Decarboxylation Of Amino Acids Research Articles

Formation of β-phenylethylamine as a consequence of lipid oxidation

The decarboxylation of phenylalanine in the presence of methyl 13-hydroperoxyoctadeca-9,11-dienoate (LOOH), 2,4-decadienal (DD), 4,5-epoxy-2-decenal (ED), 4-hydroxy-2-nonenal (HN), and 4-oxo-2-nonenal (ON) was studied both to explore the potential of lipid oxidation products (LOP) to produce amino acid decarboxylation and to understand the reaction pathways responsible for this degradation. All assayed LOP were able to decarboxylate phenylalanine, but their reactivity decreased in the following order when the reaction was carried out under non-oxidative conditions: DD>ED≈LOOH>HN≈ON. The reaction pathway is proposed to take place by decarboxylation of the imine formed between the amino group of the amino acid and the carbonylic group of the oxidized lipid. This decarboxylation is facilitated due to the formation of a relatively stable azomethine ylide after the loss of carbon dioxide from a 5-oxazolidinone intermediate. The azomethine ylide would, finally, evolve into new conjugated imines, which are the origin of both β-phenylethylamine and phenylacetaldehyde, which was also detected in the reaction mixtures. The activation energy (Ea) for the phenylalanine decarboxylation in the presence of 2,4-decadienal was 54kJ/mol. Although additional studies are needed to determine the contribution of the chemical pathways described in this study to the overall biogenic amine formation in foods, this study provides the chemical basis both to understand how biogenic amines can be produced chemically from amino acids in the presence of lipid-derived carbonyls and to propose potential inhibitors in this route.

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.

Control of biogenic amines in fermented sausages: role of starter cultures.

Biogenic amines show biological activity and exert undesirable physiological effects when absorbed at high concentrations. Biogenic amines are mainly formed by microbial decarboxylation of amino acids and thus are usually present in a wide range of foods, fermented sausages being one of the major biogenic amine sources. The use of selected starter cultures is one of the best technological measures to control aminogenesis during meat fermentation. Although with variable effectiveness, several works show the ability of some starters to render biogenic amine-free sausages. In this paper, the effect of different starter culture is reviewed and the factors determining their performance discussed.

A Commentary on Physico-chemical and Biological Activity of Biogenic Amines and Their Relationship with Safety of Cheese

This manuscript offers a comprehensive review about the bacterial amino acid decarboxylase activity and biogenic amines content of an artisanal cheese produced in Italy, named as Pecorino cheese. It is highlighted the importance of controlling primary production of raw milk, as well as cheese-making process to avoid the release of free amino-acids and viability of microorganisms possessing amino acid decarboxylating activity. Variation in other physico-chemical parameters, such as water activity or protein content, or during ripening may produce the selection of different microbial populations which could increase the formation of biogenic amines in the final product. Among the biogenic amines described in the manuscript, the formation of diamines (putrescine and cadaverine) and specially, tyramine content resulted to be present in high proportion in most of Pecorino cheeses. In some cases, mild thermal treatments applied in milk appear not to be sufficient to eliminate decarboxylase-positive bacteria in cheeses. Additionally, proteinases and peptidases released from rennet could be responsible of high content of biogenic amines. The authors concluded that the formation of biogenic amines depends on several factors and it would be necessary to study case-by-case each cheese type given the high variability in physico-chemical composition. The quality of raw milk, processing conditions, ripening, and subsequent storage before consumption may influence the biogenic amines formation. There are not so many studies made in this area and this could be particularly interesting in other cheese types, where artisanal production is commonly followed.

Prevalence and Characterization of Typical Aeromonas salmonicida Chum Salmon Isolates in Korea

Aeromonas salmonicida is an important fish pathogen commonly associated with furunculosis in salmonids. Typical A. salmonicida strains have the surface virulence A-layer protein, a major virulence determinant encoded by the vapA gene. In this study, 880 chum salmon Oncorhynchus keta were collected from the east coast of Korea during 2006-2011, including 560 wild adults and 320 artificially hatched fry pools, and the presence of typical A. salmonicida was examined by PCR using the typical A. salmonicida-specific vapA gene primers. The results demonstrated that 34.5% of the samples (304/880 samples) were PCR positive, implying that a typical A. salmonicida infection is highly prevalent among chum salmon in Korea. Twenty typical A. salmonicida isolates were recovered based on their brown pigmentation on Trypticase Soy Agar (TSA) plates, which indicates the existence of the A-layer protein. Further biochemical analyses with the four randomly selected typical A. salmonicida isolates revealed some variations in their amino acid decarboxylation and carbohydrate fermentation activity. A phylogenetic analysis based on the entire vapA gene sequence suggested that the A. salmonicida isolates from chum salmon were clustered with those isolated from Atlantic salmon in Europe. Further study is needed to resolve such an interesting relationship in detail.

Is the production of the biogenic amines tyramine and putrescine a species-level trait in enterococci?

Biogenic amines (BA) are toxic nitrogenous compounds that can be accumulated in foods via the microbial decarboxylation of certain amino acids. Lactic acid bacteria (LAB) strains belonging to different species and genera have been described as BA producers and are mainly responsible for their synthesis in fermented foods. It is generally accepted that the capacity to produced BAs is strain-dependent. However, the large number of enterococci identified as BA producers suggests that the aminogenic trait may be a species-level characteristic. Enterococcus faecalis, Enterococcus faecium and Enterococcus durans strains of different origin were analysed to determine their capacity to produce tyramine and putrescine. The presence of the genes responsible for this and the identity of their flanking regions were checked by PCR. The results suggest that tyramine biosynthesis is a species-level characteristic in E. faecalis, E. faecium and E. durans. Putrescine synthesis was found to be a species-level trait of E. faecalis, with production occurring via the agmatine deamination pathway. Some E. faecium strains of human origin also produced putrescine; this trait was probably acquired via horizontal gene transfer.

On the mechanism and rate of spontaneous decomposition of amino acids.

Spontaneous decarboxylation of amino acids is among the slowest known reactions; it is much less facile than the cleavage of amide bonds in polypeptides. Establishment of the kinetics and mechanisms for this fundamental reaction is important for gauging the proficiency of enzymes. In the present study, multiple mechanisms for glycine decomposition in water are explored using QM/MM Monte Carlo simulations and free energy perturbation theory. Simple CO(2) detachment emerges as the preferred pathway for decarboxylation; it is followed by water-assisted proton transfer to yield the products: CO(2) and methylamine. The computed free energy of activation of 45 kcal/mol, and the resulting rate-constant of 1 × 10(-21) s(-1), can be compared with an extrapolated experimental rate constant of ~2 × 10(-17) s(-1) at 25 °C. The half-life for the reaction is more than 1 billion years. Furthermore, examination of deamination finds simple NH(3)-detachment yielding α-lactone to be the favored route, though it is less facile than decarboxylation by 6 kcal/mol. Ab initio and DFT calculations with the CPCM hydration model were also carried out for the reactions; the computed free energies of activation for glycine decarboxylation agree with the QM/MM result, whereas deamination is predicted to be more favorable. QM/MM calculations were also performed for decarboxylation of alanine; the computed barrier is 2 kcal/mol higher than for glycine in qualitative accord with experiment.

Biogenic amine production by bacteria isolated from ice-preserved sardine and mackerel

The occurrence of in vitro production of biogenic amines in bacteria isolated from ice-preserved sardine and mackerel was studied. Biogenic amine production was investigated by means of amino acid decarboxylation by growth on decarboxylase differential medium, biogenic amine detection by thin-layer chromatography (TLC) and decarboxylase gene detection by PCR. Decarboxylase medium overestimate the number of biogenic amine-producer strains, as the production of amine was confirmed by TLC in only five out the 17 presumptive strains. On the producer strains, PCR was used to confirm the presence of the genes encoding the amino acid decarboxylase responsible for the synthesis of these amines. Moreover, biogenic amine-producer bacteria were molecularly identified by sequencing their 16S rRNA. Form sardine, enterobacteria producing simultaneously several biogenic amines were isolated. A Kluyvera intermedia strain producing histamine, putrescine and cadaverine, and an Enterobacter asburiae strain producing only the diamines cadaverine and putrescine were identified. From mackerel, lactic acid bacteria from the Enterococcus durans species producing tyramine were isolated. This study constitutes the first description of the presence on these putatively harmful species on ice-preserved sardine and mackerel.

Biogenic Amines in Dairy Products

Biogenic amines (BA) are organic, basic, nitrogenous compounds with biological activity, mainly formed by the decarboxylation of amino acids. BA are present in a wide range of foods, including dairy products, and can accumulate in high concentrations. In some cheeses more than 1000 mg of BA have been detected per kilogram of cheese. The consumption of food containing large amounts of these amines can have toxicological consequences. Although there is no specific legislation regarding the BA content in dairy products, it is generally assumed that they should not be allowed to accumulate. Greater knowledge of the factors involved in the synthesis and accumulation of BA should lead to a reduction in their incidence in foods. This article focuses on the factors that affect BA production, in particular environmental conditions, the microorganisms that produce them, the genetic organization and regulation of the biosynthetic pathways involved, and the available methods for detecting the presence of BA or BA-producing microorganisms in dairy products.

Molecular insights into the pathogenicity of variants associated with the aromatic amino acid decarboxylase deficiency

Dopa decarboxylase (DDC or AADC) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the decarboxylation of L-aromatic amino acids into the corresponding aromatic amines. AADC deficiency is an inborn error of neurotransmitters biosynthesis with an autosomal recessive inheritance. About 30 pathogenic mutations have been identified, but the enzymatic phenotypes causing AADC deficiency are unknown, and the therapeutic management is challenging. Here, we report biochemical and bioinformatic analyses of the human wild-type DDC and the pathogenic variants G102S, F309L, S147R and A275T whose mutations concern amino acid residues at or near the active site. We found that the mutations cause, even if to different extents, a decreased PLP binding affinity (in the range 1.4-170-fold), an altered state of the bound coenzyme and of its microenvironment, and a reduced catalytic efficiency (in the range 17-930-fold). Moreover, as compared to wild-type, the external aldimines formed by the variants with L-aromatic amino acids exhibit different spectroscopic features, do not protect against limited proteolysis, and lead to the formation, in addition to aromatic amines, of cyclic-substrate adducts. This suggests that these external Schiff bases are not properly oriented and anchored, i.e., in a conformation not completely productive for decarboxylation. The external aldimines that the variants form with D-Dopa also appear not to be correctly located at their active site, as suggested by the rate constants of PLP-L-Dopa adduct production higher than that of the wild-type. The possible therapeutic implications of the data are discussed in the light of the molecular defects of the pathogenic variants.

The reduced mobility of the biogenic amines: trimethylamine, putrescine, cadaverine, spermidine and spermine

Biogenic amines are small molecules that are formed through decarboxylation of amino acids and through biosynthesis and enzymatic processes [2, 6, 16, 21, 23]. The biogenic amines of interest include amines like trimethylamine (TMA) and histamine (HIS or 2-(1Himidazol-4-yl)ethanamine), diamines like putrescine (PUT or 1,4-diaminobutane) and cadaverine (CAD or 1,5diaminopentane), triamines like spermidine (SPD or N(3-aminopropyl)butane-1,4-diamine) and tetramines like spermine (SPM or N,N′-bis(3-aminopropyl)butane-1,4diamine). Biogenic amines are essential components in living cells and play an important role in cell proliferation and differentiation. There is a fine balance between the cellular uptake of biogenic amines and their release from the cell. However, the polyamine content in intercellular fluid and in urine may increase due to the onset of pathological condition, such as hyperplastic or neoplastic growth. Thus, polyamines can be considered as markers of such conditions. For example, elevated levels of TMA, PUT and CAD were found in vaginal discharge fluid in cases of vaginal infections that included bacterial vaginosis (BV), candidiasis (yeast infection) or trichomoniasis [4, 13]. The content of biogenic amines in food products, particularly muscle food like meat, poultry or fish, increases with spoilage, or loss of freshness, of the products [3, 14]. Several analytical methods gave been used to measure the concentration of biogenic amines in food products and body fluids (vaginal discharge, urine and saliva) [1, 5, 17– 20, 24]. Most of these techniques are labor intensive, slow, costly or lack sensitivity or specificity. Ion mobility spectrometry (IMS) has been used to diagnose vaginal infections or food freshness on the basis of measurement of biogenic amines, and also to demonstrate the suitability of IMS as an analytical technique for these compounds. However, the reduced mobility values reported in the literature are not consistent and differences of over 50% were found between different groups [7, 10, 12, 13, 22]. Most reported mobility measurements were carried out at a drift tube temperature of close to 200 °C, where clustering and humidity effects should be minimal, yet, as shown in Table 1, large variations were found. The present study attempts to explain the reasons for the discrepancies and report the reduced mobilities in air of the important biogenic amines TMA, PUT, CAD, SPD and SPD at a low drift tube temperature where the ion source is based on a corona discharge.

Kinetic isotope effects of L-Dopa decarboxylase.

A mixed centroid path integral and free energy perturbation method (PI-FEP/UM) has been used to investigate the primary carbon and secondary hydrogen kinetic isotope effects (KIEs) in the amino acid decarboxylation of L-Dopa catalyzed by the enzyme L-Dopa decarboxylase (DDC) along with the corresponding uncatalyzed reaction in water. DDC is a pyridoxal 5'-phosphate (PLP) dependent enzyme. The cofactor undergoes an internal proton transfer between the zwitterionic protonated Schiff base configuration and the neutral hydroxyimine tautomer. It was found that the cofactor PLP makes significant contributions to lowering the decarboxylation barrier, while the enzyme active site provides further stabilization of the transition state. Interestingly, the O-protonated configuration is preferred both in the Michaelis complex and at the decarboxylation transition state. The computed kinetic isotope effects (KIE) on the carboxylate C-13 are consistent with that observed on decarboxylation reactions of other PLP-dependent enzymes, whereas the KIEs on the α carbon and secondary proton, which can easily be validated experimentally, may be used as a possible identification for the active form of the PLP tautomer in the active site of DDC.

Phenotypic and genetic evaluations of biogenic amine production by lactic acid bacteria isolated from fish and fish products

In this work, biogenic amine production (histamine, tyramine and putrescine) by a collection of 74 lactic acid bacteria of aquatic origin has been investigated by means of amino acid decarboxylation by growth on decarboxylase differential medium, biogenic amine detection by thin-layer chromatography (TLC) and decarboxylase gene detection by PCR. None of the evaluated strains showed neither production of histamine and putrescine, nor presence of the genetic determinants encoding the corresponding decarboxylase activities. However, the tyrosine decarboxylase gene (tdc) was present in all the enterococcal strains, and tyramine production was detected by TLC in all of them but Enterococcus faecium BCS59 and MV5. Analysis of the tyrosine decarboxylase operon of these strains revealed the presence of an insertion sequence upstream tdc that could be responsible for their lack of tyrosine decarboxylase activity.

Oxidative decarboxylation of α-amino acids to nitriles using o-iodoxybenzoic acid in aqueous ammonia

Formation of the nitrile was the outcome of the investigation of biochemically significant oxidative decarboxylation of amino acids using o-iodoxybenzoic acid (IBX) in aqueous ammonia system.

Biogenic amines in fermented foods

Food-fermenting lactic acid bacteria (LAB) are generally considered to be non-toxic and non-pathogenic. Some species of LAB, however, can produce biogenic amines (BAs). BAs are organic, basic, nitrogenous compounds, mainly formed through decarboxylation of amino acids. BAs are present in a wide range of foods, including dairy products, and can occasionally accumulate in high concentrations. The consumption of food containing large amounts of these amines can have toxicological consequences. Although there is no specific legislation regarding BA content in many fermented products, it is generally assumed that they should not be allowed to accumulate. The ability of microorganisms to decarboxylate amino acids is highly variable, often being strain specific, and therefore the detection of bacteria possessing amino acid decarboxylase activity is important to estimate the likelihood that foods contain BA and to prevent their accumulation in food products. Moreover, improved knowledge of the factors involved in the synthesis and accumulation of BA should lead to a reduction in their incidence in foods.

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.

Stereoselective synthesis of hexahydro-3-methyl-1-arylchromeno[3,4-b]pyrrole and its annulated heterocycles as potent antimicrobial agents for human pathogens

Synthesis of a series of novel hexahydrochromenopyrrole analogues has been accomplished through an intramolecular 1,3-dipolar cycloaddition (1,3-DC reaction) of azomethine ylides, generated by the aldehyde induced decarboxylation of secondary amino acids. These compounds were screened for antibacterial and antifungal activities against six human pathogenic bacteria and three human pathogenic fungi and found to have good antimicrobial properties against most of the microorganisms.

Control of biogenic amines in food--existing and emerging approaches.

Biogenic amines have been reported in a variety of foods, such as fish, meat, cheese, vegetables, and wines. They are described as low molecular weight organic bases with aliphatic, aromatic, and heterocyclic structures. The most common biogenic amines found in foods are histamine, tyramine, cadaverine, 2-phenylethylamine, spermine, spermidine, putrescine, tryptamine, and agmatine. In addition octopamine and dopamine have been found in meat and meat products and fish. The formation of biogenic amines in food by the microbial decarboxylation of amino acids can result in consumers suffering allergic reactions, characterized by difficulty in breathing, itching, rash, vomiting, fever, and hypertension. Traditionally, biogenic amine formation in food has been prevented, primarily by limiting microbial growth through chilling and freezing. However, for many fishing based subsistence populations, such measures are not practical. Therefore, secondary control measures to prevent biogenic amine formation in foods or to reduce their levels once formed need to be considered as alternatives. Such approaches to limit microbial growth may include hydrostatic pressures, irradiation, controlled atmosphere packaging, or the use of food additives. Histamine may potentially be degraded by the use of bacterial amine oxidase or amine-negative bacteria. Only some will be cost-effective and practical for use in subsistence populations.

Emerging Infections: Shewanella – A Series of Five Cases

Background:Shewanella spp. are unusual cause of disease in humans; however, reports of Shewanella infections have been increasing. Shewanella is a ubiquitous organism that has been isolated from many foods, sewage, and both from fresh and salt water. Earlier it was named as Pseudomonas putrefaciens or Shewanella putrefaciens. There are several reports describing this organism causing human infections such as cellulitis, abscesses, bacteremia, wound infection, etc. It is oxidase and catalase-positive non-fermenter gram-negative rod that produces hydrogen sulfide.Aims:The study was conducted to identify Shewanella spp., which was wrongly reported as Pseudomonas spp.Materials and Methods:Clinical samples were cultured as per standard clinical laboratory procedure. We tested the non-lactose-fermenting colonies for oxidase positivity. Oxidase-positive colony was inoculated in triple sugar iron slant (TSI) to know the hydrogen sulfide production. Hydrogen sulfide positive colonies were further tested for citrate, urease, indole, and amino acid decarboxylation and acid and gas production from sugars.Results:Five isolates identified as Pseudomonas spp. during preliminary testing were proved to be Shewanella spp. on further testing.Conclusions:It will help in better understanding the epidemiology, pathogenesis and risk factors associated with these and prevention of the rare pathogenic organisms.

ChemInform Abstract: Nitrile-Containing Natural Products

Naturally occurring nitriles† comprise a small and surprisingly diverse set of secondary metabolites. The structures vary from simple, long-chain alkanenitriles to architecturally complex structures such as the calyculins, with new, more complex metabolites being continually reported. The number of nitrilecontaining natural products has risen from 33, in a 1981 review,3 to more than 1204 (excluding cyanogenic glycosides) and now includes metabolites from both terrestrial and marine sources.2 The increasing number of nitrile-containing natural products highlights the need for a general classification of these compounds with this review classifying metabolites according to the hybridization of the nitrile-bearing carbon. Three general categories result; alkanenitriles, a,b-unsaturated nitriles, and aromatic nitriles. This classification emphasizes the amino acid origin of the nitriles and combines some previous sub-groups into structurally-related classes. Nitrile-containing natural products are known to be derived from amino acids in plants,5 arthropods,6 bacteria and fungi1 (Scheme 1). N-Hydroxylation and decarboxylation of amino acids afford aldoximes 3 that are enzymatically converted to the corresponding nitrile 4 through a sequence that appears to be considerably more complex than a simple dehydration.7 Cyanogenic glycosides are derived from these nitriles by a stereoselective hydroxylation–glycosylation sequence.8 Glucosinolates provide another biosynthetic route to nitrilecontaining metabolites. The biosynthesis of glucosinolates 6 is closely related to that of nitriles in proceeding through an aldoxime intermediate.9 Hydrolysis of glucosinolates affords numerous metabolites, including nitriles, where the formation of nitriles results from a complex dependence on pH, co-factors, and the nature of the substituent.10 Glucosinolate hydrolysis often occurs during cooking of Brassica crops (such as cabbage, brussel sprouts, and cauliflower) leading to increased levels of nitriles from these sources.11 2 Alkanenitriles