Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein

Abstract

A southern blot analysis of the Panagrellus redivivus ornithine decarboxylase (ODC) gene suggests that it is a single-copy gene that resides on a genomic 3.2 kb EcoRI fragment. Phage clones possessing ODC gene sequences were isolated from a genomic EMBL-4 library and purified. The phage DNA inserts were analysed and a 3.2 kb EcoRI fragment containing the entire ODC gene was isolated. The nucleotide sequence analysis of this fragment reveals that the gene is interrupted by two introns of 47 and 49 bp. In the 5' non-translated region of the gene, putative AP1, VPE2 and c-Myc binding sites were identified. The ODC cDNA was expressed in a bacterial system as a His-fusion protein and the enzyme was purified by Ni(2+)-chelating affinity chromatography. The subunit molecular mass, as deduced from the cDNA and shown by SDS/PAGE, is 47.1 kDa. On the basis of gel filtration analyses it is shown that the active enzyme is a dimer. The specific enzyme activity was determined to be 4.2 mumol CO2/min/mg protein. The enzyme is dependent on pyridoxal 5-phosphate as a cofactor, and the presence of dithioerythritol or other thiol-reducing agents is essential for maximal activity. The Km value for L-ornithine was determined as 44 microM. The Ki values for putrescine, alpha-diffluoromethylornithine, alpha-hydrazino-ornithine and alpha-methylornithine were calculated as 51, 34, 0.34 and 42 microM respectively.

In the Human Malaria Parasite Plasmodium falciparum,Polyamines Are Synthesized by a Bifunctional Ornithine Decarboxylase,S-Adenosylmethionine Decarboxylase

“…Analysis of the Deduced Amino Acid Sequence in P. falciparum ODC/AdoMetDC cDNA-Pairwise sequence comparisons of the predicted plasmodial amino acid sequences of the AdoMetDC and ODC domains with the AdoMetDC and ODC sequences of the respective monofunctional proteins of mammals (24,25), nematodes (18,19), and protozoa (26,27) exhibit a moderate degree of identity. However, within the amino acid sequences of AdoMetDC and ODC from mammals, there are some highly conserved amino acids and regions, which are reported to be essential for catalytic activity, dimerization, and pro-enzyme processing (28 -33), and these are also found in the putative AdoMetDC and ODC domains of the plasmodial bifunctional protein (Fig.…”

“…The column was washed with 2 volumes of buffer A, and elution was performed with a linear gradient of 0 -0.5 M NaCl in buffer A. The eluant containing the enzyme activities was concentrated to 2 ml using a Centricon 50 microconcentrator (Amicon) and subjected to fast protein liquid chromatography on a calibrated Superdex S-200 column (1.6 cm ϫ 60 cm) equilibrated with buffer A at a flow rate of 2 ml min , Amersham Pharmacia Biotech) as described previously (18,19). The reaction mixtures contained in a final volume of 0.25 ml were: 40 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, and 50 M S-adenosylmethionine for the AdoMetDC assay or 50 M ornithine and 100 M pyridoxal 5-phosphate for the ODC assay.…”

Identification of essential active-site residues in ornithine decarboxylase of Nicotiana glutinosa decarboxylating both l-ornithine and l-lysine

“…The K i value of wild-type ODC for α-DFMO was 1.15 µM, showing a competitive inhibition pattern. ODC isolated from mouse and Panagrellus redi is had similar K i values for α-DFMO of 39 and 34 µM respectively [49,50], and K i for T. brucei was calculated to be 220 µM [51]. The fact that the K i value for α-DFMO of N. glutinosa ODC was calculated to be the lowest shows that α-DFMO is a strong competitive inhibitor of plant ODC.…”

The Plasmodium falciparum Bifunctional Ornithine Decarboxylase, S-Adenosyl-l-methionine Decarboxylase, Enables a Well Balanced Polyamine Synthesis without Domain-Domain Interaction

“…14 C]methionine and L- [1-14 C]ornithine (57 and 52 mCi/mmol, respectively, Amersham Pharmacia Biotech) as described previously (22,23). The K m values of the bifunctional rPfODC/AdoMetDC and rPfAdoMetDC domains for the respective substrates were determined by varying the concentrations of ornithine and AdoMet from 10 to 200 M. The inhibition constant (K i ) for putrescine to inhibit ODC activity was determined under standard assay conditions with varying concentrations of ornithine and the addition of 50, 100, and 200 M putrescine.…”