Pipecolic acid in the dog brain

Pipecolic acid is an intermdiary metabolite of lysine and is decarboxylated to produce piperidine, an endogenous synaptotropic substance. In the present study, the existence of pipecolic acid in the dog brain was confirmed. It was present in highest concentration in the cerebellum followed by the diencephalon and caudate nucleus, and this distribution resembles that of piperidine in dog brain. It seems to be evident that pipecolic acid is a precursor of piperidine in the brain.     

Brain uptake of pipecolic acid,amino acids,and amines following intracarotid injection in the mouse

The uptake of pipecolic acid by the mouse brain was compared to that of several amino acids and amines, following an injection of a double-labeled mixture into the carotid artery. In general, BUI (brain uptake index) values were lower in the mouse than those previously reported in the rat. The only exception was proline. Lysine, a precursor of pipecolic acid biosynthesis in brain, showed a higher BUI than pipecolic acid. The BUI ofD,l-[³H]pipecolic acid was found to be 3.39 (at 0.114 mM). This was saturable between a concentration of 0.114 and 3.44 mM. Kinetic analysis suggests the presence of two kinds of transport systems. Substances structurally related to pipecolic acid, such as nipecotic acid, isonipecotic acid,l-proline, and piperidine show a significant inhibitory effect. Among the amino acids tested, only GABA showed an inhibitory effect. Data are reported which, when considered with other findings (5), present evidence that pipecolic acid is (1) synthesized both in vitro and in vivo in the mouse brain, (2) actively transported in vivo into the brain, and (3) taken up in vitro by synaptosomal preparations.     

Conversion of Pipecolic Acid into Lysine in Penicillium chrysogenum Requires Pipecolate Oxidase and Saccharopine Reductase: Characterization of the lys7 Gene Encoding Saccharopine Reductase

Pipecolic acid is a component of several secondary metabolites in plants and fungi. This compound is useful as a precursor of nonribosomal peptides with novel pharmacological activities. In Penicillium chrysogenum pipecolic acid is converted into lysine and complements the lysine requirement of three different lysine auxotrophs with mutations in the lys1, lys2, or lys3 genes allowing a slow growth of these auxotrophs. We have isolated two P. chrysogenum mutants, named 7.2 and 10.25, that are unable to convert pipecolic acid into lysine. These mutants lacked, respectively, the pipecolate oxidase that converts pipecolic acid into piperideine-6-carboxylic acid and the saccharopine reductase that catalyzes the transformation of piperideine-6-carboxylic acid into saccharopine. The 10.25 mutant was unable to grow in Czapek medium supplemented with alpha-aminoadipic acid. A DNA fragment complementing the 10.25 mutation has been cloned; sequence analysis of the cloned gene (named lys7) revealed that it encoded a protein with high similarity to the saccharopine reductase from Neurospora crassa, Magnaporthe grisea, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Complementation of the 10.25 mutant with the cloned gene restored saccharopine reductase activity, confirming that lys7 encodes a functional saccharopine reductase. Our data suggest that in P. chrysogenum the conversion of pipecolic acid into lysine proceeds through the transformation of pipecolic acid into piperideine-6-carboxylic acid, saccharopine, and lysine by the consecutive action of pipecolate oxidase, saccharopine reductase, and saccharopine dehydrogenase.     

LYSINE METABOLISM IN THE RAT BRAIN: THE PIPECOLIC ACID-FORMING PATHWAY

Employing both the intraventricular and intraperitoneal injection techniques, ¹⁴C-l -lysine at non-overloading concentrations was found to be metabolized to l -¹⁴C-pipecolic acid at significantly high levels in the rat. Labeled pipecolic acid in the brain and liver was only found at rather low levels 24 h after intraperitoneal administration of ¹⁴C-l -lysine regardless of non-labeled lysine metabolite overload. A marked enhancement of pipecolic acid labeling was only found in the brain when ¹⁴C-l -lysine was intraventricularly administered to animals under various lysine metabolite overloads. While overloading doses of non-labeled saccharopine or α-aminoadipate did not significantly alter the labeling patterns of pipecolic acid in the brain, liver or urine when ¹⁴C-l -lysine was intraperitoneally administered, pipecolate overloading markedly reduced labeled pipecolic acid levels in the brain, liver and urine. These results indicate: pipecolic acid formation is subject to product inhibition, and saccharopine is not in the pathway of pipecolic acid synthesis from l -lysine. The labeling pattern of lysine metabolites was not significantly affected by the overloading injection of pipecolic acid when ¹⁴C-l -lysine was intraventricularly administered suggesting a blood-brain barrier for pipecolate. Besides ¹⁴C-pipecolic acid, labeled α-aminoadipic acid was also found at significant levels mostly in the brain. Labeled saccharopine was not detected in any tissues or urine samples analyzed. The ¹⁴C-l -lysine metabolic pattern of the newborn rats did not seem to be any different from the adult rats, i.e. labeled pipecolic acid was also detected in substantial quantities in the brain, liver and urine 5 h after injection. ¹⁴C-d -Lysine was mainly metabolized to l -¹⁴C-pipecolic acid through either route of administration. These experimental evidences indicate that the pipecolic acid-forming pathway is a significant route for lysine metabolism in the rat, and that the rat brain probably utilizes this pathway mainly for lysine metabolism. The present study also discusses the potential neurological significance of the pipecolic acid pathway in relation to the major lysine metabolic pathway (the saccharopine pathway).     

Polyamines in the Synthesis of Bacteriophage Deoxyribonucleic Acid II. Requirement for Polyamines in T4 Infection of a Polyamine Auxotroph

Polyamine depletion produced by exogenous arginine in Escherichia coliK-12 cultures defective in agmatine ureohydrolase activity resulted in a marked inhibition of the rates of growth and nucleic acid synthesis. Addition of putrescine or spermidine to such depleted cultures restored the control rate of growth and nucleic acid accumulation. The omission of lysine resulted in a further decrease in the rates of growth and nucleic acid synthesis in polyamine-depleted cells. The addition of exogenous cadaverine increased the rates of growth and ribonucleic acid synthesis to those observed in lysine-supplemented cultures, suggesting that lysine or a derivative of lysine serves a function similar to cadaverine. Addition of lysine to polyamine-depleted cultures at neutral pH results in the synthesis of cadaverine and a new spermidine analogue, both containing lysine carbon. This new metabolite has been isolated and identified as N-3-aminopropyl-1, 5-diaminopentane. T4D infection of the polyamine-depleted mutant resulted in a very low rate of DNA synthesis and phage maturation. The addition of putrescine or spermidine 15 min before infection restored phage DNA synthesis and phage maturation to control rates, i.e., rates observed in infected cells grown in the absence of arginine.     

Browning of furfural and amino acids, and a novel yellow compound, furpipate, formed from lysine and furfural

Furfural is an important intermediate compound of the Maillard reaction of pentose or ascorbic acid. We examined the browning of furfural and lysine by heating and found a yellow compound, called furpipate, (E)-3-(2-furylmethylidene)-3H, 4H, 5H, 6H-pyridine-2-carboxylic acid. Furpipate is a novel pipecolic acid derivative and shows absorption maxima at 375 nm and 310 nm under acidic and alkaline conditions, respectively. This compound was the major colored compound of the heated solution containing lysine and furfural.     

Effects of starvation and immobilization on imino acids in mouse brain and peripheral tissues

Effects of starvation and immobilization on the concentration of pipecolic acid and proline in mouse brain regions, liver, heart, kidney and blood plasma were analyzed. Pipecolic acid concentration in mouse brain and liver was increased after 24 or 48 h starvation, while proline concentration was not affected. Significant increases in levels of pipecolic acid were also observed in the rhombencephalon, liver and heart after 3 h immobilization. Proline in the blood plasma and kidney was decreased, while that in liver was increased, by the immobilization. Thus, the effect of such stress on concentration of pipecolic acid differed from that seen with proline. The possible involvement of pipecolic acid in synaptic mechanisms in the central nervous system and/or in pathogenesis of the diseases related to abnormal pipecolic acid metabolism should be given attention.     

Inactivation of the lys7 gene, encoding saccharopine reductase in Penicillium chrysogenum, leads to accumulation of the secondary metabolite precursors piperideine-6-carboxylic acid and pipecolic acid from alpha-aminoadipic acid

Pipecolic acid serves as a precursor of the biosynthesis of the alkaloids slaframine and swainsonine (an antitumor agent) in some fungi. It is not known whether other fungi are able to synthesize pipecolic acid. Penicillium chrysogenum has a very active alpha-aminoadipic acid pathway that is used for the synthesis of this precursor of penicillin. The lys7 gene, encoding saccharopine reductase in P. chrysogenum, was target inactivated by the double-recombination method. Analysis of a disrupted strain (named P. chrysogenum SR1-) showed the presence of a mutant lys7 gene lacking about 1,000 bp in the 3'-end region. P. chrysogenum SR1- lacked saccharopine reductase activity, which was recovered after transformation of this mutant with the intact lys7 gene in an autonomously replicating plasmid. P. chrysogenum SR1- was a lysine auxotroph and accumulated piperideine-6-carboxylic acid. When mutant P. chrysogenum SR1- was grown with L-lysine as the sole nitrogen source and supplemented with DL-alpha-aminoadipic acid, a high level of pipecolic acid accumulated intracellularly. A comparison of strain SR1- with a lys2-defective mutant provided evidence showing that P. chrysogenum synthesizes pipecolic acid from alpha-aminoadipic acid and not from L-lysine catabolism.     

Biosynthesis of slaframine, (1S,6S,8aS)-1-acetoxy-6-aminooctahydroindolizine, a parasympathomimetic alkaloid of fungal origin. 3. Origin of the pyrrolidine ring.

The phytopathogen Rhizoctonia leguminicola has previously been shown to incorporate pipecolic acid into the piperidine alkaloids 1-acetoxy-6-aminooctahydroindolizine (slaframine) and 3,4,5-trihydroxyoctahydro-1-pyrindine. In the experiments described here, resting cultures of R. leguminicola were incubated with [1-14C]- and [2-14C]malonic acid and with [1-14C]- and [2-2H]acetic acid. Both acids were incorporated into the ring systems of both alkaloids. Mass spectrometric analysis of 2H-enriched slaframine showed that the label resides in the five-membered ring and that the methyl carbon of acetate is joined to the carboxyl carbon of pipecolate. A pipecolate-dependent decarboxylation of [1-14C]malonate was demonstrated in cell-free extracts of R. leguminicola. The results account for previously unattributed carbons in the two alkaloids and suggest the formation of an eight-carbon intermediate common to both alkaloids by acylation of malonate with pipecolic acid.     

Metabolism of lysine in α-aminoadipic semialdehyde dehydrogenase-deficient fibroblasts: Evidence for an alternative pathway of pipecolic acid formation

The mammalian degradation of lysine is believed to proceed via two distinct routes, the saccharopine and the pipecolic acid routes, that ultimately converge at the level of α-aminoadipic semialdehyde (α-AASA). α-AASA dehydrogenase-deficient fibroblasts were grown in cell culture medium supplemented with either l-[α-¹⁵N]lysine or l-[ε-¹⁵N]lysine to explore the exact route of lysine degradation. l-[α-¹⁵N]lysine was catabolised into [¹⁵N]saccharopine, [¹⁵N]α-AASA, [¹⁵N]Δ¹-piperideine-6-carboxylate, and surprisingly in [¹⁵N]pipecolic acid, whereas l-[ε-¹⁵N]lysine resulted only in the formation of [¹⁵N]saccharopine. These results imply that lysine is exclusively degraded in fibroblasts via the saccharopine branch, and pipecolic acid originates from an alternative precursor. We hypothesize that pipecolic acid derives from Δ¹-piperideine-6-carboxylate by the action of Δ¹-pyrroline-5-carboxylic acid reductase, an enzyme involved in proline metabolism.     

The lysine-ketoglutarate reductase-saccharopine dehydrogenase is involved in the osmo-induced synthesis of pipecolic acid in rapeseed leaf tissues.

Higher plant responses to abiotic stresses are associated with physiological and biochemical changes triggering a number of metabolic adjustments. We focused on L-lysine catabolism, and have previously demonstrated that degradation of this amino acid is osmo-regulated at the level of lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) in Brassica napus. LKR and SDH activities are enhanced by decreasing osmotic potential and decrease when the upshock osmotic treatment is followed by a downshock osmotic one. Moreover we have shown that the B. napus LKR/SDH gene is up-regulated in osmotically-stressed tissues. The LKR/SDH activity produces alpha-aminoadipate semialdehyde which could be further converted into alpha-aminoadipate and acetyl CoA. Alternatively alpha-aminoadipate could behave as a precursor for pipecolic acid. Pipecolic acid is described as an osmoprotectant in bacteria and is co-accumulated with proline in halophytic plants. We suggest that osmo-induction of the LKR/SDH activity could be partly responsible for pipecolic acid accumulation. This proposal has been assessed in this study through pipecolic acid amounts determination in rape leaf discs subjected to various upshift and downshift osmotic treatments. Changes in pipecolic acid level actually behave as those observed for LKR and SDH activities, since it increases or decreases in rape leaf discs treated under hyper- or hypo-osmotic conditions, respectively. In addition we show that pipecolic acid level is positively correlated with the external osmotic potential as well as with the duration of the applied treatment. On the other hand pipecolic acid level is related to the availability of L-lysine and not to that of D-lysine. Collectively the results obtained demonstrate that lysine catabolism through LKR/SDH activity is involved in osmo-induced synthesis of pipecolic acid.     

Studies of lysine cyclodeaminase from Streptomyces pristinaespiralis: Insights into the complex transition NAD+ state

Lysine cyclodeaminase (LCD) catalyzes the piperidine ring formation in macrolide-pipecolate natural products metabolic pathways from a lysine substrate through a combination of cyclization and deamination. This enzyme belongs to a unique enzyme class, which uses NAD⁺ as the catalytic prosthetic group instead of as the co-substrate. To understand the molecular details of NAD⁺ functions in lysine cyclodeaminase, we have determined four ternary crystal structure complexes of LCD-NAD⁺ with pipecolic acid (LCD-PA), lysine (LCD-LYS), and an intermediate (LCD-INT) as ligands at 2.26-, 2.00-, 2.17- and 1.80 Å resolutions, respectively. By combining computational studies, a NAD⁺-mediated “gate keeper” function involving NAD⁺/NADH and Arg49 that control the binding and entry of the ligand lysine was revealed, confirming the critical roles of NAD⁺ in the substrate access process. Further, in the gate opening form, a substrate delivery tunnel between ε-carboxyl moiety of Glu264 and the α-carboxyl moiety of Asp236 was observed through a comparison of four structure complexes. The LCD structure details including NAD⁺-mediated “gate keeper” and substrate tunnel may assist in the exploration the NAD⁺ function in this unique enzyme class, and in regulation of macrolide-pipecolate natural product synthesis.     

Cadaverine, an Essential Diamine for the Normal Root Development of Germinating Soybean (Glycine max) Seeds

When the polyamine content of soybean (Glycine max) seeds was examined during the early stages of germination, the major polyamine in the cotyledons was found to be spermidine, followed by spermine; while very low concentrations of cadaverine were found. In the embryonic axes, however, cadaverine was the main polyamine and its content markedly increased 24 hours after the start of germination. When the germination of the seeds was performed in the presence of 1 millimolar α-difluoromethylornithine (DFMO), a marked decrease in the cadaverine content was found, while the other polyamines were not affected. This decrease of the cadaverine content was already noticeable after the first hours of germination. In the presence of DFMO, a pronounced elongation in the roots of the seedlings and a marked decrease in the appearance of secondary roots as compared with controls, was observed. This abnormal rooting of the seedlings caused by DFMO was almost completely reverted by the addition of 1 millimolar cadaverine. The latter also increased the appearance of secondary roots in the seedlings. The decrease in the cadaverine content produced by DFMO could be traced to a strong inhibition of lysine decarboxylase. A temporal correlation between the increase in cadaverine content and the increase in lysine decarboxylase activity was found. Both reached a maximum at the second day of germination. The activity of diamine oxidase, the cadaverine degrading enzyme, started to increase at the third day and reached a maximum between the fourth and fifth day of germination. DFMO increased the activity of diamine oxidase by about 25%. Hence, the large decrease in cadaverine content produced by DFMO has to be attributed to the in vivo suppression of lysine decarboxylase activity. Ornithine decarboxylase activity was also suppressed by DFMO, but putrescine and spermidine contents were not affected, except in the meristematic tissues. The obtained results suggest an important role for cadaverine in the normal rooting process of soybean seedlings.     

Analysis of swainsonine and its early metabolic precursors in cultures of Metarhizium anisopliae

The alpha-mannosidase inhibitor swainsonine is produced by the filamentous fungus Metarhizium anisopliae. The primary metabolite pathway from which it is derived is known to be that leading to lysine. In order to effect improvements in the yield of swainsonine it is of interest to study the changes in the intracellular levels of lysine and its biosynthetic intermediates, as well as swainsonine itself, which accompany changes in culture conditions or in the genetics of the microbe. Czapek-Dox defined medium has been used for these studies. A reversed-phase, high performance liquid chromatography procedure was developed for the analysis of lysine, saccharopine, alpha-aminoadipic acid and pipecolic acid in mycelial extracts. The method is based upon precolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC), a reagent known to be useful for the derivatization of amino-containing compounds. Elution with an acetate buffer/acetonitrile gradient effected separation of the four metabolites which were quantified by UV absorption at concentrations from 1 to 20 μg ml-1. Swainsonine concentrations were determined using a previously described enzyme-based method, but applied now to intracellular as well as extracellular samples. Analysis of mycelial extracts from the end of swainsonine accumulation in medium supplemented with L-lysine revealed the accumulation of pipecolic acid and to a lesser extent lysine compared to control mycelium. Controlling the culture medium pH to 9.0 resulted in a drop in swainsonine yield accompanied by an increase in intracellular pipecolic acid levels. Spontaneous mutants tolerant to the presence of the toxic lysine analogue 2-aminoethylcysteine (AEC) were isolated in an attempt to generate lysine over-producers, which might be expected to produce more swainsonine. Surprisingly, four independently isolated mutants produced lower yields of swainsonine, but accumulated higher levels of saccharopine. The tolerance to AEC therefore appears to be due to a reduction in the diversion of saccharopine into swainsonine biosynthesis, allowing the biosynthesis of sufficient lysine to overcome AEC competition. This revised version was published online in November 2006 with corrections to the Cover Date.     

Enhancement of hexobarbital-induced sleep by lysine and its metabolites

Lysine has been shown to be metabolized in the rat brain to pipecolic acid which is a precursor of piperidine. Lysine and its proposed metabolites in this pathway were studied for the first time for their effect on the sleeping time induced by hexobarbital in the rat. Only $\text{L}$-lysine and $\text{D}$-lysine were found to prolong sleeping time significantly without toxic effect. A 3-day pretreatment with $\text{L}$-lysine produced an even more profound sleep prolongation. In most cases sleep enhancement was accompanied by a significant shortening of the time of sleep onset. Quantification of brain hexobarbital levels in the control and treated rats indicates that prolongation of sleeping time was not produced by inhibition of hexobarbital metabolism. The sleep prolonging effect of lysine, therefore, may be a direct action of lysine, or the metabolite(s) derived $\text{in}$$\text{vivo}$ from lysine, on the central nervous system.     

A Convenient Method for the Quantitative Determination of Pipecolic Acid

The quantitative determination of pipecolic acid was examined.

The reaction of 3% ninhydrin solution in n-butanol, saturated with citrate buffer (pH 4.2), with pipecolic acid in boiling water for 3 min yielded the colored products showing λ_max at 570 mμ, but with proline hardly yielded those products. By the colorimetry proposed, it is possible to determine the amount of pipecolic acid in the sample containing proline no more than 50 times the amount of the pipecolic acid, directly from the calibration curve using pipecolic acid.

The method for removal of amino acids from the sample containing pipecolic acid and proline was examined and discussed.     

Excretion and uptake of cadaverine by CadB and its physiological functions in Escherichia coli

The functions of the putative cadaverine transport protein CadB were studied in Escherichia coli. CadB had both cadaverine uptake activity, dependent on proton motive force, and cadaverine excretion activity, acting as a cadaverine-lysine antiporter. The Km values for uptake and excretion of cadaverine were 20.8 and 303 microM respectively. Both cadaverine uptake and cadaverine-lysine antiporter activities of CadB were functional in cells. Cell growth of a polyamine-requiring mutant was stimulated slightly at neutral pH by the cadaverine uptake activity and greatly at acidic pH by the cadaverine-lysine antiporter activity. At acidic pH, the operon containing cadB and cadA, encoding lysine decarboxylase, was induced in the presence of lysine. This caused neutralization of the extracellular medium and made possible the production of CO(2) and cadaverine and aminopropylcadaverine instead of putrescine and spermidine. The induction of the cadBA operon also generated a proton motive force. When the cadBA operon was not induced, the expression of the speF-potE operon, encoding inducible ornithine decarboxylase and a putrescine-ornithine antiporter, was increased. The results indicate that the cadBA operon plays important roles in cellular regulation at acidic pH.     

The formation of cadaverine, aminopropylcadaverine and N,N bis (3-aminopropyl) cadaverine in mycorrhizal and phytopathogenic fungi

Radiolabeled products that co-chromatographed with authentic standards of cadaverine, aminopropylcadaverine (APC) and N,N bis (3-aminopropyl) cadaverine (3 APC) were isolated following the decarboxylation of a [U-¹⁴C] lysine substrate by fungal lysine decarboxylase extracts. The identity of 3 APC was confirmed by nuclear magnetic resonance (NMR) spectroscopy. The inhibition of the enzymes S-adenosylmethionine decarboxylase (AdoMetDC) and spermidine synthase led to significant reductions in the recovery of radiolabeled 3 APC. These results show that a range of ectomycorrhizal and plant pathogenic fungi can convert lysine into the higher homologues of cadaverine. These cadaverine homologues appear to be formed via the action of AdoMetDC and spermidine and spermine synthases, although the operation of an additional route for the biosynthesis of these compounds from L-aspartic-β-semialdehyde is a possibility.     

Internal pH crisis, lysine decarboxylase and the acid tolerance response of Salmonella typhimurium

Salmonella typhimurium possesses an adaptive response to acid that increases survival during exposure to extremely low pH values. The acid tolerance response (ATR) includes both log-phase and stationary-phase systems. The log-phase ATR appears to require two components for maximum acid tolerance, namely an inducible pH homeostasis system, and a series of acid-shock proteins. We have discovered one of what appears to be a series of inducible exigency pH homeostasis systems that contribute to acid tolerance in extreme acid environments. The low pH-inducible lysine decarboxylase was shown to contribute significantly to pH homeostasis in environments as low as pH 3.0. Under the conditions tested, both lysine decarboxylase and σ^s-dependent acid-shock proteins were required for acid tolerance but only lysine decarboxylase contributed to pH homeostasis. The cadBA operon encoding lysine decarboxylase and a lysine/cadaverine antiporter were cloned from S. typhimurium and were found to be 79% homologous to the cadBA operon from Escherichia coli . The results suggest that S. typhimurium has a variety of means of fulfilling the pH homeostasis requirement of the ATR in the form of inducible amino acid decarboxylases.