Agmatine deiminase from cucumber seedlings is a mono-specific enzyme: purification and characteristics

Agmatine deiminase was purified to homogeneity from cucumber seedlings. The purification procedures included treatment with DE52, ammonium sulfate precipitation, DE52 column chromatography, Superdex 200 column chromatography, and agmatine-(CNBr)-diaminohexane-CNBr-activated-Sepharose 4B column chromatography. The purified agmatine deiminase exhibited a specific activity of 242nkat/mg protein at 30 degrees C, pH 7.0, with a yield of 33%. The molecular mass of the native enzyme was 67kDa, as estimated by Superdex 200 column chromatography. On the other hand, SDS-PAGE showed that the molecular masses of the subunits with 1% SDS and 5% of 2-mercaptoethanol treatment and with additional N-glycosidase F treatment were 47 and 36kDa, respectively. These results suggest that agmatine deiminase from cucumber is a glycoprotein. The Km of the enzyme for agmatine was 16microM and arcaine was a potent competitive inhibitor of the enzyme, with a Ki of 7.1microM. The enzyme was stable for 2 months at 4 degrees C. The enzyme does not have putrescine synthase activity or the activities of its components ornithine and putrescine transcarbamylase. The characteristics of the enzyme purified from cucumber were like those of the enzyme from maize. These results indicate that agmatine deiminase is distinctly different from putrescine synthase in higher plants.     

Enzymes of agmatine degradation and the control of their synthesis in Streptococcus faecalis.

Streptococcus faecalis ATCC 11700 uses agmatine as its sole energy source for growth. Agmatine deiminase and putrescine carbamoyltransferase are coinduced by growth on agmatine. Glucose and arginine were found to exert catabolite repression on the agmatine deiminase pathway. Four mutants unable to utilize agmatine as an energy source, isolated from the wild-type strain, exhibited three distinct phenotypes. Two of these strains showed essentially no agmatine deiminase, one mutant showed negligible activity of putrescine carbamoyltransferase, and one mutant was defective in both activities. Two carbamate kinases are present in S. faecalis, one belonging to the arginine deiminase pathway, the other being induced by growth on agmatine. These two enzymes have the same molecular weight, 82,000, and seem quite different in size from the kinases isolated from other streptococci.     

Transport of diamines by Enterococcus faecalis is mediated by an agmatine-putrescine antiporter.

Enterococcus faecalis ATCC 11700 is able to use arginine and the diamine agmatine as a sole energy source. Via the highly homologous deiminase pathways, arginine and agmatine are converted into CO2, NH3, and the end products ornithine and putrescine, respectively. In the arginine deiminase pathway, uptake of arginine and excretion of ornithine are mediated by an arginine-ornithine antiport system. The translocation of agmatine was studied in whole cells grown in the presence of arginine, agmatine, or glucose. Rapid uncoupler-insensitive uptake of agmatine was observed only in agmatine-grown cells. A high intracellular putrescine pool was maintained by these cells, and this pool was rapidly released by external putrescine or agmatine but not by arginine or ornithine. Kinetic analysis revealed competitive inhibition for uptake between putrescine and agmatine. Agmatine uptake by membrane vesicles was observed only when the membrane vesicles were preloaded with putrescine. Uptake of agmatine was driven by the outwardly directed putrescine concentration gradient, which is continuously sustained by the metabolic process. Uptake of agmatine and extrusion of putrescine by agmatine-grown cells of E. faecalis appeared to be catalyzed by an agmatine-putrescine antiporter. This transport system functionally resembled the previously described arginine-ornithine antiport, which was exclusively induced when the cells were grown in the presence of arginine.     

Factors affecting the production of putrescine from agmatine by Lactobacillus hilgardii XB isolated from wine

Aims:  To elucidate and characterize the metabolic putrescine synthesis pathway from agmatine by Lactobacillus hilgardii X₁B.
Methods and Results:  The putrescine formation from agmatine by resting cells (the normal physiological state in wine) of lactic acid bacteria isolated from wine has been determined for the first time. Agmatine deiminase and N -carbamoylputrescine hydrolase enzymes, determined by HPLC and LC-Ion Trap Mass Spectrometry, carried out the putrescine synthesis from agmatine. The influence of pH, temperature, organic acids, amino acids, sugars and ethanol on the putrescine formation in wine was determined.
Conclusions:  Resting cells of Lact. hilgardii X₁B produce putrescine in wine. The putrescine production was carried out from agmatine through the agmatine deiminase system.
Significance and Impact of the Study:  These results have significance from two points of view, wine quality and toxicological and microbiological aspects, taking account that putrescine, which origin is still controversial, is quantitatively the main biogenic amine found in wine.     

Occurrence of agmatine pathway for putrescine synthesis in Selenomonas ruminatium

Selenomonas ruminantium synthesizes cadaverine and putrescine from L-lysine and L-ornithine as the essential constituents of its peptidoglycan by a constitutive lysine/ornithine decarboxylase (LDC/ODC). S. ruminantium grew normally in the presence of the specific inhibitor for LDC/ODC, DL-alpha-difluoromethylornithine, when arginine was supplied in the medium. In this study, we discovered the presence of arginine decarboxylase (ADC), the key enzyme in agmatine pathway for putrescine synthesis, in S. ruminantium. We purified and characterized ADC and cloned its gene (adc) from S. ruminantium chromosomal DNA. ADC showed more than 60% identity with those of LDC/ODC/ADCs from Gram-positive bacteria, but no similarity to that from Gram-negative bacteria. In this study, we also cloned the aguA and aguB genes, encoding agmatine deiminase (AguA) and N-carbamoyl-putrescine amidohydrolase (AguB), both of which are involved in conversion from agmatine into putrescine. AguA and AguB were expressed in S. ruminantium. Hence, we concluded that S. ruminantium has both ornithine and agmatine pathways for the synthesis of putrescine.     

Polyamine synthesis in plants: isolation and characterization of spermidine synthase from soybean (Glycine max) axes

Spermidine synthase (EC 2.5.1.16) was purified to homogeneity for the cytosol of soybean (Glycine max) axes using ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephacryl S-300, omega-aminooctyl-Sepharose and ATPA-Sepharose. The molecular mass of the enzyme estimated by gel filtration and SDS-PAGE is 74 kDa. Cadaverin and 1,6-diaminohexane could not replace putrescine as the aminopropyl acceptor. Kinetic behaviors of the substrate are consistent with a ping pong mechanism. The kinetic mechanism is further supported by direct evidence confirming the presence of an aminopropylated enzyme and identification of product, 5'-deoxy-5'-methylthioadenosine, prior to adding putrescine. The Km values for decarboxylated S-adenosylmethionine and putrescine are 0.43 microM and 32.45 microM, respectively. Optimum pH and temperature for the enzyme reaction are 8.5 and 37 degrees C, respectively. The enzyme activity is inhibited by N-ethylmaleimide and DTNB, but stimulated by Co2+, Cu2+ and Ca2+ significantly, suggesting that these metal ions could be the cellular regulators in polyamine biosynthesis.     

The gene cluster for agmatine catabolism of Enterococcus faecalis: study of recombinant putrescine transcarbamylase and agmatine deiminase and a snapshot of agmatine deiminase catalyzing its reaction

Enterococcus faecalis makes ATP from agmatine in three steps catalyzed by agmatine deiminase (AgDI), putrescine transcarbamylase (PTC), and carbamate kinase (CK). An antiporter exchanges putrescine for agmatine. We have cloned the E. faecalis ef0732 and ef0734 genes of the reported gene cluster for agmatine catabolism, overexpressed them in Escherichia coli, purified the products, characterized them functionally as PTC and AgDI, and crystallized and X-ray diffracted them. The 1.65-Angstroms-resolution structure of AgDI forming a covalent adduct with an agmatine-derived amidine reactional intermediate is described. We provide definitive identification of the gene cluster for agmatine catabolism and confirm that ornithine is a genuine but poor PTC substrate, suggesting that PTC (found here to be trimeric) evolved from ornithine transcarbamylase. N-(Phosphonoacetyl)-putrescine was prepared and shown to strongly (K(i) = 10 nM) and selectively inhibit PTC and to improve PTC crystallization. We find that E. faecalis AgDI, which is committed to ATP generation, closely resembles the AgDIs involved in making polyamines, suggesting the recruitment of a polyamine-synthesizing AgDI into the AgDI pathway. The arginine deiminase (ADI) pathway of arginine catabolism probably supplied the genes for PTC and CK but not those for the agmatine/putrescine antiporter, and thus the AgDI and ADI pathways are not related by a single "en bloc" duplication event. The AgDI crystal structure reveals a tetramer with a five-blade propeller subunit fold, proves that AgDI closely resembles ADI despite a lack of sequence identity, and explains substrate affinity, selectivity, and Cys357-mediated-covalent catalysis. A three-tongued agmatine-triggered gating opens or blocks access to the active center.     

Biogenic amine production by Lactobacillus

AIMS: The aim of this work was to demonstrate that strains of Lactobacillus may be able to produce putrescine and agmatine from one of the major amino acids present in fruit juices and wine, arginine, and from amino acid-derived ornithine. METHODS AND RESULTS: Biogenic amines were determined by HPLC. Their production in the culture medium was similar under both microaerophilic and anaerobic conditions. The presence of Mn2+ had a minimal influence on the results, whereas the addition of pyridoxal phosphate increased amine production 10-fold. Lactobacillus hilgardii X1B, isolated from wine, was able to degrade arginine by two pathways: arginine deiminase and arginine decarboxylase. The isolate was able to produce putrescine from ornithine and from agmatine. Lactobacillus plantarum strains N4 and N8, isolated from orange, utilized arginine via the arginine deiminase system. Only the N4 strain was able to produce putrescine from ornithine. CONCLUSION: It has been demonstrated that Lact. hilgardii X1B is able to produce the most important biogenic amine found in wine, putrescine, and also agmatine from arginine and ornithine, and that Lactobacillus plantarum, considered to be an innocuous spoilage micro-organism in fruit juices, is able to produce amines. SIGNIFICANCE AND IMPACT OF THE STUDY: The results have significance in relation to food poisoning caused by beverages that have been contaminated with biogenic amines.     

Inhibition of L-arginine iminohydrolase (EC 3.5.3.6) from Tetrahymena thermophila by putrescine and spermidine: feedback control of polyamine biosynthesis

L-Arginine iminohydrolase (arginine deiminase, ADI) from Tetrahymena thermophila was purified approx. 75-fold by means of gel permeation chromatography. The Km of the purified enzyme for L-arginine was 412 +/- 25 microM and L-ornithine inhibited the reaction competitively with a Ki of 985 +/- 105 microM. D-Ornithine was a weak inhibitor with a Ki of greater than 10mM. The polyamines putrescine and spermidine inhibited ADI incompetitively with a Kii of 2.8mM for putrescine and 4.3mM for spermidine. Since the concentrations required for inhibition were within the range of the normal intracellular polyamine concentrations in Tetrahymena (maximally 14mM putrescine and 4mM spermidine), it is suggested that the polyamine effects on ADI are of regulatory nature. Thus, polyamine biosynthesis in Tetrahymena thermophila is regulated not only on the level of ornithine decarboxylase activity, but also on an earlier step, the supply of ODC with substrates.     

Fermentation of Agmatine in Streptococcus faecalis: Occurrence of Putrescine Transcarbamoylase

Putrescine transcarbamoylase, EC 2.1.3.x (carbamoylphosphate:putrescine transcarbamoylase), has been purified from Streptococcus faecalis 10C1 grown on agmatine as primary energy source. The formation of N-carbamoylputrescine from putrescine and carbamoylphosphate serves as a convenient and sensitive assay for this enzymatic activity. The enzyme catalyzes both the phosphorolysis arsenolysis of N-carbamoylputrescine. Arginine does not induce the synthesis of putrescine transcarbamoylase in S. faecalis. Furthermore, the putrescine transcarbamoylase activity is easily separated from ornithine transcarbamoylase activity by gel filtration on Sephadex G-100 indicating that the two activities are associated with different proteins. The significance of this new enzyme in the fermentation of agmatine and its relation to the other known transcarbamoylases are discussed.     

Characterization and inactivation of an agmatine deiminase from Helicobacter pylori

Helicobacter pylori encodes a potential virulence factor, agmatine deiminase (HpAgD), which catalyzes the conversion of agmatine to N-carbamoyl putrescine (NCP) and ammonia - agmatine is decarboxylated arginine. Agmatine is an endogenous human cell signaling molecule that triggers the innate immune response in humans. Unlike H. pylori, humans do not encode an AgD; it is hypothesized that inhibition of this enzyme would increase the levels of agmatine, and thereby enhance the innate immune response. Taken together, these facts suggest that HpAgD is a potential drug target. Herein we describe the optimized expression, isolation, and purification of HpAgD (10-30 mg/L media). The initial kinetic characterization of this enzyme has also been performed. Additionally, the crystal structure of wild-type HpAgD has been determined at 2.1 Å resolution. This structure provides a molecular basis for the preferential deimination of agmatine, and identifies Asp198 as a key residue responsible for agmatine recognition, which has been confirmed experimentally. Information gathered from these studies led to the development and characterization of a novel class of haloacetamidine-based HpAgD inactivators. These compounds are the most potent AgD inhibitors ever described.     

Mechanistic studies of agmatine deiminase from multiple bacterial species

One subfamily of guanidino group-modifying enzymes (GMEs) consists of the agmatine deiminases (AgDs). These enzymes catalyze the conversion of agmatine (decarboxylated arginine) to N-carbamoyl putrescine and ammonia. In plants, viruses, and bacteria, these enzymes are thought to be involved in energy production, biosynthesis of polyamines, and biofilm formation. In particular, we are interested in the role that this enzyme plays in pathogenic bacteria. Previously, we reported the initial kinetic characterization of the agmatine deiminase from Helicobacter pylori and described the synthesis and characterization the two most potent AgD inactivators. Herein, we have expanded our initial efforts to characterize the catalytic mechanisms of AgD from H. pylori as well as Streptococcus mutans and Porphyromonas gingivalis. Through the use of pH rate profiles, pK(a) measurements of the active site cysteine, solvent isotope effects, and solvent viscosity effects, we have determined that the AgDs, like PADs 1 and 4, utilize a reverse protonation mechanism.     

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.     

The diverse bacterial origins of the Arabidopsis polyamine biosynthetic pathway

We functionally identified the last remaining step in the plant polyamine biosynthetic pathway by expressing an Arabidopsis thaliana agmatine iminohydrolase cDNA in yeast. Inspection of the whole pathway suggests that the arginine decarboxylase, agmatine iminohydrolase, N-carbamoylputrescine amidohydrolase route to putrescine in plants was inherited from the cyanobacterial ancestor of the chloroplast. However, the rest of the pathway including ornithine decarboxylase and spermidine synthase was probably inherited from bacterial genes present in the original host cell, common ancestor of plants and animals, that acquired the cyanobacterial endosymbiont. An exception is S-adenosylmethionine decarboxylase, which may represent a eukaryote-specific enzyme form.     

Purification and Characterization of Arginine Decarboxylase from Soybean (Glycine max) Hypocotyls

Arginine decarboxylase (EC 4.1.1.19 [EC] ) was purified from soybean,Glycine max, hypocotyls by a procedure which includes ammoniumsulfate fractionation, acetone precipitation, gel filtrationchromatography, and affinity chromatography. Using this procedure,ADC was purified to one band in non-denaturing PAGE. The purifiedADC has an M_r of 240 kDa based on gel filtration chromatographyand is a trimer of identical subunits which has an estimatedM_r of 74 kDa based on SDS-PAGE. ADC is active between 30 and50°C and has a K_m value of 46.1 µM. ADC is very sensitiveto agmatine or putrescine but not to spermidine or spermine.In the presence of 0.5 mM agmatine (or putrescine), the enzymeactivity was inhibited by 70%. However, at the same concentrationof spermidine (or spermine), the enzyme activity was inhibitedby only 10–20%. (Received April 2, 1997; Accepted August 18, 1997)