Abstract
Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to putrescine and is the rate-limiting enzyme in the polyamine biosynthesis pathway. ODC is a dimeric enzyme, and the active sites of this enzyme reside at the dimer interface. Once the enzyme dissociates, the enzyme activity is lost. In this paper, we investigated the roles of amino acid residues at the dimer interface regarding the dimerization, protein stability and/or enzyme activity of ODC. A multiple sequence alignment of ODC and its homologous protein antizyme inhibitor revealed that 5 of 9 residues (residues 165, 277, 331, 332 and 389) are divergent, whereas 4 (134, 169, 294 and 322) are conserved. Analytical ultracentrifugation analysis suggested that some dimer-interface amino acid residues contribute to formation of the dimer of ODC and that this dimerization results from the cooperativity of these interface residues. The quaternary structure of the sextuple mutant Y331S/Y389D/R277S/D332E/V322D/D134A was changed to a monomer rather than a dimer, and the K d value of the mutant was 52.8 µM, which is over 500-fold greater than that of the wild-type ODC (ODC_WT). In addition, most interface mutants showed low but detectable or negligible enzyme activity. Therefore, the protein stability of these interface mutants was measured by differential scanning calorimetry. These results indicate that these dimer-interface residues are important for dimer formation and, as a consequence, are critical for enzyme catalysis.
Highlights
Ornithine decarboxylase (ODC, EC 4.1.1.17) is universally found in organisms ranging from bacteria to humans
Previous studies have indicated that ODC exists as a stable dimer, with a dissociation constant (Kd) of 0.18 mM, whereas antizyme inhibitor (AZI) is in a rapid equilibrium between the monomer and the dimer, with a Kd value of 84 mM [49]
We explored the functional roles of the dimer-interface residues that may contribute to dimerization of the enzyme, enzyme activity and protein stability
Summary
Ornithine decarboxylase (ODC, EC 4.1.1.17) is universally found in organisms ranging from bacteria to humans. ODC and cellular polyamines play significant roles in numerous biological functions, including embryonic development, the cell cycle, and cell proliferation, differentiation and apoptosis [5,6,7,8,9,10,11]. Because of their biological roles, polyamines have been linked with several cancers [5,12,13,14,15,16,17,18,19,20,21]. ODC inhibitors and negative regulators of the polyamine pathway could be beneficial for the treatment of many cancers [3,7]
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