Unique GTP-Binding Pocket and Allostery of Uridylate Kinase from a Gram-Negative Phytopathogenic Bacterium

• Main Authors: Jhe-Le Tu, 涂誌樂
• Other Authors: Shan-Ho Chou
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/51054245596663198244

博士 === 國立中興大學 === 生物化學研究所 === 98 === 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.