Structural studies on Helicobacter pylori 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase using electrospray ionization mass spectrometry: a tetrameric complex composed of dimeric dimers.
Helicobacter pylori 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the conversion of D-arabinose-5-phosphate (A5P) and phosphoenolpyruvate (PEP) to produce KDO8P and inorganic phosphate. Since this protein is absent in mammals, it might therefore be an attractive target for the development of new antibiotics. Unlike E. coli KDO8P synthase (class I), the H. pylori counterpart is a class II enzyme, where it requires a divalent transition metal ion for catalysis. Although the metal ions have been shown to be important for catalysis, their role in the structure is not understood. Using electrospray ionization mass spectrometry (ESI-MS), the role of the metal ions in H. pylori KDO8P synthase has been investigated. This protein is found to be a tetramer in the gas phase but dissociates into the dimer with increasing declustering potential (DP2) suggesting an existence of a 'structurally specific' tetramer. An examination of mass spectra revealed that the tetrameric state of the Cd(2+)-reconstituted enzyme is less stable than those of the Zn(2+)-, Co(2+)- and Cu(2+)-enzymes. The stoichiometry of metal binding to the protein depends on the nature of the metal ion. Taken together, our data suggest that divalent metal ions play an important role in the quaternary structure of the protein and the tetrameric state may be primarily responsible for catalysis. This study demonstrates the first structural characterization and stoichiometry of metal binding in class II KDO8P synthase using electrospray ionization quadrupole time-of-flight mass spectrometry under nondenaturing conditions.
Li
Zhili
Z
Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing 100005, China. lizhili@ibms.pumc.edu.cn
Sau
Apurba Kumar
AK
D000446:Aldehyde-Lyases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D019281:Dimerization; D016480:Helicobacter pylori / Q000737:chemistry / Q000201:enzymology*; D008670:Metals / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization
D001426:Bacterial Proteins; D008670:Metals; C020011:2-dehydro-3-deoxyphosphooctonate aldolase; D000446:Aldehyde-Lyases