機制的探討，將有助於GLNBP及NahK應用於寡糖的酵素合成並進一步增加寡糖的多樣性。Bifidobacteria, gram-positive anaerobic bacteria, feature a unique lacto-N-biose I (LNB)/galacto-N-biose (GNB) pathway to utilize LNB and GNB for colonization in the gastrointestinal tract of humans. The LNB/GNB pathway is encoded in a four-gene lnpABCD operon. Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) encoded by the gene lnpA catalyzes phosphorolysis of LNB and GNB into galactose-1-phosphate (Gal-1P) and N-acetyl-D-hexoamine (GlcNAc/GalNAc) without ATP consumption. This maximizes the effectiveness of ATP production under anaerobic conditions. A novel N-acetylhexosamine 1-kinase (NahK) encoded by the gene lnpB then phosphorylates N-acetyl-D-hexoamine to N-acetylhexoamine 1-phosphate (GlcNAc-1P/GalNAc-1P). NahK has been extensively used in chemoenzymatic synthesis of carbohydrates. However, the molecular mechanisms for both GLNBP and NahK remain unclear. In this study, we wanted to determine the crystal structures of GLNBP and NahK in complex with substrates/products to gain insight into the catalytic mechanisms of these two enzymes. For GLNBP, crystal structures of GLNBP in complex with LNB/GNB were solved. Based on the structural information, GLNBP was proposed to proceed through an inverting phosphorolytic reaction by the direct nucleophilic attack of an inorganic phosphate at the anomeric carbon. For NahK, seven 3-D structures of NahK in complex with GlcNAc, GalNAc, GlcNAc-1P, GlcNAc/AMPPNP and GlcNAc-1P/ADP were solved. Based on these snapshot structures, a direct in-line phosphoryl transfer mechanism was proposed. The NahK structures presented here represent the first multiple-reaction complexes of the enzyme. The demonstration of the protein crystal structures and the elucidation of the catalytic mechanisms would provide useful information for expanding the utilizations of both GLNBP and NahK to a great extent.Contents 口試委員會審定書 i 誌謝 ii 中文摘要 iii Abstract iv Chapter 1. Introduction 1-5 1.1. Human milk oligosaccharides (HMO) and lacto-N-biose I (LNB) 1 1.2. Galacto-N-biose (GNB) 2 1.3. The LNB/GNB pathway 2 1.4. Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) 3 1.5. N-acetylhexosamine 1-kinase (NahK) 3 1.6. UDP-glucose hexose 1-phosphate uridylyl transferase (GalTBL) and UDP-glucose 4-epimerase (GalEBL) 4 1.7. Study aims 5 Chapter 2. N-acetylhexosamine 1-kinase (NahK) 6-20 2.1. Materials and Methods 6 2.1.1. Protein expression and purification 6 2.1.2. Crystallization and data collection 7 2.1.3. Structure determination and refinement 8 2.1.4. Site-directed mutagenesis 8 2.1.5. Enzymatic activity assay 10 2.1.6. Isothermal titration calorimetry (ITC) analysis 11 2.1.7. Analytical ultracentrifugation analysis 11 2.2. Results and Discussion 12 2.2.1. Structure determination 12 2.2.2. Overall structure 13 2.2.3. Structural comparison 13 2.2.4. Sugar binding site 14 2.2.5. Nucleotide binding site 15 2.2.6. Substrate specificity 16 2.2.7. Catalytic mechanism 17 2.2.8. Reaction order 18 2.3. Concluding remarks 20 Chapter 3. Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) 21-28 3.1. Materials and Methods 21 3.1.1. Gene cloning and protein purification 21 3.1.2. Crystallization and data collection 22 3.1.3. Structure determination and refinement 22 3.1.4. Enzymatic activity assay 23 3.2. Results and Discussion 24 3.2.1. Overall structure 24 3.2.2. Sugar-binding site 24 3.2.3. Substrate specificity 25 3.2.4. Reaction mechanism 26 3.2.5. Structural comparison 27 3.3. Summary 28 Figures 29-52 Figure 1. The LNB/GNB pathway in Bifidobacteria 30 Figure 2. Analytical ultracentrifugation (AUC) and gel filtration chromatography analysis for NahK 31 Figure 3. Sequence and structure alignments for NahK_JCM1217 and NahK_ATCC15697 32 Figure 4. Stereoview of the ternary NahK●GlcNAc●AMPPNP complex 33 Figure 5. The topology of NahK 33 Figure 6. Sequence alignment for NahK and choline kinases 34 Figure 7. Structural comparison of NahK, hCK and APH(2”)-IIIa 35 Figure 8. Sugar-binding site of NahK 36 Figure 9. Nucleotide binding site of NahK 37 Figure 10. LC traces. 38 Figure 11. Molecule docking of ManNAc into the sugar-binding site of NahK 39 Figure 12. Catalytic mechanism of NahK 40 Figure 13. ITC analyses of NahK 41 Figure 14. ITC analyses of NahK mutants 43 Figure 15. Gel filtration chromatography and SDS-PAGE analyses for GLNBP 45 Figure 16. Interfaces analysis of GLNBP 46 Figure 17. Overall structure of GLNBP 48 Figure 18. Sugar-binding site of GLNBP 49 Figure 19. HPAEC-PAD traces of GLNBP reactions 50 Figure 20. Sequence alignment for GLNBP and its homologues in GH112 51 Figure 21. Catalytic mechanism of GLNBP and structural comparasion 52 Tables 53-59 Table 1. Data collection, phasing and refinement statistics for NahK 54 Table 2. Relative enzymatic activities of NahK and mutants towards GlcNAc/GalNAc 55 Table 3. Summary of ITC analysis for NahK 56 Table 4. Summary of ITC analysis for NahK mutants 57 Table 5. Substrate specificity of NahK 58 Table 6. Data collection, phasing and refinement statistics for GLNBP 59 Reference 60 Appendix 694952115 bytesapplication/pdf論文公開時間：2019/07/22論文使用權限：同意有償授權(權利金給回饋學校)LNBGNB磷酸解酶激酶thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261641/1/ntu-103-D97b46016-1.pdf