- 學位論文
植物細胞分裂素生成的關鍵步驟:啤酒花腺苷酸異戊二烯基轉移酵素其晶體結構、受質特異性、與催化機制之研究
Understanding the pivotal step in cytokinin synthesis: crystal structure, substrate specificity and catalytic mechanism of adenylate isopentenyltransferase from H. lupulus
摘要
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
並列摘要
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.
參考文獻
延伸閱讀
- Chen, F. Y. (2010). 啤酒花腺苷酸異戊二烯基轉移酵素對多磷酸雙腺苷之結合與催化活性之探討 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2010.00763
- 徐瑋涏（2021）。探討聚木醣酶XynR8(N58D)形成酵素交聯聚集體的方法及對酵素生化特性的影響〔碩士論文，國立屏東科技大學〕。華藝線上圖書館。https://doi.org/10.6346/NPUST202100475
- Chang, Y. H. (2008). 調控啤酒酵母四異戊二烯焦磷酸合成酶雙元體裂解成單元體的可能機制以及人類四異戊二烯焦磷酸合成酶形成六元體的研究 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2008.10355
- Liu, Y. L. (2005). 利用定點突變方法以及突變株與受質複合體結構驗證人類麩醯胺環化酵素之催化機轉 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2005.02131
- Tung, J. Y. (2010). Crystal Structures of Starch Binding Domain of Rhizopus oryzae Glucoamylase Reveal a Polysaccharide Binding Path; Calcium Binds to LipL32, a Lipoprotein from Pathogenic Leptospira, and Modulates Fibronectin Binding [doctoral dissertation, National Tsing Hua University]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0016-2203201110475930