The arginine dihydrolase pathway is present in Giardia intestinalis

Growth of Giardia intestinalis in Diamond's TYI-S-33 medium is characterized by a rapid depletion of the arginine in the medium, and concurrent production of ornithine and ammonia. [Guanidino-14C] arginine was converted to 14CO2 by extracts of G. intestinalis suggesting the presence of the arginine dihydrolase pathway. This was confirmed by the detection of arginine deiminase, catabolic ornithine transcarbamylase, carbamate kinase and ornithine decarboxylase in giardial extracts. The findings demonstrate for the first time the existence of the arginine dihydrolase pathway in Giardia, and suggest that arginine metabolism via this pathway plays a significant role in energy metabolism by providing a site for anaerobic substrate level phosphorylation.     

Occurrence of Arginine Deiminase Pathway Enzymes in Arginine Catabolism by Wine Lactic Acid Bacteria

l-Arginine, an amino acid found in significant quantities in grape juice and wine, is known to be catabolized by some wine lactic acid bacteria. The correlation between the occurrence of arginine deiminase pathway enzymes and the ability to catabolize arginine was examined in this study. The activities of the three arginine deiminase pathway enzymes, arginine deiminase, ornithine transcarbamylase, and carbamate kinase, were measured in cell extracts of 35 strains of wine lactic acid bacteria. These enzymes were present in all heterofermentative lactobacilli and most leuconostocs but were absent in all the homofermentative lactobacilli and pediococci examined. There was a good correlation among arginine degradation, formation of ammonia and citrulline, and the occurrence of arginine deiminase pathway enzymes. Urea was not detected during arginine degradation, suggesting that the catabolism of arginine did not proceed via the arginase-catalyzed reaction, as has been suggested in some earlier studies. Detection of ammonia with Nessler's reagent was shown to be a simple, rapid test to assess the ability of wine lactic acid bacteria to degrade arginine, although in media containing relatively high concentrations (>0.5%) of fructose, ammonia formation is inhibited.     

Arginine metabolism in lactic streptococci.

Streptococcus lactis metabolizes arginine via the arginine deiminase pathway producing ornithine, ammonia, carbon dioxide, and ATP. In the four strains of S. lactis examined, the specific activities of arginine deiminase and ornithine transcarbamylase were 5- to 10-fold higher in galactose-grown cells compared with glucose- or lactose-grown cells. The addition of arginine increased the specific activities of these two enzymes with all growth sugars. The specific activity of the third enzyme involved in arginine metabolism (carbamate kinase) was not altered by the composition of the growth medium. In continuous cultures arginine deiminase was not induced, and arginine was not metabolized, until glucose limitation occurred. In batch cultures the metabolism of glucose and arginine was sequential, whereas galactose and arginine were metabolized concurrently, and the energy derived from arginine metabolism was efficiently coupled to growth. No arginine deiminase activity was detected in the nine Streptococcus cremoris strains examined, thus accounting for their inability to metabolize arginine. All nine strains of S. cremoris had specific activities of carbamate kinase similar to those found in S. lactis, but only five S. cremoris strains had ornithine transcarbamylase activity.     

Regulation of the arginine dihydrolase pathway in Clostridium sporogenes.

Arginine deiminase activity was induced during the vegetative growth of Clostridium sporogenes. The enzyme was sensitive to catabolite repression. The other enzymes of the arginine dihydrolase pathway, namely, ornithine carbamoyl-transferase and carbamate kinase, did not show such variation.     

Subcellular Localization of the Enzymes of the Arginine Dihydrolase Pathway in Trichomonas vaginalis and Tritrichomonas foetus

The enzymes of the arginine dihydrolase pathway were demonstrated in Tritrichomonas foetus and their subcellular localization determined for both T. foetus and Trichomonas vaginalis. Ornithine carbamyltransferase (anabolic and catabolic activities), ornithine decarboxylase and carbamate kinase activity were localized predominately (56–80%) in the non sedimentable fraction of both species. A large proportion (35–40%) of the arginine deiminase was, however, recovered in the large granular fraction, and this distribution was unchanged by increasing the ionic strength of the buffer. Upon density gradient centrifugation the particles containing arginine deiminase activity had an isopycnic density of 1.09 g/ml in percoll, and separated from hydrogenosomes (1.18 g/ml) and lysosomes (1.12 g/ml). Arginine deiminase was also the only enzyme of the dihydrolase pathway which demonstrated latency upon treatment of the 1.09 g/ml fraction with non-ionic detergents. The results demonstrate the presence of the arginine dihydrolase pathway in T. foetus and indicate that at least a portion of the arginine deiminase in trichomonads is membrane associated.     

Arginine catabolism in wine lactic acid bacteria: is it via the arginine deiminase pathway or the arginase-urease pathway?

The wine lactic acid bacteria Leuconostoc oenos OENO and Lactobacillus buchneri CUC-3 catabolize L-arginine to ornithine and ammonia as major end-products, with 1 mole of arginine converted into 2 moles of ammonia and 1 mole of ornithine. Some citrulline was also excreted into the medium. The excreted citrulline was reassimilated and catabolized by the lactobacillus strain, though not by the leuconostoc. Urea was not detected during arginine degradation. The activities of all three enzymes of the arginine deiminase pathway (arginine deiminase, ornithine transcarbamylase and carbamate kinase) increased significantly over time in the presence of arginine. On the other hand, arginase and urease activities were undetectable in cell extracts of cultures grown in the presence of arginine. The results show that the arginine deiminase pathway, and not the arginase-urease pathway, is the route for arginine degradation in wine lactic acid bacteria.     

Genetic and physiological characterization of Pseudomonas aeruginosa mutants affected in the catabolic ornithine carbamoyltransferase.

In Pseudomonas aeruginosa arginine can be degraded by the arginine "dihydrolase" system, consisting of arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase. Mutants of P. aeruginosa strain PAO affected in the structural gene (arcB) of the catabolic ornithine carbamoyltransferase were isolated. Firt, and argF mutation (i.e., a block in the anabolic ornithine carbamoyltransferase) was suppressed specifically by a mutationally altered catabolic ornithine carbamoyltransferase capable of functioning in the anabolic direction. The suppressor locus arcB (Su) was mapped by transduction between hisII and argA. Second, mutants having lost suppressor activity were obtained. The Su- mutations were very closely linked to arcB (Su) and caused strongly reduced ornithine carbamoyltransferase activities in vitro. Under aerobic conditions, a mutant (PA0630) which had less than 1% of the wild-type catabolic ornithine carbamoyltransferase activity grew on arginine as the only carbon and nitrogen source, at the wild-type growth rate. When oxygen was limiting, strain PA0630 grown on arginine excreted citrulline in the stationary growth phase. These observations suggest that during aerobic growth arginine is not degraded exclusively via the dihydrolase pathway.     

Amino acid utilization by the ruminal bacterium Synergistes jonesii strain 78-1

The ruminal bacterium Synergistes jonesii strain 78-1, which is able to degrade the pyridinediol toxin in the plant Leucaena leucephala, was studied for its ability to utilise amino acids. The organism used arginine, histidine and glycine from a complex mixture of amino acids, and both arginine and histidine supported growth in a semi-defined medium. The products of (U-¹⁴C)-arginine metabolism were CO₂ acetate, butyrate, citrulline and ornithine. The labelling pattern of end products from (U-¹⁴C)-histidine metabolism differed in that carbon also flowed into formate and propionate. Arginine was catabolised by the arginine deiminase pathway which was characterised by the presence of arginine deiminase, ornithine transcarbamylase and carbamate kinase. This is the first report of a rumen bacterium that uses arginine and histidine as major energy yielding substrates.     

葡萄酒苹果酸-乳酸菌精氨酸代谢研究概况

葡萄酒苹果酸-乳酸菌的精氨酸代谢会导致葡萄酒中氨基甲酸乙酯含量的增加,从而严重影响葡萄酒的饮用安全性。近年来研究表明,葡萄酒苹果酸-乳酸菌的精氨酸代谢途径是精氨酸脱亚氨基酶途径(Arginine deiminasepathway,简称ADI途径)。系统分析苹果酸-乳酸菌的ADI途径、精氨酸转运机制、ADI途径酶的调节等方面的研究进展,阐明葡萄酒苹果酸-乳酸菌的精氨酸代谢对酿造优质葡萄酒具有重要的理论和实际意义。     

Arginine catabolism by sourdough lactic acid bacteria: purification and characterization of the arginine deiminase pathway enzymes from Lactobacillus sanfranciscensis CB1

The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as Lactobacillus sanfranciscensis CB1; Lactobacillus brevis AM1, AM8, and 10A; Lactobacillus hilgardii 51B; and Lactobacillus fructivorans DD3 and DA106 showed all three enzyme activities. Lactobacillus plantarum B14 did not show CK activity. L. sanfranciscensis CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37 degrees C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of Lactobacillus sakei. L. sanfranciscensis CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in L. sanfranciscensis CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by L. sanfranciscensis CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by L. sanfranciscensis CB1 enhanced cell growth, cell survival during storage at 7 degrees C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds.     

Arginine metabolism inLactobacillus leichmannii

Lactobacillus leichmannii ATCC 4797 metabolizes arginine via the arginine dihydrolase pathway producing ornithine, ammonia, carbon dioxide, and ATP. The specific activities of arginine deiminase and ornithine transcarbamylase were two-or threefold lower (stationary growth phase) in galactose-grown cells. The addition of arginine increased the specific activities of these two enzymes with all growth sugars. When glucose was virtually exhausted from the medium, maximum activities of both enzymes were achieved. The formation of two first enzymes of the arginine dihydrolase pathway inL. leichmannii ATCC 4797 seems to be under the control of two processes: induction by arginine and repression of the induced synthesis by glucose.Dedicated to Dr. Luis F. Leloir on the occasion of his 80th birthday, 6 September 1986.     

Synthesis of the arginine dihydrolase pathway enzymes inLactobacillus buchneri

The formation of the arginine dihydrolase pathway enzymes inLactobacillus buchneri NCDO₁₁₀, a heterofermentative organism, was investigated. The specific activities of arginine deiminase, ornithine transcarbamylase, and carbamate kinase were higher in galactose-grown cells than in glucose- or sucrose-grown cells in the early stationary phase of growth. The addition of arginine to growing cells increased the specific activity of these three enzymes with all growth sugars. The specific activities of the enzymes decreased during the stationary phase of growth when the sugar-grown cells was galactose. When glucose was virtually exhausted from the medium, the activities of the three enzymes were not altered. This enzymic system was not repressed by glucose, and these results are different from those obtained withL. leichmanni, homofermentative organism.Dedicated to Dr. Luis F. Leloir on the occasion of his 80th birthday, 6 September 1986.Member of the Scientific Researcher's Career of theConsejo Nacional de Investigaciones Cientificas Ténicas (CONICET) Argentina.     

The arginine deiminase pathway in the wine lactic acid bacterium Lactobacillus hilgardii X1B: structural and functional study of the arcABC genes

The genes implicated in the catabolism of the amino acid arginine by Lactobacillus hilgardii X(1)B were investigated to assess the potential for formation of ethyl carbamate precursors in wine. L. hilgardii X(1)B can use arginine via the arginine deiminase pathway. The complete nucleotide sequence of the arc genes involved in this pathway has been determined. They are clustered in an operon-like structure in the order arcABC. No evidence was found for the presence of a homologue of the arcD gene, coding for the arginine/ornithine antiporter. The arc genes have been expressed in Escherichia coli resulting in arginine deiminase (ArcA), ornithine carbamoyltransfera (ArcB) and carbamate kinase (ArcC) activities. The results indicate the need for caution in the selection of lactic acid bacteria for conducting malolactic fermentation in wine since arginine degradation could result in high amounts of ethyl carbamate.     

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.     

Role of the arginine deiminase system in protecting oral bacteria and an enzymatic basis for acid tolerance

The arginine deiminase system was found to function in protecting bacterial cells against the damaging effects of acid environments. For example, as little as 2.9 mM arginine added to acidified suspensions of Streptococcus sanguis at a pH of 4.0 resulted in ammonia production and protection against killing. The arginine deiminase system was found to have unusual acid tolerance in a variety of lactic acid bacteria. For example, for Streptococcus rattus FA-1, the pH at which arginolysis was reduced to 10% of the maximum was between 2.1 and 2.6, or more than 1 full pH unit below the minimum for glycolysis (pH 3.7), and more than 2 units below the minimum for growth in complex medium (pH 4.7). The acid tolerance of the arginine deiminase system appeared to be primarily molecular and to depend on the tolerance of individual enzymes rather than on the membrane physiology of the bacteria; pH profiles for the activities of arginine deiminase, ornithine carbamoyltransferase, and carbamate kinase in permeabilized cells showed that the enzymes were active at pHs of 3.1 or somewhat lower. Overall, it appeared that ammonia could be produced from arginine at low pH values, even by cells with damaged membranes, and that the ammonia could then protect the cells against acid damage until the environmental pH value rose sufficiently to allow for the reestablishment of a difference in pH (delta pH) across the cell membrane.     

Role of the arginine deiminase system in protecting oral bacteria and an enzymatic basis for acid tolerance.

The arginine deiminase system was found to function in protecting bacterial cells against the damaging effects of acid environments. For example, as little as 2.9 mM arginine added to acidified suspensions of Streptococcus sanguis at a pH of 4.0 resulted in ammonia production and protection against killing. The arginine deiminase system was found to have unusual acid tolerance in a variety of lactic acid bacteria. For example, for Streptococcus rattus FA-1, the pH at which arginolysis was reduced to 10% of the maximum was between 2.1 and 2.6, or more than 1 full pH unit below the minimum for glycolysis (pH 3.7), and more than 2 units below the minimum for growth in complex medium (pH 4.7). The acid tolerance of the arginine deiminase system appeared to be primarily molecular and to depend on the tolerance of individual enzymes rather than on the membrane physiology of the bacteria; pH profiles for the activities of arginine deiminase, ornithine carbamoyltransferase, and carbamate kinase in permeabilized cells showed that the enzymes were active at pHs of 3.1 or somewhat lower. Overall, it appeared that ammonia could be produced from arginine at low pH values, even by cells with damaged membranes, and that the ammonia could then protect the cells against acid damage until the environmental pH value rose sufficiently to allow for the reestablishment of a difference in pH (delta pH) across the cell membrane.     

Arginine metabolism in wineLactobacillus plantarum: in vitro activities of the enzymes arginine deiminase (ADI) and ornithine transcarbamilase (OTCase)

This work was carried out to determine the activity of enzymes involved in arginine metabolism inLactobacillus plantarum isolated from wine and previously characterised at molecular level. The activity of the enzymes arginine deiminase and ornithine transcarbamylase was determined and citrulline and ornithine formed were analysed by HPLC analysis. Although the enzymatic activity was detected in all the strains analysed, a strong variability was observed between strains.Lactobacillus plantrum strain Lp60 is the strain with more possibilities to accumulate citrulline, precursor of the carcinogenic ethyl-carbamate, as showed by its high arginine deiminase activity and low ornithine transcarbamylase activity.     

Expression analysis of putative arcA, arcB and arcC genes partially cloned from Lactobacillus plantarum isolated from wine

AIMS: The aim of this paper was to study if homofermentative strains (Lacobacillus plantarum) capable of malolactic fermentation in wine can degrade arginine via the ADI pathway. METHODS AND RESULTS: Homofermentative lactic acid bacteria (LAB) isolated from a typical red wine were investigated for their ability to produce citrulline. Citrulline was formed suggesting that the arginine metabolism takes place via the arginine deiminase (ADI) pathway and not via the arginase/urease pathway. Ammonia was also detected with Nessler's reagent, and all the strains examined were able to produce ammonia. Identification of homofermentative LAB was performed using 16S ribosomal sequence analysis. The strains were further classified as belonging to L. plantarum species. Furthermore, the genes encoding for the three pathway enzymes (ADI, ornithine transcarbamylase, carbamate kinase) were partially cloned and gene expression was performed at two different pH values (3.6 and 4.5). CONCLUSIONS: The results suggest that citrulline production in wine, could be performed by homofermentative LAB. SIGNIFICANCE AND IMPACT OF THE STUDY: Homofermentative malolactic bacteria (L. plantarum) may degrade arginine through the ADI pathway.     

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.