Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24058
標題: 格蘭式陰性植物病原菌Uridylate kinase獨特的 GTP結合位及異位調控
Unique GTP-Binding Pocket and Allostery of Uridylate Kinase from a Gram-Negative Phytopathogenic Bacterium
作者: 涂誌樂
Tu, Jhe-Le
關鍵字: allosteric regulation;異位調控;Uridine 5'-monophosphate kinase;Xanthomonas campestris;Uridine 5’-monophosphate kinase;Xanthomonas campestris
出版社: 生物化學研究所
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摘要: 
Uridine 5’-monophosphate kinase (UMPK) 主要的功能是將UMP磷酸化產生UDP,而其活性主要是受GTP分子的異位調控以及UTP產物的抑制。由於真核生物的UMPK對UMP或CMP皆有特異性,而原核生物的UMPK僅對UMP有特異性,而且原核生物的UMPK為其生長及生存不可或缺,因此原核生物的UMPK有潛力成為抗菌藥物的標的。目前已有數個原核生物UMPK的apo form或與各種核苷酸 (包括:UMP、UDP、UTP及ATP) 的複合物結構被解出。然而,在已發表的apo-form UMPK結構中,參與結合受質ATP的loop電子密度圖無法辨識,因此很難觀察受質結合後導致的構形變化。此外,目前只有一個低解析度的UMPK與其異位調節因子GTP複合物的結構被解出,因此無法得知GTP調控UMPK的機制。本研究中,運用特殊的高磁場養晶技術以及X-ray繞射的方法,成功解析出格蘭氏陰性的植物病原菌Xanthomonas campestris (XC) 之apo-UMPK及GTP-bound UMPK複合物的結構,並且其參與ATP及UMP結合的flexible loops均清晰可見。同時,殘基R100至R127間形成一個獨特的帶正電區域,而在UMPK六聚體中心形成一個特有的GTP結合位。比較XcUMPK之GTP-binding form與apo-form的結構可發現,GTP的結合會造成XcUMPK之ATP- 及UMP-binding loops部份構形的改變。而XcUMPK的GTP結合位置,與E. coli UMPK之GTP結合位類似,但是結合的方位不同,在鹼基及五碳糖的部份約距離3.5-4.5Å,而在磷酸根的部份則約距離11-12 Å。若將XcUMPK-GTP複合物與E. coli UMPK-UTP複合物進行重疊,則可發現GTP與UMPK的結合,相較於UTP與UMPK的結合,相臨的兩個UMPK單體打開約12°,造成雙體間的交互作用更為緊密。顯然的,GTP與UMPK的結合,改變了六聚體的聚集以執行遠距離的異位調控。

Uridine 5'-monophosphate kinase (UMPK) catalyzes the reversible transfer of the γ-phosphoryl group from ATP to UMP to produce UDP. In general, UMPKs are stimulated by GTP and inhibited by UTP. In general, eukaryotic UMPKs exhibit dual specificity toward UMP or CMP, but the UMPKs of bacterial origin exhibit more dedicated UMP-specific activity and are essential for bacterial growth. Therefore, UMPKs are potential targets for developing antibacterial drug. To date, tertiary structures of a number of prokaryotic apo-form UMPKs and complexes with different nucleotides, including the UMP-, UDP-, UTP-, and ATP-binding complexes from E. coli. have been published or deposited. However, the ATP-binding loops in these UMPK structures are disordered in no-ligand bound state which is difficult to investigate the induced-fit movements by the bound ligand for these flexible loops. Also, the regulation mechanism exhibited by GTP remains to be elucidated since only one low resolution UMPK-GTP complex structure was published. We have determined the tertiary structures of the apo- and GTP-bound forms of UMPK from a Gram-negative plant pathogen Xanthomonas campestris (XC) using X-ray diffraction methodology of crystals grown in high magnetic field. The flexible ATP- and UMP-binding loops are visible in these structures. The structure revealed a unique patch (ranging from residue R100 to residue R127) of noticeably positive-charge nature which formed a GTP-binding pocket in the central hole of the UMPK hexamer. Six GTP molecules were found to bind in the central cavity of the hexamer and each GTP molecule interacted with adjacent monomers. Structure comparison of apo- and GTP-bound XcUMPK revealed moderate induced movements for the ATP- and UMP-binding loops upon GTP binding. Moreover, the GTP molecules were situated at similar positions found in the E. colUMPK structure. There are a shift of 3.5-4.5 Å in the base and ribose and a shift of 11-12 Å in the phosphate group of GTP molecule. The overall structure of GTP-bound XcUMPK, when compared to that of E. coli UTP-bound UMPK, showed a substantial rearrangement in quaternary structure and manifested by an 12° opening of the UMPK dimer and tight dimer-dimer interaction. It is likely that the binding of GTP resulting in modification of the hexameric assembly to exert long-range allosteric control on XcUMPK.
URI: http://hdl.handle.net/11455/24058
其他識別: U0005-2107201013324500
Appears in Collections:生物化學研究所

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