Modulation of ornithine decarboxylase activity in Escherichia coli by positive and negative effectors.

Abstract

Two effectors of ornithine decarboxylase (ODC; L-ornithine carboxy-lyase, IC 4.1.1.17) have been extracted from an ODC-(speC) mutant, Escherichia coli MA 255. One of these is an ODC inhibitor (M, 15,000 ± We reported (1, 2) that the activity of ornithine decarboxylase (ODC; L-ornithine carboxy-lyase, EC 4.1.1.17) can be inhibitied by a specific protein, the ODC antizyme, and proposed (3) that macromolecular activators of ODC may also exist to balance the inhibitory action of the ODC antizyme. The appearance of the ODC antizyme, a noncompetitive inhibitor of ODC in eukaryote cells, occurred in response to high concentrations of putrescine, spermidine, and spermine (1, 2). The term "antizyme" was suggested for this type of macromolecular inhibitor of an enzyme, whose synthesis is induced by the product of the reaction it inhibits (2). We now provide evidence for the existence of two macromolecules in extracts of Escherichia coli, one that activates and one that inhibits ODC.Two different ODCs exist in E. coli, a biodegradative enzyme induced by growth at low pH and a biosynthetic enzyme detectable by growth at neutral pH (4-5). The activity of both these enzymes is enhanced by GTP and by other nucleoside phosphates in vitro (6); guanosine 5'-diphosphate-3'-diphosphate is an inhibitor of the biosynthetic ODC in vitro (7). Based on this observation, Holtta et al. (7) suggested that RNA synthesis and polyamine synthesis are coordinately regulated in vivo by the intracellular level of these nucleotides. Sakai and Cohen (8) tested this hypothesis in a K+-requiring strain of E. coli and reported that there was little, if any, correspondence between the intracellular levels of putrescine and the intraThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. cellular levels of these nucleotides; this suggested a corresponding lack of regulation of ODC activity by these nucleotides in vivo in this strain of E. coli.Of the various bacterial mutants available with mutations in genes related to ODC (9), we have used the bacterial mutant strains developed by Cunningham-Rundles and Maas (10) Bacterial Strains and Media. E. coli, wild type K-12, strain 5073, MA135 (his-, trp-, thi-, proA, speB, argE), MA163 (thr-, leu-, thi-, his-, speA), MA197 (serA, thr-, leu-, thi-, speB), MA255 (thr-, leu-, thi-, speB, speC), and AB1203 (thi-, iv-, argE) were obtained from the Genetic Stock Center of Yale University. The E. coli (except MA255) were grown, with aeration at 370, in minimal medium E as described by Vogel and Bonner (12) supplemented with 1 ml of trace elements per liter (13), 0.2% D-glucose, 50 mg of amino acids per liter, and 20 ,g of thiamine per liter. The MA255 cells were grown in Luria's medium (10 g of Bactotryptone, 5 g of yeast extract, and 0.5 g of NaCl per liter) at pH 7.0, normally in the presence of 1.13 mM putrescine and 0.68 mM spermidine. Usually 1 m...

Purification and properties of agmatine ureohydrolyase, a putrescine biosynthetic enzyme in Escherichia coli

“…This copurification of an inhibitory protein is similar to that described in purifying ornithine decarboxylase, the other putrescine biosynthetic enzyme in E. coli. An acidic protein inhibitor, termed ornithine decarboxylase antizyme, was found to copurify with ornithine decarboxylase in the initial stages of purification (16).…”

“…Pathway I involves the decarboxylation of ornithine; pathway II involves the decarboxylation of arginine to agmatine, followed by the removal of urea from agmatine by agmatine ureohydrolase. Regulation of the biosynthetic enzymes if complex (16,20); their regulation involves both transcriptional and posttranslational mechanisms (3,13,14,25,26,29,30). Although the intracellular level of putrescine is constant as well as proportional to the growth rate (4), the relative use of the two polyamine biosynthetic pathways can vary considerably (23,26), depending on the nutritional supplements provided.…”

Identification, cloning, and nucleotide sequencing of the ornithine decarboxylase antizyme gene of Escherichia coli.

“…Although inhibition of ODC by Az is reversible and active ODC can be recovered from inactive ODC-Az complexes under certain conditions (8), there is also substantial evidence involving Az in ODC degradation (9,10). In Escherichia coli, the posttranslational control of the activities of the polyamine biosynthetic enzymes ODC and arginine decarboxylase (ADC; L-arginine carboxy-lyase, EC 4.1.1.…”

Adjustment of polyamine contents in Escherichia coli

“…It is believed that the regulation of ornithine decarboxylase activity is important in the regulation of polyamine biosynthesis in Escherichia coli. Ornithine decarboxylase of E. coli is stimulated by GTP (1,6) and inhibited by ppGpp (7,19), polyamines (1,23), and antizymes (13,18). It has been reported that two basic E. coli antizyme proteins are ribosomal proteins S20/L26 and L34 (18), but other major ribosomal proteins also show antizyme activity (11).…”