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

Ashraf El-Sayed,Amgad Rady,Nelly George,Bothaina Alaidaroos,Safa Aziz,Ahmed Bolbol,Mahmoud Sitohy,Marwa Yassin,Rawia Zayed,Marwa Mohamed

doi:10.3390/molecules24152756

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 (Km) for l-ornithine, l-lysine and l-arginine (0.95, 1.34 and 1.4 mM) and catalytic efficiency (kcat/Km) (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.

Highlights

Ornithine decarboxylase (ODC, EC 4.1.1.17) is a pyridoxal-50 -phosphate-dependent enzyme, catalyzing the decarboxylation of L-ornithine to the diamine putrescine as the precursor of polyamine biosynthesis [1]
Forty fungal isolates were grown on l-ornithine as sole nitrogen source and the developed fungal colonies were inspected
The optimization processes revealed the partial independence of ODC expression on the type of medium carbon and nitrogen source

Summary

Introduction

Ornithine decarboxylase (ODC, EC 4.1.1.17) is a pyridoxal-50 -phosphate-dependent enzyme, catalyzing the decarboxylation of L-ornithine to the diamine putrescine as the precursor of polyamine biosynthesis [1]. Polyamines with different cellular functions have been found in all organisms—bacteria, fungi, plants and human—the unique route for polyamine biosynthesis in fungi and animals is the de novo decarboxylation of ornithine into putrescine via ODC catalysis [3]. Putrescine is converted into spermidine by adding an aminopropyl group from the decarboxylated S-adenosylmethionine by spermidine synthase. Aminopropyl group is added to spermidine by spermine synthase to form spermine [4]. Spermine has not been reported in fungi, several orthologs of spermine synthase had been found in ascomycetous human fungal pathogens [5,6,7]. Thermo-spermine, an isomer of spermine, was confirmed to be present in fungi [8,9]

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