Purification and Characterization of Ornithine Decarboxylase from Aspergillus terreus; Kinetics of Inhibition by Various Inhibitors

Abstract

l-Ornithine decarboxylase (ODC) is the rate-limiting enzyme of de novo polyamine synthesis in humans and fungi. Elevated levels of polyamine by over-induction of ODC activity in response to tumor-promoting factors has been frequently reported. Since ODC from fungi and human have the same molecular properties and regulatory mechanisms, thus, fungal ODC has been used as model enzyme in the preliminary studies. Thus, the aim of this work was to purify ODC from fungi, and assess its kinetics of inhibition towards various compounds. Forty fungal isolates were screened for ODC production, twenty fungal isolates have the higher potency to grow on L-ornithine as sole nitrogen source. Aspergillus terreus was the most potent ODC producer (2.1 µmol/mg/min), followed by Penicillium crustosum and Fusarium fujikuori. These isolates were molecularly identified based on their ITS sequences, which have been deposited in the NCBI database under accession numbers MH156195, MH155304 and MH152411, respectively. ODC was purified and characterized from A. terreus using SDS-PAGE, showing a whole molecule mass of ~110 kDa and a 50 kDa subunit structure revealing its homodimeric identity. The enzyme had a maximum activity at 37 °C, pH 7.4-7.8 and thermal stability for 20 h at 37 °C, and 90 days storage stability at 4 °C. A. terreus ODC had a maximum affinity (K_m) for l-ornithine, l-lysine and l-arginine (0.95, 1.34 and 1.4 mM) and catalytic efficiency (k_cat/K_m) (4.6, 2.83, 2.46 × 10^-5 mM^-1·s^-1). The enzyme activity was strongly inhibited by DFMO (0.02 µg/mL), curcumin (IC₅₀ 0.04 µg/mL), propargylglycine (20.9 µg/mL) and hydroxylamine (32.9 µg/mL). These results emphasize the strong inhibitory effect of curcumin on ODC activity and subsequent polyamine synthesis. Further molecular dynamic studies to elucidate the mechanistics of ODC inhibition by curcumin are ongoing.

Keywords: Aspergillus terreus; curcumin; inhibition; kinetics; ornithine decarboxylase.

Figure 1

Morphological and molecular identification of the potent ODC-producing fungal isolates. The isolates were grown on PDA medium, incubated for 8 days at 30 °C, then photographed by the digital camera and examined by light microscope. (A) The macroscopical (left side) and microscopical (right side) views of A. terreus, F. fuijikuroi and P. crustosum. (B) The PCR amplicon of ITS region for A. terreus (lane 1), F. fuijikuroi (lane 2) and P. crustosum (lane 3), using the gDNA as PCR template, comparing to 1 kb ladder. (C) Molecular phylogenetic analyses of A. terreus, F. fujikuroi and P. crustosum constructed by the Maximum Likelihood model [28].

Figure 2

Cultural optimization of Aspergillus terreus to maximize its productivity for ODC. Different nitrogen sources (A), carbon sources (B) and initial pH values of the medium (C) were evaluated based on the ODC productivity by A. terreus.

Figure 3

Purification of ODC from the culture of A. terreus. The crude ODC was extracted from the cultures of A. terreus, concentrated by dialyzer, purified by ion-exchange and gel-filtration chromatography. The pattern of ODC purification by ion-exchange chromatography (A), and gel-filtration chromatography (B). SDS-PAGE pattern for the subunit structure of purified A. terreus ODC (C). M, protein Marker (Cat. # PG-PMT2962, 315-10 kDa), S, purified ODC sample after the last purification column.

Figure 4

Biochemical properties of ODC purified from A. terreus. Reaction temperature (A), thermal stability (B), reaction pH (C) and pH stability (D).