Decarboxylation of arginine and ornithine by arginine decarboxylase purified from cucumber (Cucumis sativus) seedlings

A purified preparation of arginine decarboxylase fromCucumis sativus seedlings displayed ornithine decarboxylase activity as well. The two decarboxylase activities associated with the single protein responded differentially to agmatine, putrescine andPi. While agmatine was inhibitory (50 %) to arginine decarboxylase activity, ornithine decarboxylase activity was stimulated by about 3-fold by the guanido arnine. Agmatine-stimulation of ornithine decarboxylase activity was only observed at higher concentrations of the amine. Inorganic phosphate enhanced arginine decarboxylase activity (2-fold) but ornithine decarboxylase activity was largely uninfluenced. Although both arginine and ornithine decarboxylase activities were inhibited by putrescine, ornithine decarboxylase activity was profoundly curtailed even at 1 mM concentration of the diamine. The enzyme-activated irreversible inhibitor for mammalian ornithine decarboxylase,viz. α-difluoromethyl ornithine, dramatically enhanced arginine decarboxylase activity (3–4 fold), whereas ornithine decarboxylase activity was partially (50%) inhibited by this inhibitor. At substrate level concentrations, the decarboxylation of arginine was not influenced by ornithine andvice-versa. Preliminary evidence for the existence of a specific inhibitor of ornithine decarboxylase activity in the crude extracts of the plant is presented. The above results suggest that these two amino acids could be decarboxylated at two different catalytic sites on a single protein.     

Putrescine Accumulation in Wine: Role of Oenococcus oeni

Putrescine, the most abundant biogenic amine in wine, was proved to be produced by Oenococcus oeni strains in wine not only from ornithine but also from arginine. In this case, putrescine may originate from strains possessing the complete enzyme system to convert arginine to putrescine or by a metabiotic association, with an exchange of ornithine, between strains capable of metabolizing arginine to ornithine but unable to produce putrescine and strains capable of producing putrescine from ornithine but unable to degrade arginine. Putrescine production by this metabiotic association occurred once the malolactic fermentation was completed, whereas conversion of ornithine to putrescine by a single culture of the ornithine decarboxylating strain concurred with the degradation of malic acid. Moreover, in the former case, putrescine formation proceeded more slowly than in the latter. Metabiosis may play an important role in the accumulation of putrescine in wine, arginine being one of the major amino acids found in wine.     

Activities of arginine and ornithine decarboxylases in various plant species

In extracts from the youngest leaves of Avena sativa, Hordeum vulgare, Zea Mays, Pisum sativum, Phaseolus vulgaris, Lactuca sativa, and four pyrrolizidine alkaloid-bearing species of Heliotropium, the activities of ornithine decarboxylase, close to V_max, ranged between traces and 1.5 nanomoles per hour per gram fresh weight when based on putrescine formed during incubation with labeled ornithine. The arginine decarboxylase activities in the same extracts ranged between 8 and 8000 nanomoles per hour per gram fresh weight being lowest in the borages and highest in oat and barley. α-Difluoromethylornithine and α-difluoromethylarginine inhibited ornithine and arginine decarboxylases, respectively, in all species. Agmatine, putrescine, spermidine, and spermine were found in all, diaminopropane in eight, and cadaverine in three species.

No correlation was observed between arginine or ornithine decarboxylase level and the levels of total polyamines. The in vitro decarboxylase activities found in the borages cannot explain the high accumulation of putrescine-derived pyrrolizidines in their youngest leaves if the pyrrolizidines are produced in situ from arginine and/or ornithine as precursors; other possibilities are discussed.

In assays of ornithine decarboxylase, an interference of decarboxylation not due to this enzyme was observed in extracts from all species. In arginine decarboxylase assays, the interfering decarboxylation as well as the interference of arginase were apparent in two species. Addition of aminoguanidine was needed to suppress oxidative degradation of putrescine and agmatine during incubation of extracts from pea, bean, lettuce, Heliotropium angiospermum, and Heliotropium indicum.

     

ADI pathway and histidine decarboxylation are reciprocally regulated in Lactobacillus hilgardii ISE 5211: proteomic evidence

Amine production by amino acid decarboxylation is a common feature that is used by lactic acid bacteria (LAB) to complement lactic fermentation, since it is coupled with a proton-extruding antiport system which leads to both metabolic energy production and the attenuation of intracellular acidity. Analogous roles are played in LAB by both malolactic fermentation (MLF) and the arginine deiminase (ADI) pathway. The present investigation was aimed at establishing reciprocal interactions between amino acid decarboxylation and the two above mentioned routes. The analyses were carried out on a Lactobacillus hilgardii strain (ISE 5211) that is able to decarboxylate histidine to histamine, which had previously been isolated from wine and whose complete genome is still unknown. The 2DE proteomic approach, followed by MALDI TOF–TOF and De Novo Sequencing, was used to study the protein expression levels. The experimental evidence has indicated that malate does not influence histidine decarboxylase (HDC) biosynthesis and that histidine does not affect the malolactic enzyme level. However, the expression of the ADI route enzymes, arginine deiminase and ornithine transcarbamylase, is down-regulated by histidine: this biosynthetic repression is more important (4-fold) in cultures that are not supplemented with arginine, but is also significant (2-fold) in an arginine supplemented medium that normally induces the ADI pathway. On the other hand, arginine partially represses HDC expression, but only when histidine and arginine are both present in the culture medium. This proteomic study has also pointed out a down-regulation exerted by histidine over sugar metabolism enzymes and a GroEL stress protein. These data, together with the reciprocal antagonism between arginine deimination and histidine decarboxylation, offer clue keys to the understanding of the accumulation of lactate, amine, ammonia and ethylcarbamate in wine, with consequent implications on different health risk controls.     

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.     

Evidence of two functionally distinct ornithine decarboxylation systems in lactic acid bacteria

Biogenic amines are low-molecular-weight organic bases whose presence in food can result in health problems. The biosynthesis of biogenic amines in fermented foods mostly proceeds through amino acid decarboxylation carried out by lactic acid bacteria (LAB), but not all systems leading to biogenic amine production by LAB have been thoroughly characterized. Here, putative ornithine decarboxylation pathways consisting of a putative ornithine decarboxylase and an amino acid transporter were identified in LAB by strain collection screening and database searches. The decarboxylases were produced in heterologous hosts and purified and characterized in vitro, whereas transporters were heterologously expressed in Lactococcus lactis and functionally characterized in vivo. Amino acid decarboxylation by whole cells of the original hosts was determined as well. We concluded that two distinct types of ornithine decarboxylation systems exist in LAB. One is composed of an ornithine decarboxylase coupled to an ornithine/putrescine transmembrane exchanger. Their combined activities results in the extracellular release of putrescine. This typical amino acid decarboxylation system is present in only a few LAB strains and may contribute to metabolic energy production and/or pH homeostasis. The second system is widespread among LAB. It is composed of a decarboxylase active on ornithine and l-2,4-diaminobutyric acid (DABA) and a transporter that mediates unidirectional transport of ornithine into the cytoplasm. Diamines that result from this second system are retained within the cytosol.     

Regulation of arginine-ornithine exchange and the arginine deiminase pathway in Streptococcus lactis.

Streptococcus lactis metabolizes arginine by the arginine deiminase (ADI) pathway. Resting cells of S. lactis grown in the presence of galactose and arginine maintain a high intracellular ornithine pool in the absence of arginine and other exogenous energy sources. Addition of arginine results in a rapid release of ornithine concomitant with the uptake of arginine. Subsequent arginine metabolism results intracellularly in high citrulline and low ornithine pools. Arginine-ornithine exchange was shown to occur in a 1-to-1 ratio and to be independent of a proton motive force. The driving force for arginine uptake in intact cells is supplied by the ornithine and arginine concentration gradients formed during arginine metabolism. These results confirm studies of arginine and ornithine transport in membrane vesicles of S. lactis (A. J. M. Driessen, B. Poolman, R. Kiewiet, and W. N. Konings, Proc. Natl. Acad. Sci. USA, 84:6093-6097). The activity of the ADI pathway appears to be affected by the internal concentration of (adenine) nucleotides. Conditions which lower ATP consumption (dicyclohexylcarbodiimide, high pH) decrease the ADI pathway activity, whereas uncouplers and ionophores which stimulate ATP consumption increase the activity. The arginine-ornithine exchange activity matches the ADI pathway most probably by adjusting the intracellular levels of ornithine and arginine. Regulation of the ADI pathway and the arginine-ornithine exchanger at the level of enzyme synthesis is exerted by glucose (repressor, antagonized by cyclic AMP) and arginine (inducer). An arginine/ornithine antiport was also found in Streptococcus faecalis DS5, Streptococcus sanguis 12, and Streptococcus milleri RH1 type 2.     

嗜盐四联球菌arc operon多拷贝基因在精氨酸代谢中的作用

【背景】嗜盐四联球菌(Tetragenococcus halophilus)是一类存在于发酵食品中的耐盐乳酸菌,研究其精氨酸(arginine,Arg)代谢对解析食品发酵过程中氨基甲酸乙酯(ethyl carbamate,EC)前体积累机制和保障食品安全具有重要意义。【目的】研究酱醪来源嗜盐四联球菌精氨酸脱亚氨基(arginine deiminase,ADI)途径的基因构成,揭示这些基因对菌株精氨酸代谢和氨基甲酸乙酯前体瓜氨酸(citrulline,Cit)利用与积累的影响。【方法】采用PCR扩增与测序分析不同菌株的ADI途径基因组成,通过比较ADI途径关键基因转录水平和关键酶活性,探究环境因素对嗜盐四联球菌代谢氨基酸能力的影响及各拷贝基因参与氨基酸代谢的功能。【结果】酱醪来源嗜盐四联球菌基因组中ADI途径基因类型主要有两大类：以菌株R23为代表含有完整arc操纵子(operon)基因且具有最多基因拷贝数;以菌株C3为代表缺失arcA和arcB但含有多拷贝arcB和arcC。基因组中有arcA的菌株才具有利用精氨酸能力,并通过利用精氨酸生成瓜氨酸。体系中精氨酸含量和乙醇与脂肪酸的存在均可影响嗜盐四联球菌利用精氨酸积累中间产物瓜氨酸。当精氨酸含量大于5 g/L或体系中含有乙醇与脂肪酸时,嗜盐四联球菌会利用精氨酸积累中间产物瓜氨酸。脂肪酸和乙醇对ADI途径的3个关键酶均有显著抑制作用,可使精氨酸脱亚氨基酶(arginine deiminase,ADI)、鸟氨酸氨甲酰基转移酶(ornithine transcarbamylase,OTC)和氨甲酰磷酸激酶(carbamate kinase,CK)的活性分别降低41.0%、46.4%和60.0%。嗜盐四联球菌中arcB转录水平分别是其拷贝arcB₁和arcB₂的10.5倍和29.8倍,arcC的转录水平分别是arcC₁、arcC₂、arcC₃的17.6、20.3、23.9倍,说明arcB和arcC在瓜氨酸代谢中起主要作用。【结论】精氨酸含量和乙醇加脂肪酸是影响嗜盐四联球菌代谢精氨酸能否积累瓜氨酸的关键环境因素。嗜盐四联球菌arc operon的多拷贝基因中,arcB和arcC基因在瓜氨酸代谢中起主要作用。     

MINIATURIZED ANALYTICAL METHOD FOR THE DETECTION OF AMINE PRODUCTION BY MICROORGANISMS

Histamine, the result of histidine decarboxylation, has been associated with allergic reactions due to the consumption of certain foods. Other biogenic amines, such as putrescine and cadaverine, have been related to quality deterioration in foods. A quantitative miniaturized method for the detection of biogenic amines produced by microorganisms in culture media, was designed. The reaction takes place in microplates containing microquantities of inoculated media and reagents. Amine production is determined spectrophotometrically by monitoring changes in the acid phase of the pH indicator at 405 nm. Using the following amino acids: histidine, phenylalanine, tyrosine, tryptophan, arginine, lysine and ornithine, 44 microorganisms were tested for amine production. Sensitivity of the method is 10 μM of amine.     

Dual Role for the Tyrosine Decarboxylation Pathway in Enterococcus faecium E17: Response to an Acid Challenge and Generation of a Proton Motive Force

In this work we investigated the role of the tyrosine decarboxylation pathway in the response of Enterococcus faecium E17 cells to an acid challenge. It was found that 91% of the cells were able to remain viable in the presence of tyrosine when they were incubated for 3 h in a complex medium at pH 2.5. This effect was shown to be related to the tyrosine decarboxylation pathway. Therefore, the role of tyrosine decarboxylation in pH homeostasis was studied. The membrane potential and pH gradient, the parameters that compose the proton motive force (PMF), were measured at different pHs (pH 4.5 to 7). We obtained evidence showing that the tyrosine decarboxylation pathway generates a PMF composed of a pH gradient formed due to proton consumption in the decarboxylation reaction and by a membrane potential which results from electrogenic transport of tyrosine in exchange for the corresponding biogenic amine tyramine. The properties of the tyrosine transporter were also studied in this work by using whole cells and right-side-out vesicles. The results showed that the transporter catalyzes homologous tyrosine/tyrosine antiport, as well as electrogenic heterologous tyrosine-tyramine exchange. The tyrosine transporter had properties of a typical precursor-product exchanger operating in a proton motive decarboxylation pathway. Therefore, the tyrosine decarboxylation pathway contributes to an acid response mechanism in E. faecium E17. This decarboxylation pathway gives the strain a competitive advantage in nutrient-depleted conditions, as well as in harsh acidic environments, and a better chance of survival, which contributes to higher cell counts in food fermentation products.     

Lactic acid bacteria isolated from apples are able to catabolise arginine

We investigated the potentiality of lactic acid bacteria (LAB) isolated from two apples variety to utilize arginine at different initial pH values. Apples surface contained average levels of bacteria ranging from log 2.49 ± 0.53 to log 3.73 ± 0.48 cfu/ml for Red Delicious and Golden Delicious varieties, respectively. Thirty-one strains able to develop in presence of arginine at low pH were phenotypically and genotipically identified as belonging to Lactobacillus, Pediococcus and Leuconostoc genera. In general, they did not produce ammonia from arginine when cultivated in basal medium with arginine (BMA) at pH 4.5 or 5.2. When this metabolite was quantified only six strains belonging to Leuconostoc dextranicum, Lactobacillus brevis and Lactobacillus plantarum species formed higher ammonia amounts in BMA as compared to control. This was correlated with arginine utilization and it was more pronounced at pH 4.5 than 5.2. Analysis of citrulline production confirmed the arginine utilization in these bacteria by the arginine deiminase (ADI) pathway. Maxima citrulline production was observed for Lactobacillus brevis M15 at the two pH values. In this strain ammonia was formed at higher rate than citrulline, which was detected in concentration lower than 1 mM. Thus, main LAB species found on apple surfaces with abilities to degrade arginine by the ADI pathway under different conditions were reported here at the first time. The results suggested that the ADI pathway in apples LAB might not be mainly relevant for their survival in the acid natural environmental, despite leading to the ammonia formation, which may contribute to the increase in pH, coping the acid stress.     

Polyamine Metabolism in Acanthamoeba: Polyamine Content and Synthesis of Ornithine,Putrescine, and Diaminopropane1

Five polyamines which could be separated by high performance liquid chromatography were found in Acanthamoeba castellanii (strain Neff). These included in order of decreasing abundance: 1,3-diaminopropane, spermidine, spermine, norspermidine, and putrescine. Only diaminopropane and norspermidine had been found previously. Spermine was present in cultures grown in broth, but not in defined medium. Radioactive substrates were used to establish that putrescine was synthesized by decarboxylation of ornithine, ornithine was synthesized from arginine or citrulline, and diaminopropane was synthesized from spermidine. The presence of ornithine decarboxylase (EC 4.1.1.17), arginase (EC 3.5.3.1), and urease (EC 3.5.1.5) and the absence of arginine decarboxylase (EC 4.1.1.19) were established. A scheme for polyamine biosynthesis in A. castellanii is proposed.     

Biogenic Amine Production by Oenococcus oeni

The biogenic amine-producing capability of several Oenococcus oeni strains, originally isolated from different Italian wines, was determined. The amine-producing capability was quali-quantitatively variable among the strains: out of the 44 strains investigated under optimal growth conditions, more than 60% were able to produce histamine, at concentrations ranging from 1.0 to 33 mg/L, and about 16% showed the additional capability to form both putrescine and cadaverine, to different extents and variable relative proportions. The amine-producing behavior of the strains was confirmed under stress culture conditions, while performing malolactic fermentation. In wine, one randomly chosen strain was very effective in forming putrescine from ornithine. The formation of putrescine from arginine by some strains has been also demonstrated. Consequently, O. oeni can really and significantly contribute to the overall biogenic amine content of wines. Practical consequences of these findings are discussed. Received: 2 August 2001 / Accepted: 28 August 2001     

Catabolism of arginine byCarnobacterium spp. isolated from vacuum-packed sugar-salted fish

The ability ofCarnobacterium spp. originally isolated from vacuum-packed, sugar-salted fish to catabolize arginine was examined. All strains were able to produce citrulline, ornithine, and NH₃ from arginine, presumably by the arginine deiminase pathway. The metabolism of arginine was concurrent with acid production from glucose for one strain ofCarnobacterium sp. but delayed for one strain ofCarnobacterium piscicola. The arginine catabolism was not inhibited in the presence of 2% glucose for three strains of carnobacteria during growth in test broth and/or shrimp extract. Growth as well as arginine catabolism was delayed for two strains of carnobacteria by lowering the temperature from 9°C to 4°C. A similar result was obtained by incubating one strain ofC. piscicola in CO₂. None of the compoundsl-citrulline,l-ornithine hydrochloride, and (NH₄)₂SO₄ had any effect on growth or arginine catabolism of this strain. Neither did pH of the medium affect the time for initiation of arginine catabolism.     

Determination of lactic acid bacteria producing biogenic amines in wine by quantitative PCR methods

Aims: To develop rapid methods allowing enumeration of lactic acid bacteria producing biogenic amines in wines and to analyse wine samples by the methods. Methods and Results: Methods based on quantitative PCR targeting bacterial genes involved in histamine, tyramine and putrescine production were developed and applied to detect and quantify the bacteria producing these biogenic amines in wine. Analysis of 102 samples revealed low populations of the targeted bacteria in grape must samples, an increased bacteria biomass in wine samples after alcoholic fermentation, reaching the highest population levels (above 10⁶ cells ml^?1) during spontaneous malolactic fermentation. A minimum of 10³ ml^?1 producing cells was required for production of more than 1 mg l^?1 of biogenic amines. Accumulation of putrescine in wine was correlated with the presence of bacteria carrying an ornithine decarboxylation pathway. Trials of winemaking showed that the use of selected bacteria for inducing malolactic fermentation was efficient to limit the proliferation of undesirable bacteria and the production of biogenic amines. Conclusion: Methods using quantitative PCR are efficient to enumerate biogenic amines‐producing cells in wine. Significance and Impact of the Study: The methods can help to better control and to improve winemaking conditions in order to avoid biogenic amine production.     

Putrescine production by engineered Corynebacterium glutamicum

Here, we report the engineering of the industrially relevant Corynebacterium glutamicum for putrescine production. C. glutamicum grew well in the presence of up to 500 mM of putrescine. A reduction of the growth rate by 34% and of biomass formation by 39% was observed at 750 mM of putrescine. C. glutamicum was enabled to produce putrescine by heterologous expression of genes encoding enzymes of the arginine- and ornithine decarboxylase pathways from Escherichia coli. The results showed that the putrescine yield by recombinant C. glutamicum strains provided with the arginine-decarboxylase pathway was 40 times lower than the yield by strains provided with the ornithine decarboxylase pathway. The highest production efficiency was reached by overexpression of speC, encoding the ornithine decarboxylase from E. coli, in combination with chromosomal deletion of genes encoding the arginine repressor ArgR and the ornithine carbamoyltransferase ArgF. In shake-flask batch cultures this strain produced putrescine up to 6 g/L with a space time yield of 0.1 g/L/h. The overall product yield was about 24 mol% (0.12 g/g of glucose).     

Three-Component Lysine/Ornithine Decarboxylation System in Lactobacillus saerimneri 30a

Lactic acid bacteria play a pivotal role in many food fermentations and sometimes represent a health threat due to the ability of some strains to produce biogenic amines that accumulate in foods and cause trouble following ingestion. These strains carry specific enzymatic systems catalyzing the uptake of amino acid precursors (e.g., ornithine and lysine), the decarboxylation inside the cell, and the release of the resulting biogenic amines (e.g., putrescine and cadaverine). This study aimed to identify the system involved in production of cadaverine from lysine, which has not been described to date for lactic acid bacteria. Strain Lactobacillus saerimneri 30a (formerly called Lactobacillus sp. 30a) produces both putrescine and cadaverine. The sequencing of its genome showed that the previously described ornithine decarboxylase gene was not associated with the gene encoding an ornithine/putrescine exchanger as in other bacteria. A new hypothetical decarboxylation system was detected in the proximity of the ornithine decarboxylase gene. It consisted of two genes encoding a putative decarboxylase sharing sequence similarities with ornithine decarboxylases and a putative amino acid transporter resembling the ornithine/putrescine exchangers. The two decarboxylases were produced in Escherichia coli, purified, and characterized in vitro, whereas the transporter was heterologously expressed in Lactococcus lactis and functionally characterized in vivo. The overall data led to the conclusion that the two decarboxylases and the transporter form a three-component decarboxylation system, with the new decarboxylase being a specific lysine decarboxylase and the transporter catalyzing both lysine/cadaverine and ornithine/putrescine exchange. To our knowledge, this is an unprecedented observation of a bacterial three-component decarboxylation system.     

Isolation and characterization of lactic acid bacteria strains with ornithine producing capacity from natural sea salt

Two lactic acid bacteria (LAB) having ornithine-producing capacity were isolated from Korean natural sea salt. They were Gram-positive, short rod-type bacteria, and able to grow anaerobically with CO₂ production. The isolates grew well on MRS broth at 30–37°C and a pH of 6.5–8.0. The optimum temperature and pH for growth are 37°C and pH 7.0. The isolates fermented D-ribose, D-galactose, D-lactose, D-maltose, Dcellobiose, D-tagatose, D-trehalose, sucrose, D-melezitose, gentiobiose, D-glucose but not D-melibiose, inositol, and L-sorbose. The 16S rDNA sequences of the two isolates showed 99.5% and 99.6% homology with the Weissella koreensis S5623 16S rDNA (Access no. AY035891). They were accordingly identified and named as Weissella koreensis MS1-3 and Weissella koreensis MS1-14, and produced intracellular ornithine at levels of 72 mg/100 g cell F.W. and 105 mg/100 g cell F.W. and extracellular ornithine at levels of 4.5 mg/100 ml and 4.6 mg/100 ml medium, respectively, by culturing in MRS broth supplemented with 1% arginine. High cell growth was maintained in MRS broth with a NaCl concentration of 0–6%. These results show for the first time that Korean natural sea salts contain lactic acid bacteria Weissella koreensis strains having ornithine producing capacity.     

Ornithine dissimilation byTreponema denticola

Treponema denticola convertedl-ornithine, a product ofl-arginine catabolism, to putrescine via a decarboxylation reaction and to proline via a deamination reaction. Ornithine decarboxylation byT. denticola extracts was stimulated by pyridoxal 5′-phosphate. In the absence of pyridoxal 5′-phosphate, (NH₄)₂SO₄-fractionated extracts converted ornithine to proline and ammonia. This activity was not stimulated by α-keto acids, nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide or ADP. Neither ornithine δ-transaminase (l-ornithine: 2-oxoacid aminotransferase, EC 2.6.1.13) nor Δ¹ reductase [l-proline: NAD(P) 5-oxidoreductase, EC 1.5.1.2.] activity was detectable in cell extracts. These results indicate that formation of proline from ornithine inT. denticola is catalyzed by an enzyme system analogous to the ornithine cyclase (deaminating) ofClostridium sporogenes. Exogenous ornithine inhibited the growth ofT. denticola. Thus, in addition to generating putrescine and proline, the ornithine dissimilatory pathways may serve to prevent accumulation of inhibitory concentrations of ornithine in the spirochete's environment.     

Assaying ornithine and arginine decarboxylases in some plant species

A release of ¹⁴CO₂ not related to ornithine decarboxylase activity was found in crude leaf extracts from Lycopersicon esculentum, Avena sativa, and especially from the pyrrolizidine alkaloid-bearing Heliotropium angiospermum when incubated with [1-¹⁴C]- or [U-¹⁴C]ornithine. The total ¹⁴CO₂ produced was about 5- to 100-fold higher than that due to ornithine decarboxylase activities calculated from labeled putrescine (Put) found by thin-layer electrophoresis in the incubation mixtures. Partial purification with (NH₄)₂SO₄ did not eliminate completely the interfering decarboxylation. When incubated with labeled arginine, a very significant ¹⁴CO₂ release not related to arginine decarboxylase activity was observed only in extracts from H. angiospermum leaves, especially in Tris·HCl buffer. Under the assay conditions, these extracts exhibited oxidative degradation of added Put and agmatine (Agm) and also revealed a high arginase activity. Amino-guanidine at 0.1 to 0.2 millimolar prevented Put degradation and greatly decreased oxidative degradation of Agm; ornithine at 15 to 20 millimolar significantly inhibited arginase activity. A verification of the reliability of the standard ¹⁴CO₂-based method by assessing labeled Put and/or Agm—formed in the presence of added aminoguanidine and/or ornithine when needed—is recommended especially when crude or semicrude plant extracts are assayed.

When based on Put and/or Agm formed at 1.0 to 2.5 millimolar of substrate, the activities of ornithine decarboxylase and arginine decarboxylase in the youngest leaves of the tested species ranged between 1.1 and 3.6 and 1 and 1600 nanomoles per hour per gram fresh weight, respectively. The enzyme activities are discussed in relation to the biosynthesis of pyrrolizidine alkaloids.