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 poly-ubiquitination. 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 sequence of yeast and mammalian AZ differ considerably. In our current work we have studied the biochemical and biophysical characteristics of the proteins, and the resulting ODC: AZ complex. Surface plasmon resonance analyses show that the association constant (KA) between yeast AZ and yeast ODC is 6 x 107 (M-1). The circular dichroism (CD) spectra of the two individual proteins in different ratios and of the ODC: AZ complex show no significant change in the secondary or tertiary structure of either ODC or AZ after binding to each other as determined by far-UV and near UV CD analyses. The CD spectra also show that the secondary structure of yeast antizyme is different from the published NMR structure of rat antizyme. Using purified His-tagged AZ as a binding partner, we have purified the ODC: AZ complex and show that it has no ODC activity. The native molecular weight of the complex is 90 kDa, which suggests a one to one stoichiometric binding of AZ and ODC in vitro. We have recently communicated this part of studies for publication. Further studies are currently going on in collaboration with Dr. David Davies'laboratory (Laboratory of Molecular Biology) 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 two proteins separately. We have also developed a strain to overexpresss ODC and antizyme in the same E. coli expression system so that when we induce the proteins both AZ and ODC will co-express and bind together in the same cell in vivo. For better purification of the complex, we have introduced another tag (Flag) that will facilitate further purification of AZ:ODC complex. We will use this co-expression system to co-purify the complex for crystallization.
| 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 |
