Decarboxylation Of L-ornithine Research Articles

Kinetic and solvent deuterium isotope effects in the oxidation of putrescine catalysed by enzyme diamine oxidase

In this study, the kinetic isotope effects and solvent isotope effects in the reaction of the deamination of [(1R)-2H ] putrescine – catalysed by enzyme diamine oxidase (EC 1.4.3.6) – were determined using a non-competitive spectroscopic method. Putrescine, stereospecifically labelled with deuterium, was obtained by enzymatic decarboxylation of l-ornithine that was carried out in a fully deuteriated incubation medium.

Stabilization and immobilization of Trypanosoma brucei ornithine decarboxylase for the biobased production of 1,4-diaminobutane

Using the biorefinery concept, L-arginine could become widely available from biomass waste streams via the nitrogen storage polypeptide cyanophycin. In our pursuit to develop a route from biobased L-arginine to 1,4-diaminobutane, one of the monomers in nylon-4,6, we were previously successful in the stabilization and immobilization of Bacillus subtilisarginase. In the present study, we investigated the stabilization and immobilization of Trypanosoma bruceiornithine decarboxylase (EC 4.1.1.17) (TbODC) for its application in the decarboxylation of L-ornithine, the final step in the envisioned route towards 1,4-diaminobutane. The stability observed for TbODCin vitro was substantially improved upon addition of dithiothreitol (DTT), which not only has a stabilizing, but also an activating effect. For optimal TbODC performance, the pH should be controlled at pH 8 and the ionic strength should be kept to a minimum. The temperature for optimal productivity is 40 °C. Immobilization of TbODC on Sepabeads EC-HFA was most successful, leading to an almost three-fold improvement in operational stability as compared to the soluble enzyme. Overall, we demonstrated that by optimization of reaction conditions and covalent immobilization the productivity of TbODC was vastly improved, opening up possibilities for its application in the biobased production of 1,4-diaminobutane.

Mouse Ornithine Decarboxylase-like Gene Encodes an Antizyme Inhibitor Devoid of Ornithine and Arginine Decarboxylating Activity

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a labile protein that is regulated by interacting with antizymes (AZs), a family of polyamine-induced proteins. Recently, a novel human gene highly homologous to ODC, termed ODC-like or ODC-paralogue (ODCp), was cloned, but the studies aimed to determine its function rendered contradictory results. We have cloned the mouse orthologue of human ODCp and studied its expression and possible function. mRNA of mouse Odcp was found in the brain and testes, showing a conserved expression pattern with regard to the human gene. Transfection of mouse Odcp in HEK 293T cells elicited an increase in ODC activity, but no signs of arginine decarboxylase activity were evident. On the other hand, whereas the ODCp protein was mainly localized in the mitochondrial/membrane fraction, ODC activity was found in the cytosolic fraction and was markedly decreased by small interfering RNA against human ODC. Co-transfection experiments with combinations of Odc, Az1, Az2, Az3, antizyme inhibitor (Azi), and Odcp genes showed that ODCp mimics the action of AZI, rescuing ODC from the effects of AZs and prevented ODC degradation by the proteasome. A direct interaction between ODCp and AZs was detected by immunoprecipitation experiments. We conclude that mouse ODCp has no intrinsic decarboxylase activity, but it acts as a novel antizyme inhibitory protein (AZI2).

N1-Aminopropylagmatine, a New Polyamine Produced as a Key Intermediate in Polyamine Biosynthesis of an Extreme Thermophile, Thermus thermophilus

In the extreme thermophile Thermus thermophilus, a disruption mutant of a gene homologous to speB (coding for agmatinase = agmatine ureohydrolase) accumulated N1-aminopropylagmatine (N8-amidino-1,8-diamino-4-azaoctane, N8-amidinospermidine), a new compound, whereas all other polyamines produced by the wild-type strain were absent from the cells. Double disruption of speB and speE (polyamine aminopropyltransferase) resulted in the disappearance of N1-aminopropylagmatine and the accumulation of agmatine. These results suggested the following. 1) N1-Aminopropylagmatine is produced from agmatine by the action of an enzyme coded by speE. 2) N1-Aminopropylagmatine is a metabolic intermediate in the biosynthesis of unique polyamines found in the thermophile. 3) N1-Aminopropylagmatine is a substrate of the SpeB homolog. They further suggest a new biosynthetic pathway in T. thermophilus, by which polyamines are formed from agmatine via N1-aminopropylagmatine. To confirm our speculation, we purified the expression product of the speB homolog and confirmed that the enzyme hydrolyzes N1-aminopropylagmatine to spermidine but does not act on agmatine.

Enterocyte metabolism during early adaptation after extensive intestinal resection in a rat model

Objective A better knowledge of intestinal adaptation after resection is required to improve the nutritional support that is given to patients. The aim of this study was to understand the metabolic changes underlying early adaptation after massive intestinal resection. Methods Rats were assigned to either 80% intestinal resection or transection. All animals received the same intragastric nutrition. On day 8, plasma glutamine turnover was measured. Substrate use was determined on isolated enterocytes that were incubated in the presence of D-[U- 14C] glucose (2 mmol/L), L-[U- 14C] glutamine (2 mmol/L), L-[U- 14C] arginine (1 mmol/L), or L-[1- 14C] ornithine (1 mmol/L). Results Plasma glutamine turnover was similar in both groups. The rate of enterocyte glutamine use was significantly increased in the resection group, although the maximal glutaminase activity was unchanged. Glutathione generation was enhanced 3-fold in remnant intestine as compared with transected intestine ( P <.05). L-ornithine decarboxylation was increased markedly in resected animals ( P <.05), without any detectable change of maximal ornithine decarboxylase activity. Conclusion The early phase of intestinal adaptation after resection induces changes in enterocyte glutamine and ornithine metabolism that may be related, in part, to increased de novo polyamine synthesis. This observation suggests that a supplementation of artificial nutrition by nutrients that lead to the generation of trophic agents may be of potential interest.

Synthesis of putrescine under possible primitive Earth conditions.

The synthesis of putrescine was accomplished by decarboxylation of L-ornithine when this amino acid was heated in aqueous solution and in the absence of oxygen. Chromatographic, radioisotopic, and enzymatic techniques were used to demonstrate that one mole of non-radioactive putrescine and one mole of 14CO2 was formed during the heating of L-(l-14C)-ornithine. This work indicates that the synthesis of putrescine can occur starting with ornithine and in conditions that are presumed could have existed on the primitive Earth. The possible significance of these results in the prebiotic molecular evolution is briefly discussed.

Putrescine biosynthesis in Tetrahymena thermophila.

The putrescine-biosynthesis pathway in Tetrahymena thermophila was delineated by studying crude extracts prepared from exponentially growing cultures. A pyridoxal phosphate-stimulated ornithine decarboxylase activity competitively inhibited by putrescine was detected. CO2 was also liberated from L-arginine, but analyses by t.l.c. and enzyme studies suggested that the activity was not due to arginine decarboxylase, nor could enzyme activities converting agmatine into putrescine be detected. We conclude that the decarboxylation of L-ornithine is probably the only major route for putrescine biosynthesis in this organism during exponential growth.

Stereochemistry of ornithine decarboxylase reaction.

To determine the steric course of the reaction of bacterial ornithine decarboxylase [EC 4.1.1.17], we have carried out the decarboxylation of L-ornithine in 2H2O and that of DL-[2-2H]ornithine in H2O, and obtained putrescine bearing a single deuterium atom in the C-1 position. The stereochemistry of [1-2H]putrescine was established by conversion to 1-(2-pyrrolidinyl)-2-propanone with acetoacetate and the pro-S hydrogen-specific diamine oxidase from pea seedlings. Analysis of deuterium content by gas chromatography-mass spectrometry showed that the deuterium label was fully retained during the conversion of [1-2H]putrescine produced by the decarboxylation of L-ornithine in 2H2O to 1-(2-pyrrolidinyl)-2-propanone, in contrast with the considerable loss of label from [1-2H]putrescine which was produced by the decarboxylation of DL-[2-2H]ornithine in H2O. The extent of loss of the deuterium label was in good agreement with the estimated value based on the isotope effect in the diamine oxidase reaction. These results indicate that the introduced deuterium (or hydrogen) is in the pro-R position at C-1 of putrescine, and consequently the ornithine decarboxylase reaction proceeds with retention of configuration.

Inhibition of rat embryonic development by the intrauterine administration of α-difluoromethylornithine

Decarboxylation of L-ornithine by L-ornithine decarboxylase (ODC; E.C.4.1.1.17.) is the initial step in the biosynthesis of putrescine. ODC activity is generally low in most tissues, marked increases are associated with rapid tissue growth and particularly with mammalian embryogenesis. 0.5 mg/kg of DL-α-difluoromethylornithine (DFMO) (irreversible inhibitor of ODC) was administered to uterine horns of Long-Evans adult rats during the 4th day of pregnancy. As control material, saline (0.15 M) was administered to contralateral uterine horn. The animals were sacrificed on different days, the uterine horns were removed and the number of implanted embryos were counted. DNA, RNA, protein and dry weight content in implantation sites (5th day of pregnancy) indicated that decidualization following DFMO took place normally but that embryonic growth was arrested in the treated horn. When 100μg of putrescine were added together with DFMO, the embryotoxic effect was absent.

Studies on polyamine biosynthesis in Euglena gracilis

Euglene gracilis (strain Z) was found to contain five polyamines which could be separated by high-pressure cation-exchange chromatography. 1,3-Diaminopropane, putrescine, norspermidine ( N-(3- aminopropyl)-1,3- diaminopropane ), spermidine and norspermine ( N,N′- bis(aminopropyl)-1,3- diaminopropane ) were identified. Biosynthesis of putrescine in E. gracilis proceeds through decarboxylation of l-ornithine, no arginine decarboxylase (EC 4.1.1.19) activity could be detected. The properties of the enzymes ornithine decarboxylase (EC 4.1.1.17) and S- adenosylmethionine decarboxylase (EC 4.1.1.50) in this alga were found to be similar to those of the enzymes isolated from animal tissues or yeast cells. A biosynthetic scheme is proposed which relates the different polyamines occurring in E. gracilis.

Chemically defined media for growing anaerobic bacteria of the genus Veillonella.

A chemically defined medium for Veillonella parvula and V. alcalescens is described. Some nutritional aspects of the two strains used were examined: the optimum concentration of reducing agents, the requirements for amino acids, diamines, vitamins and other growth factors, and the conditions needed for well balanced nutrition. No specific requirements for single amino acids were observed. A combination of L-cysteine, DL-aspartic acid, L-glutamic acid, L-serine and L-tyrosine, promoted growth. In V. alcalescens, serine could substitute both arginine and tryptophan (or histidine). No growth was obtained with ammonium salts as the sole N source. Decarboxylation of L-ornithine, L-lysine and L-arginine was not demonstrated in the Veillonella parvula strain, which required putrescine or cadaverine for growth. Spermine, spermidine, L-lysine, L-ornithine and L-arginine, could not substitute putrescine in Veillonella parvula. Veillonella alcalescens, which does not require putrescine in the medium, was able to decarboxylate L-ornithine while forming putrescine.

Mechanisms and regulation of polyamine and putrescine biosynthesis in male genital glands and other tissues of mammals

The experimental results presented in this paper are largely concerned with enzymes involved in the biosynthesis of putrescine, spermidine, and spermine in the rat ventral prostate gland. The occurrence and possible functions of polyamines in male accessory genital glands and their secretions are first reviewed. Evidence is presented that the decarboxylation of l-ornithine by a specific soluble, pyridoxal phosphate-dependent enzyme is the principal pathway for the biosynthesis of putrescine in the prostate. Biochemical reactions involved in the formation and utilization of l-ornithine in this gland are discussed in relation to the provision of this amino acid for polyamine synthesis. Spermidine is synthesized in the ventral prostate by a soluble, putrescine-activated S-adenosylmethionine decarboxylase system which forms CO 2, spermidine, and thiomethyladenosine in stoichiometric amounts. This enzyme system has been partially purified, and many of its properties are described. Although decarboxylated S-adenosylmethionine (prepared with an enzyme from E. coli) serves well as a propylamine donor for spermidine synthesis by the prostatic enzyme system, the latter could not be fractionated into a distinct S-adenosylmethionine decarboxylase and a separate “propylamine transferase” as previously described in micro-organisms by C. W. Tabor. It is proposed that spermidine synthesis in the prostate gland may not involve free decarboxylated S-adenosylmethionine as an intermediate. Spermine synthesis from spermidine and S-adenosyl-methionine (or its decarboxylated derivative) was demonstrated with soluble prostatic extracts; the mechanism(s) of spermine biosynthesis are discussed. Enzymes degrading putrescine, spermidine, and spermine could not be demonstrated in rat ventral prostate. Large and rapid increases in the activity of both ornithine decarboxylase and the putrescine-dependent S-adenosylmethionine decarboxylating system occur during the early phases of prostatic growth induced by testosterone in orchiectomized rats. The role of polyamines in the androgen-dependent functional differentiation of the prostate is considered.