Critical Factors Determining Dimerization of Human Antizyme Inhibitor

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 M). Finally, a quadruple mutant, S331Y/D389Y/S277R/E332D, behaved as a dimer with a K d value of ϳ0.1 M, 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.Polyamines (putrescine, spermidine, and spermine) have been shown to have both structural and regulatory roles in protein and nucleic acid biosynthesis and function (1-3). Ornithine decarboxylase (ODC, 3 EC 4.1.1.17) is a central regulator of cellular polyamine synthesis (reviewed in Refs. 1, 4, 5). This enzyme catalyzes the pyridoxal 5-phosphate (PLP)-dependent decarboxylation of ornithine to putrescine, and it is the first and rate-limiting enzyme in polyamine biosynthesis (2, 3, 6, 7). ODC and polyamines play important roles in a number of biological functions, including embryonic development, cell cycle, proliferation, differentiation, and apoptosis (8 -15). They also have been associated with human diseases and a variety of cancers (16 -26). Because the regulation of ODC and polyamine content is critical to cell proliferation (11), as well as in the origin and progression of neoplastic diseases (23, 24), ODC has been identified as an oncogenic enzyme, and the inhibitors of ODC and the polyamine pathway are important targets for therapeutic intervention in many cancers (6,11).ODC is ubiquitously found in organisms ranging from bacteria to humans. It contains 461 amino acid residues in each monomer and is a 106-kDa homodimer with molecular 2-fold symmetry (27, 28). Importantly, ODC activity req...

Novel Interaction of Ornithine Decarboxylase with Sepiapterin Reductase Regulates Neuroblastoma Cell Proliferation

“…So far, OAZs are the only proteins described that physically interact with ODC, thereby regulating its enzymatic activity 24; 25; 26 . Under physiological conditions, ODC functions as a homodimer which generates two active sites at the dimer interface that contain residues contributed by each subunit 27 . ODC cycles between monomeric and dimeric forms and ODC activity resides exclusively in the dimeric form.…”

Molecular cloning and mRNA expression analysis of antizyme inhibitor 1 in the ovarian follicles of the Sichuan white goose

“…This study revealed that the goose Azin1 protein had a molecular mass of 50 kDa, and a theoretical pI of 4.79, similar to that of mammals (Murakami et al, 1996;Liu et al, 2011). A previous study showed that the human AZIN1 gene encodes 448 amino acids (Su et al, 2009), while the goose AZIN1 encodes 450 amino acids. The predictive Azin1 amino acid sequence showed that two amino acid deletions had occurred in the domain of pyridoxal-dependent decarboxylase in the human Azin1 protein.…”

Discovery of novel inhibitors of human S-adenosylmethionine decarboxylase based on in silico high-throughput screening and a non-radioactive enzymatic assay

“…In weak basic solutions, low-level CO 2 exists primarily as bicarbonate (HCO 3 - ). The PEPC-MDH method was widely used for detecting CO 2 produced by decarboxylases (ODC, for example)119202132343536373839. In this method, the first step, catalyzed by Phosphoenolpyruvate Decarboxylase (PEPC), is the bicarbonate condenses with phosphoenol pyruvate to form oxalate, and in the second step, oxalate is enzymatically reduced by Malate Dehydrogenase (MDH, using an NADH cofactor) to form malate and NAD + .…”

“…Recently, Smithson et al 1 presented an optimized protocol based on this method for screening inhibitors of decarboxylases in the polyamine pathway, and they still used ODC as an example, although the authors claimed that it should be suitable for AdoMetDC too. This is quite surprising to us, since the PEPC-MDH method has actually been used on ODC in many previous studies32373839. Therefore, we supposed that some conditions could have hampered the use of this method in assessing AdoMetDC activity.…”