臺灣博碩士論文加值系統

細胞分裂素是重要的植物賀爾蒙，其生物合成最開始是從二甲烯丙烯基焦磷酸（dimethylallyl pyrophosphate, DMAPP)上的異戊烯基，轉移到腺嘌呤的氨基N6上，藉著腺苷酸異戊二烯基轉移酵素(adenylate isopentenyltransferase, AIPT)或轉移核醣核酸異戊二烯基轉移酵素(tRNA-IPT)。植物腺苷酸異戊二烯基轉移酵素使用三磷酸腺苷(ATP)或二磷酸腺苷(ADP)作為異戊二烯基接受者，而細菌和轉移核醣核酸的異戊二烯基轉移酵素則分別以單磷酸腺苷和轉移核醣核酸的特定位置作為接受者。在這裡，解析出來自啤酒花的腺苷酸異戊二烯基轉移酵素和三磷酸腺苷 (AIPT-ATP)複合體結晶結構，此結構類似於先前所發表的農桿菌的腺苷酸異戊二烯基轉移酵素和酵母菌的轉移核醣核酸異戊二烯基轉移酵素。這個酵素與NTP結合激素(NTP-binding kinase)有結構同源的關係，但是它會額外形成一個反應通道來讓兩個受質三磷酸腺苷(ATP)與二甲烯丙烯基焦磷酸(DMAPP)結合。利用等溫滴定微量熱法(ITC)去測量不同核苷酸與酵素的結合能力，得到了以下的關係ATP>dATP~ADP>GTP>CTP>UTP。然而當進一步測量這些不同核苷酸與酵素的活性時，發現它們的活性大小為ATP>ADP>dATP>CTP>>GTP≈UTP≈0，不同於之前結合能力的關係。此外除了二甲烯丙烯基焦磷酸(DMAPP(C5))，這個酵素也展現出對二異戊二烯基焦磷酸(Geranyl pyrophosphate, GPP(C10))有活性，表示其可以把它當作轉移反應的供給者。在結構中，兩個鹼性的胺基酸側鏈Lys275和Lys220會與三磷酸腺苷(ATP)的

Cytokinins are important plant hormones, and their biosynthesis most begins with the transfer of isopentenyl group from dimethylallyl diphosphate (DMAPP) to the N6-amino group of adenine by either adenylate isopentenyltransferase (AIPT) or tRNA-IPT. Plant AIPTs use ATP/ADP as an isopentenyl acceptor and bacterial AIPTs prefer AMP, whereas tRNA-IPTs act on specific sites of tRNA. The crystal structure of an AIPT-ATP complex from Humulus lupulus (HlAIPT) presented here is similar to the previous structures of Agrobacterium AIPT and yeast tRNA-IPT. The enzyme is structurally homologous to the NTP-binding kinase family of proteins but forms a solvent-accessible channel that binds to the donor substrate DMAPP, which is directed toward the acceptor substrate ATP/ADP. When measured with isothermal titration calorimetry, some nucleotides displayed different binding affinities to HlAIPT with an order of ATP > dATP ~ ADP > GTP > CTP > UTP. However, further measurements of activity using these nucleotides showed that the order becomes ATP > ADP > dATP > CTP >> GTP ≈ UTP ≈ 0. In addition to DMAPP (C5), HlAIPT also showed remarkable activity for GPP (C10) as the donor substrate. Two basic residues Lys275 and Lys220 in HlAIPT interact with the β and γ-phosphate of ATP. By contrast, the interactions are absent in Agrobacterium AIPT because they are replaced by the acidic residues Asp221 and Asp171. Despite its structural similarity to the yeast tRNA-IPT, HlAIPT has evolved with a different binding strategy for adenylate. Finally, by screening a series of dinucleotide polyphosphates, it was found, surprisingly, that the binding affinities of some diadenosine polyphosphates (A(p)4A, A(p)5A and A(p)6A) are even higher than those of the original substrates (ATP and ADP). These results may imply that novel substrates of HlAIPT have been found in plant.

中文摘要…………………...……………………..…...……………………….........I
Abstract……..…..………………………………..………………...……………..III
Abbreviations…………..……...……………..…..…………………….………....V Chapter 1 Introduction
1-1 Hormonal regulation of plant growth and development……………………..2
1-2 Cytokinins are a group of phytohormones that play a crucial role in
plant development process………………………………………...…………3
1-3 Different CKs showing the different binding affinity to receptors…………..4
1-4 Cytokinin signaling in plant cell……………………………………………..4
1-5 Biosynthesis of cytokinin…………………………………………………….6
1-5-1 Free adenine-IPT pathway…………………………………………8
1-5-2 tRNA-IPT pathway………………………………………………...9
1-6 Agrobacterium tumefaciens and the plant…………………………………..10
1-7 The motive of this study…………………………………………………….11
Chapter 2 Materials and Methods
2-1 Chemicals…………………………………………………………………….15
2-2 Construct of full length, truncated HlAIPT and single point mutation………15
2-3 Protein expression and purification…………………………………………..15
2-4 Crystallization condition……………………………………………………..17
2-5 Data collection,
2-6 Structure determination, structure refinement and model building…...……..17
2-7 Isothermal titration calorimetry…...…………………………………………18
2-8 AIPT activity assay…...……………………….……………………………..19
2-9 Mass spectroscopy………...…………………………………………………20
2-10 Phylogenetic tree……………………………………………………………21
2-11 PDB accession code………………………………………………………...21
Chapter 3 Results
3-1 Improvement of process of purification and dialysis………………………...23
3-2 Co-crystallization HlAIPT and truncated HlAIPT with different substrates...23
3-3 The overall structure of H. lupus AIPT………………………………………24
3-3-1 The structure of Active site of HlAIPT……………………………….25
3-3-2 The role of basic side chain Lys275 of HlAIPT…………………...…26
3-4 Nucleotide binding assay…………………………………………………….27
3-4-1 Different binding affinity for ATP, dATP and ADP with HlAIPT……29
3-4-2 Different binding affinity for CTP, GTP and UTP with HlAIPT…….29
3-5 Enzymatic activity for different prenyl acceptors and donors……………….30
3-6 Relationship between nucleotide binding and activity of HlAIPT…………..31
3-7 Different nucleotide derivatives showing binding affinity to HlAIPT………34
3-8 Enzymativ activity on diadenosine polyphosphates…………………………35
Chapter 4 Discussions
4-1 Mg2+ ion interfering ATP binding to HlAIPT………………………………37
4-2 Comparison of active site of the H. lupus AIPT and Agrobacterium AIPT
Nucleotide binding site……………………………………………………...38
4-3 Comparison of DMAPP binding site……………………………………….39
4-4 Structural comparison of H. lupus AIPT with tRNA-IPT showing different binding mode……………………………………..…………………………40
4-5 Modeling of HlAIPT/ATP/DMASPP/Mg2+ complex………………………42
4-6 The proposed catalytic mechanism…………………………………………43
4-7 Model of complex structure (HlAIPT/A(p)5A)……………………………..44
4-8 Implication of activity of diadenosine polyphosphates to HlAIPT…………45
4-9 The role of a conserved aspartic acid……………………………………….46
Chapter 5 Tables
Table 1. Data collection and refinement statistics………………………………49
Table 2. Thermodynamic parameters of interactions between AIPT and nucleotides……………………………………………………………..50
Table 3. Substrate specificity of the recombinant HlAIPT and the mutants……51
Table 4. Thermodynamic parameters of interactions between HlAIPT and nucleotide derivatives…………………………...……………………..52
Chapter 6 Figures
Figure 1. SDS-PAGE of HlAIPT and photograph of HlAIPT crystal used for data collection……………………………………………………………..54
Figure 2. Overall structure of HlAIPT………………………………….………55
Figure 3. Comparison of HlAIPT with other enzymes…………………………56
Figure 4. Surface charge distribution in the nucleotide binding site……………57
Figure 5. Phylogenetic tree of the IPT enzymes………………………………..59
Figure 6. The reaction channel of HlAIPT……………………………………...60
Figure 7. The active site of HlAIPT…………………………………………….61
Figure 8. Multiple sequence alignment of AIPTs……………………………….62
Figure 9. Typical ITC trace for the interaction between AIPT and nucleotides...64
Figure 10. A HlAIPT/GTP model……………………………………………….66
Figure 11. Analysis of the AIPT’s activity and product on ATP and DMAPP….67
Figure 12. Analysis of the AIPT’s activity and product on CTP and DMAPP…68
Figure 13. Analysis of the AIPT’s activity and product on ATP and GPP……...69
Figure 14. Analysis of the AIPT’s product on GTP and GPP……………….….70
Figure 15. Comparison of different conformation between DMAPP and its isomer IPP…………………...…………………….……………………...…….71
Figure 16. Model of the complex (HlAIPT/ATP/GPP/Mg2+)…………………..72
Figure 17. ITC analysis of ATP binding to HlAIPT…………………………….73
Figure 18 Comparison of HlAIPT with Agrobacterium AIPT………………….74
Figure 19. The possible residues controlling the substrate specificity………….75
Figure 20. Comparison of HlAIPT with tRNA-IPT…………………………….76
Figure 21. Modeling of HlAIPT/ATP/DMASPP/Mg2+ complex……………….77
Figure 22. The proposed catalytic mechanism………………………………….78
Figure 23. Nucleotide Analog showing binding affinity to HlAIPT……………79
Figure 24. Typical ITC trace for the interaction between AIPT and diadenosine polyphosphates…………………………………………………….80
Figure 25. Analysis of the AIPT’s activity and product on A(p)3A and DMAPP…………………………………………………………....82
Figure 26. Analysis of the AIPT’s product on A(p)4A, A(p)5A and A(p)6A…..83
Figure 27. Model of complex structure (HlAIPT/A(p)5A)……………………..84
Figure 28.Schematic diagram of the AIPT reaction for diadenosine
Polyphosphates……………………………………………………...85
Figure 29. The role of a conserved aspartic acid………………………………..86
Chapter 7 References
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