title: Functional roles of the dimer-interface residues in human ornithine decarboxylase.
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 Kd 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.
authors.affiliation: Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan; Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan; Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan.
authors.forename: Chien-Yun
authors.initials: CY
authors.lastname: Lee
authors.affiliation: Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan; Institute of Microbiology and Immunology and Division of Allergy, Immunology and Rheumatology, Chung Shan Medical University and Hospital, Taichung, Taiwan.
authors.forename: Yi-Liang
authors.initials: YL
authors.lastname: Liu
authors.affiliation: Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan.
authors.forename: Chih-Li
authors.initials: CL
authors.lastname: Lin
authors.affiliation: Institute of Microbiology and Immunology and Division of Allergy, Immunology and Rheumatology, Chung Shan Medical University and Hospital, Taichung, Taiwan.
authors.forename: Guang-Yaw
authors.initials: GY
authors.lastname: Liu
authors.affiliation: Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan; Institute of Genomics and Bioinformatics, National Chung-Hsing University, Taichung, Taiwan; Agricultural Biotechnology Center (ABC), National Chung-Hsing University (NCHU), Taichung, Taiwan.
authors.forename: Hui-Chih
authors.initials: HC
authors.lastname: Hung
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D019281:Dimerization; D006801:Humans; D008958:Models, Molecular; D009955:Ornithine Decarboxylase / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D009955:Ornithine Decarboxylase