Mutational analysis of the antizyme-binding element reveals critical residues for the function of ornithine decarboxylase

• Published in: Biochimica et biophysica acta Vol. 1830; no. 11; pp. 5157 - 5165
• Main Authors: Ramos-Molina, Bruno, Lambertos, Ana, López-Contreras, Andrés J., Peñafiel, Rafael
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2013
• Subjects: Amino Acid Sequence; amino acid sequences; Antizyme; Antizyme-binding element; Binding Sites; biochemical pathways; cell growth; Cell Line; DNA Mutational Analysis - methods; eukaryotic cells; HEK293 Cells; Humans; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutant Proteins - genetics; Mutant Proteins - metabolism; mutants; Ornithine decarboxylase; Ornithine Decarboxylase - genetics; Ornithine Decarboxylase - metabolism; Polyamines; Protein Binding; Protein Conformation; proteins; Proteins - genetics; Proteins - metabolism; Protein–protein interaction; site-directed mutagenesis; AZIN; Antizyme-binding element; AZBE; Ornithine decarboxylase; Polyamines; Protein–protein interaction; HEK; ODC; PLP; Antizyme; AZ; HA; DFMO; α-difluoromethylornithine; pyridoxal phosphate; antizyme inhibitor; hemagglutinin; human embryonic kidney
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2013.07.003

Ornithine decarboxylase (ODC), the key enzyme in the polyamine biosynthetic pathway, is highly regulated by antizymes (AZs), small proteins that bind and inhibit ODC and increase its proteasomal degradation. Early studies delimited the putative AZ-binding element (AZBE) to the region 117-140 of ODC. The aim of the present work was to study the importance of certain residues of the region 110-142 that includes the AZBE region for the interaction between ODC and AZ1 and the ODC functionality. Computational analysis of the protein sequences of the extended AZBE site of ODC and ODC paralogues from different eukaryotes was used to search for conserved residues. The influence of these residues on ODC functionality was studied by site directed mutagenesis, followed by different biochemical techniques. The results revealed that: a) there are five conserved residues in ODC and its paralogues: K115, A123, E138, L139 and K141; b) among these, L139 is the most critical one for the interaction with AZs, since its substitution decreases the affinity of the mutant protein towards AZs; c) all these conserved residues, with the exception of A123, are critical for ODC activity; d) substitutions of K115, E138 or L139 diminish the formation of ODC homodimers. These results reveal that four of the invariant residues of the AZBE region are strongly related to ODC functionality. This work helps to understand the interaction between ODC and AZ1, and describes various new residues involved in ODC activity, a key enzyme for cell growth and proliferation. •Sequence analysis of AZBE domains reveals conserved residues in eukaryote species.•K115, A123, E138, L139 and K141 remain invariant in ODC and its paralogues.•The substitutions of conserved residues, excepting A123, affect ODC activity.•L139 is critical for the interaction with AZ, homodimerization and enzyme activity.•E138 and K115 are involved in the formation of ODC homodimers.