Final steps in the catabolism of nicotine

New enzymes of nicotine catabolism instrumental in the detoxification of the tobacco alkaloid by Arthrobacter nicotinovorans pAO1 have been identified and characterized. Nicotine breakdown leads to the formation of nicotine blue from the hydroxylated pyridine ring and of gamma-N-methylaminobutyrate (CH(3)-4-aminobutyrate) from the pyrrolidine ring of the molecule. Surprisingly, two alternative pathways for the final steps in the catabolism of CH(3)-4-aminobutyrate could be identified. CH(3)-4-aminobutyrate may be demethylated to gamma-N-aminobutyrate by the recently identified gamma-N-methylaminobutyrate oxidase. In an alternative pathway, an amine oxidase with noncovalently bound FAD and of novel substrate specificity removed methylamine from CH(3)-4-aminobutyrate with the formation of succinic semialdehyde. Succinic semialdehyde was converted to succinate by a NADP(+)-dependent succinic semialdehyde dehydrogenase. Succinate may enter the citric acid cycle completing the catabolism of the pyrrolidine moiety of nicotine. Expression of the genes of these enzymes was dependent on the presence of nicotine in the growth medium. Thus, two enzymes of the nicotine regulon, gamma-N-methylaminobutyrate oxidase and amine oxidase share the same substrate. The K(m) of 2.5 mM and k(cat) of 1230 s(-1) for amine oxidase vs. K(m) of 140 microM and k(cat) of 800 s(-1) for gamma-N-methylaminobutyrate oxidase, determined in vitro with the purified recombinant enzymes, may suggest that demethylation predominates over deamination of CH(3)-4-aminobutyrate. However, bacteria grown on [(14)C]nicotine secreted [(14)C]methylamine into the medium, indicating that the pathway to succinate is active in vivo.     

Action of inhibitors on monoamine and diamine metabolism in the rat.

The effects of a series of inhibitors of monoamine oxidase (EC 1.4.3.4) and diamine oxidase (EC 1.4.3.6) and of two chelating agents were studied in rats, with respect to the catabolism of labeled pentylamine and putrescine to radioactive carbon dioxide. D-Tranylcypromine, clorgyline, and deprenyl inhibited oxidation of the monoamine, with essentially no effect on putrescine, under our test conditions. Aminoguanidine inhibited putrescine but not pentylamine oxidation. Iproniazid, isoniazid, and Lilly 51641 affected the catabolism of both amines. Pargyline inhibited putrescine oxidation, apparently in a reversible manner.     

Putrescine catabolism in mammalian brain

In contrast with putrescine (1,4-diaminobutane), which is a substrate of diamine oxidase, monoacetylputrescine is oxidatively deaminated both in vitro and in vivo by monoamine oxidase. The product of this reaction is N-acetyl-gamma-aminobutyrate. The existence of a degradative pathway in mammalian brain for putrescine is shown, which comprises acetylation of putrescine, oxidative deamination of monoacetylputrescine to N-acetyl-gamma-aminobutyrate, transformation of N-acetyl-gamma-aminobutyrate to gamma-aminobutyrate and degradation of gamma-aminobutyrate to CO(2) via the tricarboxylic acid cycle.     

Genetic manipulation of the metabolism of polyamines in poplar cells. The regulation of putrescine catabolism

We investigated the catabolism of putrescine (Put) in a non-transgenic (NT) and a transgenic cell line of poplar (Populus nigra x maximowiczii) expressing a mouse (Mus musculus) ornithine (Orn) decarboxylase (odc) cDNA. The transgenic cells produce 3- to 4-fold higher amounts of Put than the NT cells. The rate of loss of Put from the cells and the initial half-life of cellular Put were determined by feeding the cells with [U-(14)C]Orn and [1,4-(14)C]Put as precursors and following the loss of [(14)C]Put in the cells at various times after transfer to label-free medium. The amount of Put converted into spermidine as well as the loss of Put per gram fresh weight were significantly higher in the transgenic cells than the NT cells. The initial half-life of exogenously supplied [(14)C]Put was not significantly different in the two cell lines. The activity of diamine oxidase, the major enzyme involved in Put catabolism, was comparable in the two cell lines even though the Put content of the transgenic cells was severalfold higher than the NT cells. It is concluded that in poplar cells: (a) exogenously supplied Orn enters the cells and is rapidly converted into Put, (b) the rate of Put catabolism is proportional to the rate of its biosynthesis, and (c) the increased Put degradation occurs without significant changes in the activity of diamine oxidase.     

Role of pyridoxine in the metabolism of putrescine in the rat.

1. The ability of rats to metabolize radioactive putrescine to 14CO2 in vivo has been studied. 2. Animals made deficient in pyridoxine exhibit a significantly lower rate of catabolism of the diamine. 3. There dose not appear to be an important interaction between the effects of the deficiency and those stemming from treatment of the animals with the diamine oxidase inhibitor aminoguanidine. 4. These results favour the concept of a role of pyridoxal cofactor in the metabolism of diamines, presumably at the diamine oxidase stage.     

Genomic organisation,activity and distribution analysis of the microbial putrescine oxidase degradation pathway

The catalytic action of putrescine specific amine oxidases acting in tandem with 4-aminobutyraldehyde dehydrogenase is explored as a degradative pathway in Rhodococcus opacus. By limiting the nitrogen source, increased catalytic activity was induced leading to a coordinated response in the oxidative deamination of putrescine to 4-aminobutyraldehyde and subsequent dehydrogenation to 4-aminobutyrate. Isolating the dehydrogenase by ion exchange chromatography and gel filtration revealed that the enzyme acts principally on linear aliphatic aldehydes possessing an amino moiety. Michaelis–Menten kinetic analysis delivered a Michaelis constant (K_M = 0.014 mM) and maximum rate (V_max = 11.2 μmol/min/mg) for the conversion of 4-aminobutyraldehyde to 4-aminobutyrate. The dehydrogenase identified by MALDI-TOF mass spectrometric analysis (E value = 0.031, 23% coverage) belongs to a functionally related genomic cluster that includes the amine oxidase, suggesting their association in a directed cell response. Key regulatory, stress and transport encoding genes have been identified, along with candidate dehydrogenases and transaminases for the further conversion of 4-aminobutyrate to succinate. Genomic analysis has revealed highly similar metabolic gene clustering among members of Actinobacteria, providing insight into putrescine degradation notably among Micrococcaceae, Rhodococci and Corynebacterium by a pathway that was previously uncharacterised in bacteria.     

Transport and metabolism of agmatine in rat hepatocyte cultures.

Rat hepatocytes in culture take up [14C]-agmatine by both a high-affinity transport system [KM = 0.03 mM; Vmax = 30 pmol x min x (mg protein)-1] and a low-affinity system. The high-affinity system also transports putrescine, but not cationic amino acids such as arginine, and the polyamines spermidine and spermine. The rate of agmatine uptake is increased in cells deprived of polyamines with difluoromethylornithine. Of the agmatine taken up, 10% is transformed into polyamines and 50% is transformed into 4-guanidinobutyrate, as demonstrated by HPLC and MS. Inhibition by aminoguanidine and pargyline shows that this is due to diamine oxidase and an aldehyde dehydrogenase. 14C-4-aminobutyrate is also accumulated in the presence of an inhibitor of 4-aminobutyrate transaminase.     

Pathway and enzyme redundancy in putrescine catabolism in Escherichia coli

Putrescine as the sole carbon source requires a novel catabolic pathway with glutamylated intermediates. Nitrogen limitation does not induce genes of this glutamylated putrescine (GP) pathway but instead induces genes for a putrescine catabolic pathway that starts with a transaminase-dependent deamination. We determined pathway utilization with putrescine as the sole nitrogen source by examining mutants with defects in both pathways. Blocks in both the GP and transaminase pathways were required to prevent growth with putrescine as the sole nitrogen source. Genetic and biochemical analyses showed redundant enzymes for γ-aminobutyraldehyde dehydrogenase (PatD/YdcW and PuuC), γ-aminobutyrate transaminase (GabT and PuuE), and succinic semialdehyde dehydrogenase (GabD and PuuC). PuuC is a nonspecific aldehyde dehydrogenase that oxidizes all the aldehydes in putrescine catabolism. A puuP mutant failed to use putrescine as the nitrogen source, which implies one major transporter for putrescine as the sole nitrogen source. Analysis of regulation of the GP pathway shows induction by putrescine and not by a product of putrescine catabolism and shows that putrescine accumulates in puuA, puuB, and puuC mutants but not in any other mutant. We conclude that two independent sets of enzymes can completely degrade putrescine to succinate and that their relative importance depends on the environment.     

Putrescine, a source of gamma-aminobutyric acid in the adrenal gland of the rat.

Putrescine is the major source of gamma-aminobutyric acid (GABA) in the rat adrenal gland. Diamine oxidase, and not monoamine oxidase, is essential for GABA formation from putrescine in the adrenal gland. Aminoguanidine, a diamine oxidase inhibitor, decreases the GABA concentration in the adrenal gland by more than 70% after 4 h, and almost to zero in 24 h. Studies using [14C]putrescine confirm that [14C]GABA is the major metabolite of putrescine in the adrenal gland. Inhibition of GABA transaminase by amino-oxyacetic acid does not change the GABA concentration in the adrenal gland, as compared with the brain, where the GABA concentration rises. With aminoguanidine, the turnover time of GABA originating from putrescine in the adrenal gland is 5.6 h, reflecting a slower rate of GABA metabolism compared with the brain. Since GABA in the adrenal gland is almost exclusively derived from putrescine, the role of GABA may relate to the role of putrescine as a growth factor and regulator of cell metabolism.     

Enzymes and pathways of polyamine breakdown in microorganisms.

The information currently available on the breakdown of spermidine and putrescine by microorganisms is reviewed. Two major metabolic routes have been described, one for the free bases via delta 1-pyrroline (4-aminobutyraldehyde), the other via N-acetyl derivatives. In both pathways oxidases or aminotransferases are the key enzymes in removing the nitrogen atoms. The two routes converge at 4-aminobutyrate, which is then metabolized via succinate. The degradation of putrescine in Escherichia coli has been well characterized at both genetic and biochemical levels, but for other bacteria much less information is available. The C3 moiety of spermidine is broken down via beta-alanine, but the metabolism of this compound and its precursors is poorly understood. In yeasts, a catabolic route for spermidine and putrescine via N-acetyl derivatives has been described in Candida boidinii, and the evidence for its occurrence in other species is reviewed. Except for the terminal step of this pathway, the same group of enzymes can metabolize both the C3 and C4 moieties of spermidine. It is likely that other routes of polyamine catabolism also exist in both bacteria and yeasts.     

Distribution and properties of human intestinal diamine oxidase and its relevance for the histamine catabolism

High activities of diamine oxidase (EC 1.4.3.6) were measured in the intestinal tract of human subjects and of several mammalian species. The enzyme was localized in the mucosa and was distributed primarily in the cytoplasm; the only exception being the guinea-pig where it was located in the particulate fraction. Despite its instability the enzyme from human colonic mucosa was purified 80-fold. During the purification a soluble monoamine oxidase (EC 1.4.3.4) was separated from diamine oxidase. The pH optima of diamine oxidase for putrescine and histamine were 6.6-7.0 and 6.4-6.6, respectively. Short-chain aliphatic diamines were deaminated with the highest reaction velocity, but histamine and N tau-methylhistamine were also excellent substrates. The Km for putrescine was 8.3 x 10(-5) M, for histamine 1.9 x 10(-5) M and for N tau-methylhistamine 9.7 x 10(-5) M. Typical substrates of monoamine oxidase were not deaminated by the enzyme. Aminoguanidine strongly inhibited human intestinal diamine oxidase (IC50 = 1.1 x 10(-8) M). Because of its properties the intestinal diamine oxidase is considered to play a protective role against histamine in diseases such as ischaemic bowel syndrome, mesenteric infarction and ulcerative colitis.     

Modification of diamine oxidase activity in vitro by metabolites of asparagine and differences in asparagine decarboxylation in normal and virus-transformed baby hamster kidney cells.

1. The oxidation of putrescine in vitro by pig kidney diamine oxidase (EC 1.4.3.6) was increased in the presence of 2-oxosuccinamic acid and malonamic acid. 2. It was inhibited by 3-aminopropionamide, oxaloacetate and pyruvate. 3. 2-Oxosuccinamate was derived from asparagine in virus-transformed baby hamster kidney (BHK) cells growing in tissue culture. 4. Asparagine was decarboxylated more efficiently by transformed than by normal BHK cells. 5. In BHK cells transformed by polyoma virus (Py BHK), 2-oxosuccinamate is the most likely immediate precursor of the 14CO2 arising from [U-14C]asparagine, and there was some evidence for its formation in an asparagine-dependent clone of BHK cells before and after their transformation by hamster sarcoma virus (respectively Asn- and HSV Asn-). 6. The relationship between 2-oxosuccinamate and pyruvate and the possible roles of these two substances in controlling cellular diamine oxidase activity are discussed.     

Induction of diamine oxidase activity in rat kidney during compensatory hypertrophy

Diamine oxidase (EC 1.4.3.6) activity, measured as [¹⁴C]Δ¹-pyrroline formation from [¹⁴C] putrescine, was studied in homogenates of rat kidney during compensatory hypertrophy after unilateral nephrectomy. Acetaldehyde and to a lesser degree phenobarbital, at concentrations which did not modify the activity of a preparation of hog kidney diamine oxidase, increased Δ¹-pyrroline formation in kidney homogenate, which suggests that aldehyde-metabolizing enzymes present in this tissue may interfere with yield of Δ¹-pyrroline and that the use of acetaldehyde may give better information on kidney diamine oxidase activity. Other inhibitors of aldehyde-metabolizing enzymes such as chloral hydrate, disulfiram, and pyrazole cannot be used for diamine oxidase determination since they stimulated or depressed this enzyme activity. In rat kidney undergoing compensatory hypertrophy the levels of putrescine, spermidine, and spermine increased rapidly and were followed by an increase in diamine oxidase activity that presented a first peak on day 2 and a second peak on day 6. The administration of cycloheximide or actinomycin D to nephrectomized rats prevented the increase in diamine oxidase activity. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 14 h in normal and hypertrophic kidney. These results suggest that the increase in diamine oxidase activity in renal hypertrophy was due to the synthesis of new enzyme rather than to a slowing of its degradation.     

Monoamine oxidase in adult Hymenolepis diminuta (Cestoda).

A membrane-bound monoamine oxidase (EC 1.4.3.4) was demonstrated in homogenates of Hymenolepis diminuta. The enzyme oxidized a variety of biologically active amines (in decreasing order: dopamine, adrenaline, noradrenaline, tryptamine, tyramine, octopamine), there was, however, no activity with 5-hydroxytryptamine or benzylamine. No diamine oxidase (EC 1.4.3.6.) could be detected in H. diminuta (using histamine, cadaverine or putrescine as substrates). The monoamine oxidase from H. diminuta was not inhibited by azide, hydroxylamine or semicarbazide, but was inhibited by cupferron, alpha-alpha dipyridyl and iodoacetamide, and by the specific monoamine oxidase inhibitors pargyline, nialamide and iproniazid. Several anthelmintics were also found to be inhibitors of monoamine oxidase. The possible roles of monoamine oxidase in H. diminuta are discussed.     

Oxidative deamination of biogenic amines by intestinal amine oxidases: histamine is specifically inactivated by diamine oxidase.

The ability of the gut to inactivate various amines by oxidative deamination was tested with a 130-fold purified amine oxidase preparation from dog small intestine. Of 34 amines tested, putrescine, benzylamine, cadaverine, and serotonin were the most favourable substrates. Histamine was inactivated rapidly by this enzyme preparation, too. Histamine derivatives methylated at the imidazole nucleus were also deaminated, whereas Nalpha-methylhistamine was only a poor substrate and Nalpha, Nalpha-dimethylhistamine was not a substrate at all. Using a second procedure for the purification of amine oxidases from gut, the separation of a soluble monoamine oxidase from diamine oxidase was achieved by gel filtration on Sephadex G-200. The diamine oxidase deaminated putrescine (Km = 1.3 x 10(-4)M) and histamine (Km = 6.6 x 10(-5)M), but not serotonin, and was inhibited by aminoguanidine, but not by pargyline. The soluble monoamine oxidase inactivated serotonin (Km = 4.5 x 10(-4)M), but not histamine and putrescine and was inhibited by pargyline, but not by aminoguanidine. It was concluded that in dog small intestine (as well as in rabbit small intestine) only diamine oxidase was capable of inactivating histamine by oxidative deamination.     

Enzymes and pathways of polyamine breakdown in microorganisms

Abstract The information currently available on the breakdown of spermidine and putrescine by microorganisms is reviewed. Two major metabolic routes have been described, one for the free bases via δ¹-pyrroline (4-aminobutyraldehyde), the other via N -acetyl derivatives. In both pathways oxidases or aminotransferases are the key enzymes in removing the nitrogen atoms. The two routes converge at 4-aminobutyrate, which is then metabolized via succinate. The degradation of putrescine in Escherichia coli has been well characterized at both genetic and biochemical levels, but for other bacteria much less information is available. The C₃ moiety of spermidine is broken down via β-alanine, but the metabolism of this compound and its precursors is poorly understood. In yeasts, a catabolic route for spermidine and putrescine via N -acetyl derivatives has been described in Candida boidinii , and the evidence for its occurrence in other species is reviewed. Except for the terminal step of this pathway, the same group of enzymes can metabolize both the C₃ and C₄ moieties of spermidine. It is likely that other routes of polyamine catabolism also exist in both bacteria and yeasts.     

Enzymes of agmatine degradation and the control of their synthesis in Klebsiella aerogenes.

The degradation of agmatine to succinate by Klebsiella aerogenes occurs in five steps. The enzyme catalyzing the first step, agmatinase, is induced by agmatine. The enzymes catalyzing the second and third steps, putrescine aminotransferase and 4-aminobutyraldehyde dehydrogenase, are induced by putrescine and also by their product, 4-aminobutyrate. The enzymes catalyzing the fourth and fifth steps, 4-aminobutyrate aminotransferase and succinate semialdehyde dehydrogenase, are induced by 4-aminobutyrate. This compound also serves as gratuitous inducer of the catabolic acetylornithine aminotransferase. The formation of the enzymes responsible for agmatine degradation is regulated not only by induction, but also by catabolite repression and activation by glutamine synthetase.     

Fermentative degradation of putrescine by new strictly anaerobic bacteria

Three strains of new strictly anaerobic, Grampositive, non-sporeforming bacteria were isolated from various anoxic sediment samples with putrescine as sole carbon and energy source. Optimal growth in carbonate-buffered defined medium occurred at 37°C at pH 7.2–7.6. The DNA base ratio of strain NorPut1 was 29.6±1 mol% guanine plus cytosine. In addition to a surface layer and the peptidoglycan layer, the cell wall contained a second innermost layer with a periodic arrangement of subunits. All strains fermented putrescine to acetate, butyrate, and molecular hydrogen; the latter originated from both oxidative putrescine deamination and 4-aminobutyraldehyde oxidation. In defined mixed cultures with methanogens or homoacetogenic bacteria, methane or additional acetate were formed due to interspecies hydrogen transfer. Also 4-aminobutyrate and 4-hydroxybutyrate were fermented to acetate and butyrate, but no hydrogen was released from these substrates. No sugars, organic acids, other primary amines or amino acids were used as substrates. Neither sulfate, thiosulfate, sulfur, nitrate nor fumarate was reduced. Most of the enzymes involved in putrescine degradation could be demonstrated in cell-free extracts. A pathway of putrescine fermentation via 4-aminobutyrate and crotonyl-CoA with subsequent dismutation to acetate and butyrate is suggested.     

On the role of GABA in vertebrate polyamine metabolism

4-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in vertebrate brain, is formed not only by decarboxylation of glutamic acid but also directly from putrescine. Two pathways can be shown to operate in vertebrates: oxidative deamination by diamine oxidase and transformation of putrescine into monoacetylputrescine with subsequent oxidative deamination of this intermediate by monoamine oxidase. Monoacetylation and oxidation degradation of the acetyl derivatives is most probably a common pathway of the polyamines. The formation of spermic acid and putreanine from spermine and spermidine, respectively, seems analogous to the reaction of putrescine with diamine oxidase. Apart from metabolic transformation of the polyamines to GABA, there are indirect interrelations with potential regulatory functions. A variety of agents able to influence brain GABA metabolism induce changes of the activity of the decarboxylases involved in polyamine metabolism and alterations of cerebral putrescine concentrations. These interrelations could be important in the control of local cerebral protein metabolism. The excessive transformation of putrescine to GABA in early neural development suggests a role in cellular differentiation.     

IN VIVO FORMATION AND CATABOLISM OF [14C]HISTAMINE IN MOUSE BRAIN

Abstract— The formation of histamine in brain was studied in mice injected with l -[¹⁴C]-histidine (ring 2-¹⁴C) intravenously (i.v.) or intracerebrally; [¹⁴C]histamine appeared rapidly and exhibited a rapid rate of turnover. Drugs known to block various pathways of histamine catabolism were tested for effects on brain–[¹⁴C]histamine and [¹⁴C]-methyl-histamine in mice given (1) [¹⁴C]histamine i.v., (2) [¹⁴C]histamine intracerebrally, and (3) l -[¹⁴C]histidine i.v. Blood-borne histamine did not enter brain; brain histamine was formed locally by decarboxylation of histidine Methylhistamine did cross the blood-brain barrier. Methylation was the major route of histamine catabolism in mouse brain and some of the methylhistamine formed was destroyed by monoamine oxidase. No evidence for catabolism by the action of diamine oxidase was found.