Pipecolic acid biosynthesis in Rhizoctonia leguminicola. I. The lysine saccharopine, delta 1-piperideine-6-carboxylic acid pathway

The biosynthesis of pipecolic acid from L-lysine in the fungal parasite, Rhizoctonia leguminicola has been reinvestigated. Pipecolate is then utilized to form the toxic octahydroindolizine alkaloids, slaframine and swainsonine. Incorporation studies of L-versus D-[U-14C]lysine into R. leguminicola metabolites confirmed earlier findings that L-lysine is the predominant substrate for pipecolate formation and D-lysine for alpha-N-acetyllysine (concerned in lysine catabolism). However [alpha-15N]lysine, not [epsilon-15N]lysine as previously reported, labeled pipecolate. Such findings implied that delta 1-piperideine-6-carboxylate, not delta 1-piperideine-2-carboxylate, was formed from lysine and was the immediate precursor of pipecolate. Evidence from cell-free enzyme systems established the following biosynthetic events: L-lysine A----saccharopine B----delta 1-piperideine-6-carboxylate C----pipecolate. Products of reactions A and C were identified from biological and chemical considerations. Reaction B was carried out by a previously undescribed flavin enzyme termed saccharopine oxidase. The product of reaction B, which reacted with p-dimethylaminobenzaldehyde, was reduced with Na-CNB2H3. Its NMR spectrum was identical with that of deuteriated pipecolate prepared from authentic delta 1-piperideine-6-carboxylate, but not from authentic delta 1-piperideine-2-carboxylate. Reaction B represents a branching of primary lysine metabolism from saccharopine to a secondary pathway leading to pipecolate and to octahydroindolizine alkaloids in R. leguminicola.     

A 15N NMR study on D-lysine metabolism in Neurospora crassa

The metabolic relationship of D-lysine, L-lysine, and L-pipecolic acid has been investigated in Neurospora crassa. Kinetic experiments show that radioactivity from D-lysine is efficiently incorporated into L-pipecolic acid and that this metabolite is converted to L-lysine. The alpha-amino group from D-[alpha-15N]lysine is lost in the course of its conversion to L-pipecolic acid and is trapped by pyruvate and alpha-keto glutarate to give L-[alpha-15N]alanine and L-[alpha-15N]glutamic acid. These amino acids are devoid of any label, however, when D-[epsilon-15N]lysine is applied to the fungus. As determined by mass and 15N NMR spectrometry the label from D-[epsilon-15N]lysine migrate via L-pipecolic acid into the alpha position of L-lysine, i.e. D-[epsilon-15N]lysine is converted to L-[alpha-15N]lysine. L-Pipecolic acid functions as an intermediate in this conversion.     

Multiple and interconnected pathways for L-lysine catabolism in Pseudomonas putida KT2440

L-lysine catabolism in Pseudomonas putida KT2440 was generally thought to occur via the aminovalerate pathway. In this study we demonstrate the operation of the alternative aminoadipate pathway with the intermediates D-lysine, L-pipecolate, and aminoadipate. The simultaneous operation of both pathways for the use of L-lysine as the sole carbon and nitrogen source was confirmed genetically. Mutants with mutations in either pathway failed to use L-lysine as the sole carbon and nitrogen source, although they still used L-lysine as the nitrogen source, albeit at reduced growth rates. New genes were identified in both pathways, including the davB and davA genes that encode the enzymes involved in the oxidation of L-lysine to delta-aminovaleramide and the hydrolysis of the latter to delta-aminovalerate, respectively. The amaA, dkpA, and amaB genes, in contrast, encode proteins involved in the transformation of Delta1-piperidine-2-carboxylate into aminoadipate. Based on L-[U-13C, U-15N]lysine experiments, we quantified the relative use of pathways in the wild type and its isogenic mutants. The fate of 13C label of L-lysine indicates that in addition to the existing connection between the D- and L-lysine pathways at the early steps of the catabolism of L-lysine mediated by a lysine racemase, there is yet another interconnection at the lower end of the pathways in which aminoadipate is channeled to yield glutarate. This study establishes an unequivocal relationship between gene and pathway enzymes in the metabolism of L-lysine, which is of crucial importance for the successful colonization of the rhizosphere of plants by this microorganism.     

Pipecolic acid biosynthesis in Rhizoctonia leguminicola. II. Saccharopine oxidase: a unique flavin enzyme involved in pipecolic acid biosynthesis

The fungal parasite Rhizoctonia leguminicola produces two indolizidine alkaloids, slaframine and swainsonine, of physiological interest. These alkaloids are biosynthesized from pipecolic acid which in turn is derived from L-lysine in this fungus as shown in the accompanying paper (Wickwire, B.M., Harris, C.M., Harris, T.M., and Broquist, H.P. (1989) J. Biol. Chem. 265, 14742-14747): L-lysine----saccharopine----delta 1----piperideine-6- carboxylate----pipecolate. This paper concerns the discovery, purification, and properties of a flavoenzyme, termed saccharopine oxidase, which carries out the oxidative cleavage of saccharopine as follows: Saccharopine + O2----delta 1-piperidine-6-carboxylate + glutamate + H2O2 The enzyme was purified 2,000-fold to homogeneity (polyacrylamide gel electrophoresis) in 14% yield from R. leguminicola mycelia, and had a native molecular mass of about 45,000 daltons by gel filtration (fast protein liquid chromatography Superose). Evidence for the presence of a flavin in the enzyme was drawn from these considerations: (a) the enzyme, while oxidatively cleaving saccharopine, concomitantly reduces 2,6-dichlorophenolindophenol; (b) the purified enzyme has a fluorescence spectrum typical of flavins; and (c) the enzyme requires oxygen and produces hydrogen peroxide. Good correlation was shown with purified saccharopine oxidase between disappearance of saccharopine with the concomitant appearance of delta 1-piperideine-6-carboxylate plus glutamate. The enzyme has a pH optimum about 6 and a Km for saccharopine of 0.128 mM. The enzyme apparently exists in R. leguminicola to shunt saccharopine, a major lysine metabolite, into a secondary pathway of lysine metabolism leading to pipecolate and subsequently to slaframine and swainsonine.     

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.     

Organization of Enzymes in the Common Aromatic Synthetic Pathway: Evidence for Aggregation in Fungi

Centrifugation in sucrose density gradients of partially purified extracts from six species of fungi, i.e., Rhizopus stolonifer, Phycomyces nitens, Absidia glauca (Phycomycetes), Aspergillus nidulans (Ascomycetes), Coprinus lagopus, and Ustilago maydis (Basidiomycetes), indicate that the five enzymes catalyzing steps two to six in the prechorismic acid part of the polyaromatic synthetic pathway sediment together. The sedimentation coefficients for these enzymes are very similar in the six species and are comparable to those previously observed for the multienzyme complexes (arom aggregates) of Neurospora crassa and Saccharomyces cerevisiae. These results are interpreted as indicating the presence in each of these fungi of arom aggregates, presumably encoded by arom gene clusters similar to those in N. crassa and S. cerevisiae. Evidence has also been obtained for the presence in two species (A. nidulans and U. maydis) and the absence in the other four species of a second dehydroquinase isozyme which is distinguishable from the synthetic activity on the basis of both thermostability tests and S values. This second dehydroquinase, which is apparently involved in the catabolism of quinic acid via a pathway similar to that in N. crassa, is inducible in A. nidulans (as it is in N. crassa), but constitutive in U. maydis. These comparative findings are discussed in relation to the organization, evolution, and possible functional relationships of synthetic and catabolic aromatic pathways in fungi.     

Influence of Linoleate Hydroperoxide Minor Isomers on GLC Analysis of Oleate Hydroperoxides

The distribution of the methylcitric acid cycle and the modified ^-oxidation pathway for propionate catabolism was surveyed in yeasts and filamentous fungi, mainly by comparing the activities of the key enzymes. All the six tested species of filamentous fungi belonging to five genera and 21 species of yeasts belonging to eleven genera were found to catabolize propionate through the methylcitric acid cycle, with the exception of Candida rugosa and one group of strains of C. catenulata, which catabolize propionate through the ß-oxidation pathway. From the observed diversity of propionate catabolism among closely related strains or species, it was assumed that different minor pathways evolved from universal metabolic pathways, such as the citric acid cycle and the ^-oxidation pathway for fatty acids, in later stages of an evolutionary history.     

Carnitine biosynthesis in Neurospora crassa: enzymatic conversion of lysine to epsilon-N-trimethyllysine.

The enzymatic conversion of L-lysine, epsilon-N-trimethyl-L-lysine the first series of reactions in the biosynthesis of carnitine in Neurospora crassa, proceeds via sequential methylation of free L-lysine, epsilon-N-methyl-L-lysine, and epsilon -N-dimethyl-L-lysine. The latter two compounds have been shown to be intermediates in the biosynthesis of carnitine by radioisotope dilution and incorporation experiments in growing cultures of N. crassa 33933 (lys-) and 38706 (met-). Methionine but not choline, has been recognized as an effective methyl donor in vivo. Inclusion of choline in the growth medium of strain 33933 does, however, enhance incorporation of the methyl groups of L-[methyl-3H]methionine into carnitine in an apparent "sparing" effect on methionine synthesis. Studies in cell-free extracts of the lysine auxotroph strain 33933 of N. crassa have established that lysine and epsilon-N-methyl and epsilon-N-dimethyllysine are enzymatically methylated, with S-adenosyl-L-methionine as the methyl group donor. The enzyme system appears to have no essential cofactors. Lysine does not induce synthesis of the enzyme system in the wild-type strain 262, whereas both carnitine and epsilon-N-trimethyllysine repress its synthesis in strain 33933.     

Lysine epsilon-aminotransferase, the initial enzyme of cephalosporin biosynthesis in actinomycetes

Streptomyces clavuligerus, Streptomyces lipmanii and Nocardia (formerly Streptomyces) lactamdurans are Gram-positive mycelial bacteria that produce medically important beta-lactam antibiotics (penicillins and cephalosporins including cephamycins) that are synthesized through a series of reactions starting from lysine, cysteine and valine. L-lysine epsilon-aminotransferase (LAT) is the initial enzyme in the two-step conversion of L-lysine to L-alpha-aminoadipic acid, a specific precursor of all penicillins and cephalosporins. Whereas S. clavuligerus uses LAT for cephalosporin production, it uses the cadaverine pathway for catabolism when lysine is the nitrogen source for growth. Although the cadaverine path is present in all examined streptomycetes, the LAT pathway appears to exist only in beta-lactam-producing strains. Genetically increasing the level of LAT enhances the production of cephamycin. LAT is the key rate-limiting enzyme in cephalosporin biosynthesis in S. clavuligerus strain NRRL 3585. This review will summarize information on this important enzyme.     

Lysine Catabolism in Barley (Hordeum vulgare L.)

Lysine catabolism in seedlings of barley (Hordeum vulgare L. var. Emir) was studied by direct injection of the following tracers into the endosperm of the seedlings: aspartic acid-3-(14)C, 2-aminoadipic acid-1-(14)C, saccharopine-(14)C, 2,6-diaminopimelic acid-1-(7)-(14)C, and lysine-1-(14)C. Labeled saccharopine was formed only after the administration of either labeled 2,6-diaminopimelic acid or labeled lysine to the seedlings. The metabolic fate of the other tracers administered also supported a catabolic lysine pathway via saccharopine, and apparently proceeding by a reversal of some of the biosynthetic steps of the 2-aminoadipic acid pathway known from lysine biosynthesis in most fungi. Pipecolic acid seems not to be on the main pathway of l-lysine catabolism in barley seedlings.     

Catabolism of L-lysine by Pseudomonas aeruginosa.

Pseudomonas aeruginosa PACI grows poorly on L-lysine as sole source of carbon but mutant derivatives which grow rapidly were readily isolated. Studies with one such mutant, P. aeruginosa PAC586, supported the existence of a route for L-lysine catabolism which differes from those reported previously in other species of Pseudomonas. The postulated route, the cadaverine or decarboxylase pathway, is initiated by the decarboxylation of L-lysine and involves the following steps: L-lysine leads to cadverine leads to I-piperideine leads 5-aminovalerate leads to glutarate semialdehyde leads glutarate. Evidence for this pathway is based on the characterization of the pathway reactions and the induction of the corresponding enzymes by growth on L-lysine. The first three enzymes were also induced by growth on cadaverine and to a lesser extent by 5-aminovalerate. No evidence was obtained for the presence of pathways involving L-lysine 2-monooxygenase or L-pipecolate dehydrogenase, but another potential route for L-lysine catabolism initiated by L-lysine 6-aminotransferase was detected. Studies with mutants unable to grow on L-lysine supported the existence of more than one catabolic pathway for L-lysine in this organism and indicated that all routes converge on a pathway for glutarate catabolism which generates acetyl-CoA. Pipecolate catabolism also appeared to converge on the glutarate pathway in P. AERUGINOSA. The results suggested that the growth rate of the parental strain is limited by the rate of transport and/or decarboxylation of L-lysine. The cadaverine pathway was present, but not so highly induced, in the parental strain P. aeruginosa PACI. Pseudomonas fluorescens contained enzymes of both the cadaverine (decarboxylase) and oxygenase pathways, strains of P. putida (biotypes A and B) contained enzymes of the oxygenase pathway but not the decarboxylase pathway and P. multivorans appeared deficient in both. All these species possessed L-lysine aminotransferase activity.     

Stereospecific abstraction of epsilon-pro-R-hydrogen of L-lysine by L-lysine epsilon-dehydrogenase from Agrobacterium tumefaciens

The stereochemical aspects of the L-lysine epsilon-dehydrogenase reaction were examined with (6R)-L-[6-3H]lysine and (6S)-DL-[6-3H]lysine. When (6S)-DL-[6-3H]lysine was used as a substrate, the tritium was found in the product, delta 1-piperideine-6-carboxylate. In contrast, the radioactivity from (6R)-L-[6-3H]lysine was not retained in the product. Thus, the pro-R hydrogen at the prochiral C-6 carbon of L-lysine is specifically abstracted by the enzyme: the enzyme behaves stereochemically as an amino acid D-dehydrogenase.     

The davDT operon of Pseudomonas putida, involved in lysine catabolism, is induced in response to the pathway intermediate delta-aminovaleric acid

Pseudomonas putida KT2440 is a soil microorganism that attaches to seeds and efficiently colonizes the plant's rhizosphere. Lysine is one of the major compounds in root exudates, and P. putida KT2440 uses this amino acid as a source of carbon, nitrogen, and energy. Lysine is channeled to delta-aminovaleric acid and then further degraded to glutaric acid via the action of the davDT gene products. We show that the davDT genes form an operon transcribed from a single sigma70-dependent promoter. The relatively high level of basal expression from the davD promoter increased about fourfold in response to the addition of exogenous lysine to the culture medium. However, the true inducer of this operon seems to be delta-aminovaleric acid because in a mutant unable to metabolize lysine to delta-aminovaleric acid, this compound, but not lysine, acted as an effector. Effective induction of the P. putida P(davD) promoter by exogenously added lysine requires efficient uptake of this amino acid, which seems to proceed by at least two uptake systems for basic amino acids that belong to the superfamily of ABC transporters. Mutants in these ABC uptake systems retained basal expression from the davD promoter but exhibited lower induction levels in response to exogenous lysine than the wild-type strain.     

Nitrate regulation of alpha-aminoadipate reductase formation and lysine inhibition of its activity in Penicillium chrysogenum and Acremonium chrysogenum

alpha-Aminoadipate reductase (alpha-AAR) is a key enzyme in the branched pathway for lysine and beta-lactam biosynthesis of filamentous fungi since it competes with alpha-aminoadipyl-cysteinyl-valine synthetase for their common substrate L-alpha-aminoadipic acid. The alpha-AAR activity in two penicillin-producing Penicillium chrysogenum strains and two cephalosporin-producing Acremonium chrysogenum strains has been studied. The alpha-AAR activity peaked during the growth-phase preceding the onset of antibiotic production, which coincides with a decrease in alpha-AAR activity, and was lower in high penicillin- or cephalosporin-producing strains. The alpha-AAR required NADPH for enzyme activity and could not use NADH as electron donor for reduction of the alpha-aminoadipate substrate. The alpha-AAR protein of P. chrysogenum was detected by Western blotting using anti-alpha-AAR antibodies. The mechanism of lysine feedback regulation in these two filamentous fungi involves inhibition of the alpha-AAR activity but not repression of its synthesis by lysine. This is different from the situation in yeasts where lysine feedback inhibits and represses alpha-AAR. Nitrate has a strong negative effect on alpha-AAR formation as shown by immunoblotting studies of alpha-AAR. The nitrate effect was reversed by lysine.     

Comparative biosynthesis of ornithine and lysine by Mycoplasma and L forms

Smith, Paul F. (University of South Dakota, Vermillion). Comparative biosynthesis of ornithine and lysine by Mycoplasma and L forms. J. Bacteriol. 92:164-169. 1966.-Seven species of Mycoplasma, two L forms not requiring salt and their parent bacteria, and two yeasts were examined for enzymes involved in the biosynthesis of ornithine and lysine. All organisms tested, except two species of Mycoplasma and the yeasts, decarboxylated meso-alpha, epsilon-diaminopimelic acid. None of the Mycoplasma species or L forms was capable either of reducing alpha-aminoadipic acid to its semialdehyde or of incorporating alpha-aminoadipic acid-6-C(14) into lysine. All organisms, except the yeasts and Mycoplasma sp. caprine strain 14, acetylated glutamic acid, and all organisms possessed N(alpha)-acetyl-l-ornithine:2-oxo-glutarate aminotransferase activity. N(alpha)-acetylornithase activity was negligible in all organisms except Proteus and its L form. No transacetylation between acetylglutamic acid and ornithine, and vice versa, was demonstrable in any of the organisms. Mycoplasma species appear to possess the bacterial pathway to lysine. Ornithine does not appear to arise from glutamic acid.     

Molecular mechanisms of peroxisome biogenesis in yeasts

Peroxisomes contain oxidases, which generate hydrogen peroxide, and catalase, which degrades this toxic compound. Another characteristic function of each eukaryotic peroxisome, from yeast to man, is fatty acid β-oxidation. However, a variety of other metabolic pathways are also located in peroxisomes. In fungi, peroxisomes contain enzymes involved in catabolism of unusual carbon and nitrogen sources (methanol, purines, D-amino acids, pipecolynic acid, sarcosine, glycolate, spermidine, etc.), as well as biosynthesis of lysine in yeasts and penicillin in mycelial fungi. Impairment of the peroxisome structure and functions causes many human disorders. Similar defects were identified in yeast mutants defective in peroxisome biogenesis. Peroxisome biogenesis has been actively studied using unicellular and multicellular model systems over the last two decades. It was observed that many aspects of peroxisome biogenesis and proteins involved in the process display striking similarity among all eukaryotes from yeasts to humans. Yeasts provide a convenient model system for this kind of research. The review summarizes the data on the molecular events of peroxisome biogenesis, the functions of peroxine proteins, the import of peroxisomal matrix and membrane proteins, and the mechanisms of peroxisome division and inheritance.     

Metabolic and secretory responses of parotid cells to cationic amino acids. Oxidation of the amino acids and interference with the oxidation of D-glucose or endogenous nutrients.

Cationic amino acids were recently found to stimulate amylase release from rat parotid cells. The possible relevance of their oxidative catabolism to such a secretory stimulation was investigated. D-Glucose, which was efficiently metabolized in parotid cells and which augmented O2 uptake above basal value, failed to affect basal or stimulated amylase release. L-Arginine, L-lysine and L-histidine failed to stimulate the oxidation of either exogenous D-[6-14C]glucose or endogenous nutrients in cells pre-labelled with [U-14C]palmitate or L-[U-14C]glutamine. The oxidation of L-[U-14C]arginine, L-[U-14C]ornithine, L-[U-14C]lysine and L-[U-14C]histidine, all tested at a 10 mM concentration, was much lower than that of D-[U-14C]glucose (5.6 mM). These findings argue against the view that the stimulation of amylase release by cationic amino acids would be related to their role as a source of energy in the parotid cells.     

Inhibition and repression of homocitrate synthase by lysine in Penicillium chrysogenum

Homocitrate synthase in the first enzyme of the lysine biosynthetic pathway. It is feedback regulated by L-lysine. Lysine decreases the biosynthesis of penicillin (determined by the incorporation of [14C]valine into penicillin) by inhibiting and repressing homocitrate synthase, thereby depriving the cell of alpha-aminoadipic acid, a precursor of penicillin. Lysine feedback inhibited in vivo the biosynthesis and excretion of homocitrate by a lysine auxotroph, L2, blocked in the lysine pathway after homocitrate. Neither penicillin nor 6-aminopenicillanic acid exerted any effect at the homocitrate synthase level. The molecular mechanism of lysine feedback regulation in Penicillium chrysogenum involved both inhibition of homocitrate synthase activity and repression of its synthesis. In vitro studies indicated that L-lysine feedback inhibits and represses homocitrate synthase both in low- and high-penicillin-producing strains. Inhibition of homocitrate synthase activity by lysine was observed in cells in which protein synthesis was arrested with cycloheximide. Maximum homocitrate synthase activity in cultures of P. chrysogenum AS-P-78 was found at 48 h, coinciding with the phase of high rate of penicillin biosynthesis.     

1-Deoxygalactonojirimycin-lysine hybrids as potent D-galactosidase inhibitors

Cyclization by double reductive amination of L-arabino-hexos-5-ulose with suitably protected D- as well as L-lysine derivatives provided 1-deoxygalactonojirimycin lysine hybrids without any observable epimer formation at C-5. Modifications on the lysine moiety by acylation gave access to lipophilic derivatives which exhibited excellent D-galactosidase inhibitory activities.     

Catabolism of lysine in Penicillium chrysogenum leads to formation of 2-aminoadipic acid, a precursor of penicillin biosynthesis.

Penicillium chrysogenum L2, a lysine auxotroph blocked in the early steps of the lysine pathway before 2-aminoadipic acid, was able to synthesize penicillin when supplemented with lysine. The amount of penicillin produced increased as the level of lysine in the media was increased. The same results were observed in resting-cell systems. Catabolism of [U-14C]lysine by resting cells and batch cultures of P. chrysogenum L2 resulted in the formation of labeled saccharopine and 2-aminoadipic acid. Formation of [14C]saccharopine was also observed in vitro when cell extracts of P. chrysogenum L2 and Wis 54-1255 were used. Saccharopine dehydrogenase and saccharopine reductase activities were found in cell extracts of P. chrysogenum, which indicates that lysine catabolism may proceed by reversal of the two last steps of the lysine biosynthetic pathway. In addition, a high lysine:2-ketoglutarate-6-aminotransferase activity, which converts lysine into piperideine-6-carboxylic acid, was found in cell extracts of P. chrysogenum. These results suggest that lysine is catabolized to 2-aminoadipic acid in P. chrysogenum by two different pathways. The relative contribution of lysine catabolism in providing 2-aminoadipic acid for penicillin production is discussed.