| Enolase is a key evolutionary conserved glycolytic metalloenzyme in the cytoplasm of prokaryotic and eukaryotic cells, which catalyzes the dehydration of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP). The enzyme can also locate on the surface of a variety of cells, including carcinoma cells, hematopoietic cells, neuronal cells and pathogenic bacteria. Cell surface enolase serves as a receptor of plasminogen, by which enhancing pericellular fibrinolytic activity and leading extracellular matrix degradation. Staphylococcus aureus (S.aureus) is a typical gram positive bacterium with strong pathogenicity, causing frequent and severe large scale infection all over the world. In addition, S.aureus enolase also appeared in RNA degradosome.In part I, we present the crystal structures of Sa_enolase with and without PEP bound at 1.6 A and 2.45 A resolution, respectively. The structure reveals an octameric arrangement, however, both dimer and octamer conformations were observed in solution. Furthermore, enzyme activity assays show that only the octamer variant is catalytically active. Isothermal Titration Calorimetry (ITC) assay reveals that Sa_enolase octameric enolase is capable of binding 2-PG, while the interaction between substrate and dimeric enolase is too weak to be detected.Rh1B is one of the five DEAD box RNA helicase in Escherichia coli, which is ATP dependent. The ATPase and RNA unwinding activities of Rh1B require the interaction with RNaseE. The crystal structure of the Drosophila melanogaster DEADbox protein Vasa in complex with single-strand RNA and non-hydrolysable ATP analogue provides a clue to unveil the mechanism of RNA duplex unwinding. However, the role of RNaseE and the activity of ATPase of Rh1B are still unclear.In part â…¡, full length E.coli Rh1B and its different truncations, Rh1B/RNaseE complex, and Rh1B/RNaseE/RraA complex were expressed, purified for crystallization. Crosslinking assays and proteolysis were used for purification of Rh1B complex. |
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