幽門螺旋桿菌尿嘧啶核苷酸激酶GTP複合物晶體結構及其異位調控機制

Crystal Structure of Helicobacter pylori Uridine Monophosphate Kinase GTP Complex Reveals an Allosteric Regulation

陳品臻(Pin-Chen Chen)

指導教授 ： 孫玉珠

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2010年)

摘要

Bacterial uridine monophosphate kinase(UMPK), an essential cytosolic enzyme (EC2.7.4.22), plays an important role in the biosynthesis of pyrimidine nucleotides. UMPK catalyzes the reaction which converts the substrate, UMP, to the product, UDP; meanwhile, it accompanies ATP hydrolyzed to ADP. The reaction can be allosterically regulated by activator, GTP, and inhibited by UTP. The crystal structure of UMPK from H. pylori (HpUMPK) in complex with GTP (HpUMPK-GTP complex) was determined by molecular replacement method at 1.8 Å resolution. The HpUMPK-GTP complex formed as a hexamer with two trimers, packing antiparallelly in crystal. HpUMPK also functions as a hexamer in solution. There are six GTP molecules per hexamer and the GTP is situated between molecules of a trimer and near the center of trimer. The interactions between HpUMPK and GTP are from β3 strand, α4 helix and the β3-α4 loop.
The quaternary structures of HpUMPK-GTP and HpUMPK-UDP (from our previous study) complexes are packed by two kinds of dimer, noncrystallogrophy dimer and cross-link dimer. The HpUMPK-GTP complex formed as a perfect hexamer and stablized by GTP; however, the HpUMPK-UDP complex packed as a pseudo hexamer with a loose oligomerization interaction.
To compare with these two complexes, there are some dominated conformational changes while GTP binds to the HpUMPK. First, the α7 helices, near the ATP binding site, form a few hydrogen bonds between cross-link dimer. Meanwhile, the β6 strand was developed and this phenomenon indicates the stability increasing of the ATP binding site. On the other hand, the α2 of the UDP binding site would undergo the secondary structural variable when GTP bound. These GTP induced conformational changes may promote the UDP released, turn over the reaction, increase the enzymic activity and act as an activator for UMPK. The previous comparison demonstrates that how the GTP distant allosteric regulate the HpUMPK function.

關鍵字

幽門螺旋桿菌 ； 異位調控機制 ； 尿嘧啶核苷酸激

參考文獻

2. Covacci, A., Telford, J. L., Del Giudice, G., Parsonnet, J. & Rappuoli, R. (1999). Helicobacter pylori virulence and genetic geography. Science 284, 1328-33.

4. Yan, H. & Tsai, M. D. (1999). Nucleoside monophosphate kinases: structure, mechanism, and substrate specificity. Adv Enzymol Relat Areas Mol Biol 73, 103-34, x.

5. Jong, A., Yeh, Y. & Ma, J. J. (1993). Characteristics, substrate analysis, and intracellular location of Saccharomyces cerevisiae UMP kinase. Arch Biochem Biophys 304, 197-204.

6. Serina, L., Bucurenci, N., Gilles, A. M., Surewicz, W. K., Fabian, H., Mantsch, H. H., Takahashi, M., Petrescu, I., Batelier, G. & Barzu, O. (1996). Structural properties of UMP-kinase from Escherichia coli: modulation of protein solubility by pH and UTP. Biochemistry 35, 7003-11.

7. Yamanaka, K., Ogura, T., Niki, H. & Hiraga, S. (1992). Identification and characterization of the smbA gene, a suppressor of the mukB null mutant of Escherichia coli. J Bacteriol 174, 7517-26.