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.     

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.     

A rapid procedure for detection of bacterial amino acid decarboxylases

The detection of decarboxylases of arginine, glutamic acid, histidine, lysine, ornithine, phenylalanine, tryptophan, and tyrosine in bacteria by thin-layer chromatography on polyamide sheets is described. The bacteria were grown on agar medium plates supplemented with eight amino acids at pH 5.5 for induction of amino acid decarboxylases, then transferred to amine-production media. The decarboxylation products in the spent media (amines and/or γ-amino-n-butyric acid) were dansylated and the dansyl derivatives were separated by thin-layer chromatography on polyamide sheets. This method requires only two separate incubations of the decarboxylase-induced bacteria in amine-production media for 1 h at 37°C for simultaneous detection of eight bacterial amino acid decarboxylases using 0.4 μl of the spent media.     

Stereochemistry of the reaction catalysed by glutamic acid decarboxylase from a higher plant (Hordeum vulgare)

The decarboxylation of (2S)-glutamic acid to yield γ-aminobutyric acid catalysed by L-glutamic acid decarboxylase (EC 4.1.1.15) from Hordeum vulgare proceeds with net retention. The result is interpreted in terms of a single progenitor hypothesis of the pyridoxal phosphate enzymes and confirms that not only bacteria and animals but also plant decarboxylases catalyse the biosynthesis of biogenic amines from amino acids with net retention.     

Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella

During the course of infection, Salmonella enterica serovar Typhimurium must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments. Three low pH inducible amino acid decarboxylases were annotated in the genome of S. Typhimurium, AdiA, CadA and SpeF, which are specific for arginine, lysine and ornithine, respectively. In this study, we characterized and compared the contributions of those enzymes in response to acidic challenges. Individual mutants as well as a strain deleted for the three genes were tested for their ability (i) to survive an extreme acid shock, (ii) to grow at mild acidic pH and (iii) to infect the mouse animal model. We showed that the lysine decarboxylase CadA had the broadest range of activity since it both had the capacity to promote survival at pH 2.3 and growth at pH 4.5. The arginine decarboxylase AdiA was the most performant in protecting S. Typhimurium from a shock at pH 2.3 and the ornithine decarboxylase SpeF conferred the best growth advantage under anaerobiosis conditions at pH 4.5. We developed a GFP-based gene reporter to monitor the pH of the environment as perceived by S. Typhimurium. Results showed that activities of the lysine and ornithine decarboxylases at mild acidic pH did modify the local surrounding of S. Typhimurium both in culture medium and in macrophages. Finally, we tested the contribution of decarboxylases to virulence and found that these enzymes were dispensable for S. Typhimurium virulence during systemic infection. In the light of this result, we examined the genomes of Salmonella spp. normally responsible of systemic infection and observed that the genes encoding these enzymes were not well conserved, supporting the idea that these enzymes may be not required during systemic infection.     

Participation of the enzymes involved in the biosynthesis of biogenic amines in biochemical interactions between wheat (Triticum aestivum; Poaceae) and bird cherry-oat aphid (Rhopalosiphum padi; Aphididae)

The studies concerned changes in the activities of ornithine decarboxylase (ODC), lysine decarboxylase (LDC) and tyrosine decarboxylase (TyDC) in tissues of wheat (Triticum aestivum L.) infested with bird cherry-oat aphid (Rhopalosiphum padi L.).Obtained results showed that the activities of the enzymes were stimulated in the less susceptible wheat Kontesa cv. infested by the aphids. In the case of the more susceptible Tonacja cv., on most occasions a decrease in the enzyme activities occurred. Such responses were especially clear for TyDC in both analysed cvs., and for LDC and ODC in the case of Kontesa cv. Thus it may be concluded that amino acid decarboxylation plays an important part in the biochemical defence developed in wheat tissues in response to R. padi infestation. The changes in the activities of the decarboxylases were dependent on the wheat genotype as well as the duration of the infestation.     

A continual spectrophotometric assay for amino acid decarboxylases

A spectrophotometric method for assaying the activity of three amino acid decarboxylases is reported. This method makes use of the coupled reaction of the decarboxylase with phosphoenolpyruvate carboxylase and malate dehydrogenase. The assay is simple and rapid and allows continuous monitoring of the reaction progress. The kinetic parameters obtained using this method for diaminopimelate decarboxylase, lysine decarboxylase, and arginine decarboxylase are comparable to values obtained by radiochemical methods.     

Lysine decarboxylase in Pseudomonas aeruginosa]

The research of lysine, ornithine and arginine decarboxylases has been made for 50 strains of fluorescent Pseudomonas (P. aeruginosa, P. fluorescens, P. putida). By thin layer chromatography, all the strains of Pseudomonas aeruginosa and the fifth of the strains of P. putida had lysine decarboxylase activity at alcaline pH (optimal pH 8) ; Pseudomonas fluorescens did not produce this decarboxylase. Arginine and ornithine decarboxylase are absent for all the strains of fluorescent Pseudomonas.     

Regulation of polyamine biosynthesis by antizyme and some recent developments relating the induction of polyamine biosynthesis to cell growth

This review considers the role of antizyme, of amino acids and of protein synthesis in the regulation of polyamine biosynthesis.The ornithine decarboxylase of eukaryotic ceils and ofEscherichia coli coli can be non-competitively inhibited by proteins, termed antizymes, which are induced by di-and poly- amines. Some antizymes have been purified to homogeneity and have been shown to be structurally unique to the cell of origin. Yet, the E. c o l i antizyme and the rat liver antizyme cross react and inhibit each other's biosynthetic decarboxylases. These results indicate that aspects of the control of polyamine biosynthesis have been highly conserved throughout evolution.Evidence for the physiological role of the antizyme in mammalian cells rests upon its identification in normal uninduced cells, upon the inverse relationship that exists between antizyme and ornithine decarboxylase as well as upon the existence of the complex of ornithine decarboxylase and antizyme in vivo. Furthermore, the antizyme has been shown to be highly specific; its Keq for ornithine decarboxylase is 1.4 x 1011 M-1. In addition, mammalian ceils contain an anti-antizyme, a protein that specifically binds to the antizyme of an ornithine decarboxylase-antizyme complex and liberates free ornithine decarboxylase from the complex. In B. coli , in which polyamine biosynthesis is mediated both by ornithine decarboxylase and by arginine decarboxylase, three proteins (one acidic and two basic) have been purified, each of which inhibits both these enzymes. They do not inhibit the biodegradative ornithine and arginine decarboxylases nor lysine decarboxylase. The two basic inhibitors have been shown to correspond to the ribosomal proteins S₂₀/L₂₆ and L₃₄, respectively. The relationship of the acidic antizyme to other known B. coli proteins remains to be determined.     

Quinolinate dehydrogenase and 6-hydroxyquinolinate decarboxylase involved in the conversion of quinolinic acid to 6-hydroxypicolinic acid by<Emphasis Type="Italic"> Alcaligenes</Emphasis> sp. strain UK21

In the conversion of quinolinic acid to 6-hydroxypicolinic acid by whole cells of Alcaligenes sp. strain UK21, the enzyme reactions involved in the hydroxylation and decarboxylation of quinolinic acid were examined. Quinolinate dehydrogenase, which catalyzes the first step, the hydroxylation of quinolinic acid, was solubilized from a membrane fraction, partially purified, and characterized. The enzyme catalyzed the incorporation of oxygen atoms of H₂O into the hydroxyl group. The dehydrogenase hydroxylated quinolinic acid and pyrazine-2,3-dicarboxylic acid to form 6-hydroxyquinolinic acid and 5-hydroxypyrazine-2,3-dicarboxylic acid, respectively. Phenazine methosulfate was the preferred electron acceptor for quinolinate dehydrogenase. 6-Hydroxyquinolinate decarboxylase, catalyzing the nonoxidative decarboxylation of 6-hydroxyquinolinic acid, was purified to homogeneity and characterized. The purified enzyme had a molecular mass of approximately 221 kDa and consisted of six identical subunits. The decarboxylase specifically catalyzed the decarboxylation of 6-hydroxyquinolinic acid to 6-hydroxypicolinic acid, without any co-factors. The N-terminal amino acid sequence was homologous with those of bacterial 4,5-dihydroxyphthalate decarboxylases.     

Decarboxylation of homoarginine and lysine by an enzyme from Lathyrus sativus seedlings

Homoarginine decarboxylase has been purified ca 110-fold from Lathyrus sativus seedlings and resolved from arginine decarboxylase by DEAE-Sephadex column chromatography. The enzyme was less active than arginine decarboxylase and was highly labile. This preparation decarboxylated l-lysine in addition to L-homoarginine. The purified enzyme preparation had an absolute requirement for exogenous Mn²⁺ or Fe²⁺ for both the enzyme activities. The pH and temperature optima for decarboxylation of both homoarginine and lysine were the same viz. 8·4 and 41° respectively. The K_m value l-homoarginine was 3·33 mM and for l-lysine was 0·88 mM. Arginine and homoarginine decarboxylases appear to be different and separable entities having different physico-chemical characteristics, despite the fact that their respective guanido amino acid substrates undergo similar metabolic conversion to guanido- and diamines in this plant system.     

The enzymatic activities of the Escherichia coli basic aliphatic amino acid decarboxylases exhibit a pH zone of inhibition

The stringent response regulator ppGpp has recently been shown by our group to inhibit the Escherichia coli inducible lysine decarboxylase, LdcI. As a follow-up to this observation, we examined the mechanisms that regulate the activities of the other four E. coli enzymes paralogous to LdcI: the constitutive lysine decarboxylase LdcC, the inducible arginine decarboxylase AdiA, the inducible ornithine decarboxylase SpeF, and the constitutive ornithine decarboxylase SpeC. LdcC and SpeC are involved in cellular polyamine biosynthesis, while LdcI, AdiA, and SpeF are involved in the acid stress response. Multiple mechanisms of regulation were found for these enzymes. In addition to LdcI, LdcC and SpeC were found to be inhibited by ppGpp; AdiA activity was found to be regulated by changes in oligomerization, while SpeF and SpeC activities were regulated by GTP. These findings indicate the presence of multiple mechanisms regulating the activity of this important family of decarboxylases. When the enzyme inhibition profiles are analyzed in parallel, a "zone of inhibition" between pH 6 and pH 8 is observed. Hence, the data suggest that E. coli utilizes multiple mechanisms to ensure that these decarboxylases remain inactive around neutral pH possibly to reduce the consumption of amino acids at this pH.     

The effect of a mealybug infestation on the activity of amino acid decarboxylases in orchid leaves

Changes in the activity of lysine decarboxylase (LDC), tyrosine decarboxylase (TyDC), and ornithine decarboxylase (ODC) within orchid (Phalaenopsis × hybridum ‘Innocence’) leaves, infested by two mealybug species: Pseudococcus longispinus (Targ. Tozz.) and Pseudococcus maritimus (Ehrh.) were quantified. The pattern of changes was dependent on the insect species and duration of infestation. P. longispinus feeding increased LDC and TyDC activity after one week during the total period of observations. This species inhibited ODC activity after one week but increased later. P. maritimus decreased LDC activity in orchid leaves at all studied terms. TyDC action also went up during the first week of the infestation and was reduced after two weeks, while ODC was decreased after one day and induced later. The mechanism for the participation of analysed amino acid decarboxylases in local and/or systemic steps of orchid responses to mealybug infestation is discussed.     

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.     

Construction of lac fusions to the inducible arginine-and lysine decarboxylase genes of Escherichia coli K12

The induction of several amino acid decarboxylases under anaerobic conditions at low pH has been known for many years, but the mechanism associated with this type of regulation has not been elucidated. To study the regulation of the biodegradative arginine and lysine decarboxylases of Escherichia coli K12, Mudlac fusions to these genes were isolated. Mudlac fusion strains deficient for lysine decarboxylase or arginine decarboxylase were identified using decarboxylase indicator media and analysed for their regulation of beta-galactosidase expression. The position of the Mudlac fusion in lysine decarboxylase-deficient strains has been mapped to the cadA gene at 93.7 minutes, while the Mudlac fusions exhibiting a deficiency in the inducible arginine decarboxylase have been mapped to 93.4 minutes.     

Novel sites in the p65 subunit of NF-kappaB interact with TFIIB to facilitate NF-kappaB induced transcription

The citM gene from Lactococcus lactis CRL264 was demonstrated to encode for an oxaloacetate decarboxylase. The enzyme exhibits high levels of similarity to malic enzymes (MEs) from other organisms. CitM was expressed in Escherichia coli, purified and its oxaloacetate decarboxylase activity was demonstrated by biochemical and genetic studies. The highest oxaloacetate decarboxylation activity was found at low pH in the presence of manganese, and the K_m value for oxaloacetate was 0.52 ± 0.03 mM. However, no malic activity was found for this enzyme. Our studies clearly show a new group of oxaloacetate decarboxylases associated with the citrate fermentation pathway in gram-positive bacteria. Furthermore, the essential catalytic residues were found to be conserved in all members of the ME family, suggesting a common mechanism for oxaloacetate decarboxylation.     

解淀粉芽孢杆菌Q-426酚酸脱羧酶的克隆表达及酶学性质鉴定*

目的:生物法脱羧制备4-乙烯基衍生物具有诸多优势和良好的发展前景,研究解淀粉芽孢杆菌Q-426酚酸脱羧酶(BaPAD-Q-426)的酶学性质,为其进一步应用提供理论基础。方法:从解淀粉芽孢杆菌中克隆酚酸脱羧酶基因;以pET-28a(+)为载体,将重组质粒转化至E. coli BL21(DE3)中,实现酚酸脱羧酶BaPAD-Q-426的高效表达,利用Ni-NTA亲和层析进行纯化,并进行酶学性质鉴定。结果:酚酸脱羧酶BaPAD-Q-426在pH 7.0~9.0范围内保持良好的pH稳定性,最适pH为8.0;在25~40℃范围内保持着较高的酶活性,最适温度为35℃,在4℃时保持30 min后该酶依然保持95%以上的酶活性;K⁺对BaPAD-Q-426的酶活具有明显促进作用,酶活力提高60%;该酶在石油醚中具有良好的耐受能力,在40%石油醚存在下,仍保留50%以上的酶活力;BaPAD-Q-426的最适底物为阿魏酸,酶活力达到19.5 IU/mL。结论:与其他来源的酚酸脱羧酶相比,BaPAD-Q-426在低温时具有更好的稳定性,在弱碱性环境下对阿魏酸的催化脱羧能力最强。     

A Study by High Voltage Electrophoresis of the Amino Acid Decarboxylases and Arginine Dihydrolase of Bacteria Isolated from the Alimentary Tract of Pigs

S ummary . The use of high voltage paper electrophoresis for studies on the breakdown of amino acids by bacteria is described. Examination of a number of different isolates from the alimentary tract of the pig showed that the decarboxylase activity was restricted to Escherichia coli and one strain of Lactobacillus fermenti. In some isolates studied the optimum pH of activity differed from those previously reported for similar systems, being higher for ornithine, glutamic acid and lysine decarboxylases. The heterofermentative lactobacilli all converted arginine to ornithine and this may contribute to the final level of putrescine in the gut by providing a substrate for the ornithine decarboxylase of E. coli.     

Assay for aliphatic amino acid decarboxylases by high-performance liquid chromatography

A sensitive and rapid assay for aliphatic amino acid decarboxylases based on separation of the product from the substrate by ion-pairing reversed-phase high-performance liquid chromatography and subsequent fluorometric detection has been developed. The resolution of substrates and products of seven amino acid decarboxylases, namely, arginine, aspartate, 2,6-diaminopimelate, histidine, glutamate, lysine, and ornithine decarboxylase, is complete within 15 to 35 min of isocratic elution. The limit of detection for the product is 40 pmol. The applicability of the procedure was assessed with glutamate decarboxylase. The formation of the product 4-aminobutyrate proved to be linear with time and protein concentration. The method allows the time course of the reaction to be followed in a single assay and works well with crude extracts of bacteria or tissues.     

Functional classification of amino acid decarboxylases from the alanine racemase structural family by phylogenetic studies

Arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) are involved in the biosynthesis of putrescine, which is the precursor of other polyamines in animals, plants, and bacteria. These pyridoxal-5'-phosphate-dependent decarboxylases belong to the alanine racemase (AR) structural family together with diaminopimelate decarboxylase (DapDC), which catalyzes the final step of lysine biosynthesis in bacteria. We have constructed a multiple-sequence alignment of decarboxylases in the AR structural family and, based on the alignment, inferred phylogenetic trees. The phylogenetic tree consists of 3 distinct clades formed by ADC, DapDC, and ODC that diverged from an ancestral decarboxylase. The ancestral decarboxylase probably was able to recognize several substrates, and in archaea and bacteria, ODC may have retained the ability to bind other amino acids. Previously, a paralogue of ODC has been proposed to account for ADC activity detected in mammalian cells. According to our results, this appears unlikely, emphasizing the need for more caution in functional assignment made using sequence data and illustrating the continuing value of phylogenetic analysis in clarifying relationships and putative functions.