This project aims to investigate the unique regulation of ornithine decarboxylase (ODC), the key rate-limiting enzyme of polyamine biosynthesis, involving a unique protein termed antizyme. Antizyme binds to ODC, inhibits ODC activity and facilitates the degradation of ODC by the 26S proteasome without polyubiquitination. Saccharomyces cerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the degradation of ornithine decarboxylase (ODC). Despite the presence of conserved domains in the frameshifting site, the amino acid sequences of yeast and mammalian AZ differ considerably. Despite many studies on the interaction of AZ and ODC, the ODC: AZ complex has not been purified from any source and thus clear information about the stoichiometry of the complex were still lacking as well as details in the structural interactions. In our initial study, we have reported the biochemical and biophysical characteristics of the proteins, and the resulting ODC: AZ complex. The far UV CD spectrum of the full-length antizyme shows that the yeast protein consists of 51% beta-sheet, 19% alpha-helix, and 24% coils. Surface plasmon resonance analyses show that theassociation constant (KA) between yeast AZ and yeast ODC is 6x 107 (M-1). Using purified His-tagged AZ as a binding partner, we have purified the ODC: AZ inhibitory complex. The isolated complex has no ODC activity. The molecular weight of the complex is 90 kDa, which indicates a one to one stoichiometric binding of AZ and ODC in vitro. Comparison of the circular dichroism (CD) spectra of the two individual proteins and of the ODC: AZ complex shows a change in the secondary structure in the complex. Currently, in collaboration with Dr. David Davies'laboratory, and Dr. Udesh de Silva (Laboratory of Molecular Biology) we are trying to crystallize the AZ:ODC complex. For this study, we have developed a method to rapidly co-purify the AZ:ODC complex from crude extracts without purifying the two proteins separately. We have also developed a strain to overexpress ODC and antizyme in the same E. coli expression system so that when we induce the proteins both AZ and ODC co-express and bind together in the same cell in vivo. Using this preparation we obtained several hits in our crystal screens for both antizyme and antizyme-ODC complex. In a related project, we are studying in detail the protein-protein interaction of yeast antizyme and ornithine decarboxylase by deuterium exchange. For this we have purified yeast antizyme, ornithine decarboxylase and antizyme-ODC complex from the E. coli expression systems and analyze the interaction by deuterium exchange followed by mass spectrometric analyses to find out the interacting residues in antizyme and ODC. For this study we have sent protein samples to the NIC-Frederick LPAT-Mass Spectroscopy facility (Dr. Timothy Veenstra and Dr. Ming Zhou). H/D exchange experiments have been performed in D2O buffer and the peptides were analyzed by mass spectrometry. Preliminary data from these analyses show many interacting residues from both the proteins in the antizyme-ODC complex. These studies are now being repeated and based on these studies mutants will be generated by site directed mutagenesis to further confirm the interacting residues in each molecule and to create a molecular model to show details of the yeast antizyme and ODC interaction.
|Chattopadhyay, Manas K; Fernandez, Cristina; Sharma, Deepak et al. (2011) Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: purification and characterization of the inhibitory complex. Biochem Biophys Res Commun 406:177-82|