Characterization of ornithine decarboxylase and regulation by its antizyme in Thermus thermophilus

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis was highly purified from the thermophilic bacterium Thermus thermophilus. The enzyme preparation showed a single band on SDS-polyacrylamide gel electrophoresis, a pH optimum of 7.5 and a temperature optimum at 60°C. The native enzyme which is phosphorylated could, upon treatment with alkaline phosphatase, lose all activity. The inactive form could be reversibly activated by nucleotides in the order of NTP>NDP>NMP. When physiological polyamines were added to the purified enzyme in vitro, spermine or spermidine activated ODC by 140 or 40%, respectively, while putrescine caused a small inhibition. The basic amino acids lysine and arginine were competitive inhibitors of ODC, while histidine did not affect the enzyme activity. Among the phosphoamino acids tested, phosphoserine was the most effective activator of purified ODC. Polyamines added at high concentration to the medium resulted in a delay or in a complete inhibition of the growth of T. thermophilus, and in a decrease of the specific activity of ornithine decarboxylase. The decrease of ODC activity resulted from the appearance of a non-competitive inhibitor of ODC, the antizyme (Az). The T. thermophilus antizyme was purified by an ODC-Sepharose affinity column chromatography, as well as by immunoprecipitation using antibodies raised against the E. coli antizyme. The antizyme of E. coli inhibited the ODC of T. thermophilus, and vice versa. The fragment of amino acids 56-292 of the E. coli antizyme, produced as a fusion protein of glutathione S-transferase, did not inhibit the ODC of E. coli or T. thermophilus.     

The old and new biochemistry of polyamines

Polyamines are ubiquitous positively charged amines found in all organisms. These molecules play a crucial role in many biological functions including cell growth, gene regulation and differentiation. The three major polyamines produced in all mammalian cells are putrescine, spermidine and spermine. The intracellular levels of these polyamines depend on the interplay of the biosynthetic and catabolic enzymes of the polyamine and methionine salvage pathway, as well as the involvement of polyamine transporters. Polyamine levels are observed to be high in cancer cells, which contributes to malignant transformation, cell proliferation and poor patient prognosis. Considering the critical roles of polyamines in cancer cell proliferation, numerous anti-polyaminergic compounds have been developed as anti-tumor agents, which seek to suppress polyamine levels by specifically inhibiting polyamine biosynthesis, activating polyamine catabolism, or blocking polyamine transporters. However, in terms of the development of effective anti-cancer therapeutics targeting the polyamine system, these efforts have unfortunately resulted in little success. Recently, several studies using the iron chelators, O-trensox and ICL670A (Deferasirox), have demonstrated a decline in both iron and polyamine levels. Since iron levels are also high in cancer cells, and like polyamines, are required for proliferation, these latter findings suggest a biochemically integrated link between iron and polyamine metabolism.     

The mouse Gm853 gene encodes a novel enzyme: Leucine decarboxylase

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthesis of polyamines. ODC-antizyme inhibitors (AZINs) are homologous proteins of ODC, devoid of enzymatic activity but acting as regulators of polyamine levels. The last paralogue gene recently incorporated into the ODC/AZINs family is the murine Gm853, which is located in the same chromosome as AZIN2, and whose biochemical function is still unknown. By means of transfection assays of HEK293T cells with a plasmid containing the coding region of Gm853, we show here that unlike ODC, GM853 was a stable protein that was not able to decarboxylate l-ornithine or l-lysine and that did not act as an antizyme inhibitor. However, GM853 showed leucine decarboxylase activity, an enzymatic activity never described in animal cells, and by acting on l-leucine (Km = 7.03 × 10^? 3 M) it produced isopentylamine, an aliphatic monoamine with unknown function. The other physiological branched-chain amino acids, l-valine and l-isoleucine were poor substrates of the enzyme. Gm853 expression was mainly detected in the kidney, and as Odc, it was stimulated by testosterone. The conservation of Gm853 orthologues in different mammalian species, including primates, underlines the possible biological significance of this new enzyme. In this study, we describe for the first time a mammalian enzyme with leucine decarboxylase activity, therefore proposing that the gene Gm853 and its protein product should be named as leucine decarboxylase (Ldc, LDC).     

Functional characterization of the histidine kinase of the E. coli two-component signal transduction system AtoS–AtoC

The Escherichia coli AtoS–AtoC two-component signal transduction system regulates the expression of the atoDAEB operon genes, whose products are required for short-chain fatty acid catabolism. In this study purified his-tagged wild-type and mutant AtoS proteins were used to prove that these proteins are true sensor kinases. The phosphorylated residue was identified as the histidine-398, which was located in a conserved Η-box since AtoS carrying a mutation at this site failed to phosphorylate. This inability to phosphorylate was not due to gross structural alterations of AtoS since the H398L mutant retained its capability to bind ATP. Furthermore, the H398L mutant AtoS was competent to catalyze the trans-phosphorylation of an AtoS G-box (G565A) mutant protein which otherwise failed to autophosphorylate due to its inability to bind ATP. The formation of homodimers between the various AtoS proteins was also shown by cross-linking experiments both in vitro and in vivo.