Abstract
Ornithine decarboxylase (ODC) is the first enzyme involved in polyamine biosynthesis, and it catalyzes the decarboxylation of ornithine to putrescine. ODC is a dimeric enzyme, whereas antizyme inhibitor (AZI), a positive regulator of ODC that is homologous to ODC, exists predominantly as a monomer and lacks decarboxylase activity. The goal of this paper was to identify the essential amino acid residues that determine the dimerization of AZI. The nonconserved amino acid residues in the putative dimer interface of AZI (Ser-277, Ser-331, Glu-332, and Asp-389) were substituted with the corresponding residues in the putative dimer interface of ODC (Arg-277, Tyr-331, Asp-332, and Tyr-389, respectively). Analytical ultracentrifugation analysis was used to determine the size distribution of these AZI mutants. The size-distribution analysis data suggest that residue 331 may play a major role in the dimerization of AZI. Mutating Ser-331 to Tyr in AZI (AZI-S331Y) caused a shift from a monomer configuration to a dimer. Furthermore, in comparison with the single mutant AZI-S331Y, the AZI-S331Y/D389Y double mutant displayed a further reduction in the monomer-dimer K(d), suggesting that residue 389 is also crucial for AZI dimerization. Analysis of the triple mutant AZI-S331Y/D389Y/S277R showed that it formed a stable dimer (K(d) value = 1.3 microm). Finally, a quadruple mutant, S331Y/D389Y/S277R/E332D, behaved as a dimer with a K(d) value of approximately 0.1 microm, which is very close to that of the human ODC enzyme. The quadruple mutant, although forming a dimer, could still be disrupted by antizyme (AZ), further forming a heterodimer, and it could rescue the AZ-inhibited ODC activity, suggesting that the AZ-binding ability of the AZI dimer was retained.
Highlights
Polyamines have been shown to have both structural and regulatory roles in protein and nucleic acid biosynthesis and function [1,2,3]
X-ray structures of the Ornithine decarboxylase (ODC) enzyme reveal that this dimer contains two active sites, both of which are formed at the interface between the N-terminal domain of one monomer, which provides residues involved in pyridoxal 5-phosphate (PLP) interactions, and the C-terminal domain of the other subunit, which provides the residues that interact with substrate [27, 32,33,34,35,36,37,38,39,40,41]
ODC is a key enzyme in the biosynthesis of cellular polyamines, and antizyme inhibitor (AZI) is a positive regulator of ODC activity
Summary
Polyamines (putrescine, spermidine, and spermine) have been shown to have both structural and regulatory roles in protein and nucleic acid biosynthesis and function [1,2,3]. ODC and polyamines play important roles in a number of biological functions, including embryonic development, cell cycle, proliferation, differentiation, and apoptosis (8 –15). They have been associated with human diseases and a variety of cancers (16 –26). AZI is capable of direct interaction with cyclin D1, preventing its degradation, and this effect is at least partially independent of AZ function [60, 61]. These results demonstrate a role for AZI in the positive regulation of cell proliferation and tumorigenesis
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