Relationship of nitrogen to the onset and suppression of ligninolytic activity and secondary metabolism in Phanerochaete chrysosporium

Ligninolytic activity in the white-rot fungus Phanerochaete chrysosporium was previously found not to be induced by lignin, but to develop in cultures in response to nitrogen starvation. Added NH ₄ ⁺ suppressed existing activity. The present study examined amino acid profiles and protein concentrations during onset of ligninolytic activity (synthetic ¹⁴C-lignin¹⁴CO₂) in nitrogen-limited cultures, and defined some characteristics of subsequent suppression by added nutrient nitrogen. During the transition between depletion of medium nitrogen and the onset of ligninolytic activity, total free intracellular amino acids increased, then rapidly decreased; changes in glutamate concentration played a major role. Intracellular protein concentration fluctuated in a manner roughly converse to that of the concentration of free amino acids. Protein turnover was rapid (5–7%/h) during the transition period. Glutamate, glutamine, and histidine were the most effective of 14 nitrogenous compounds in suppressing ligninolytic activity after its onset. The suppressive effect was not mediated through carbon (glucose)-catabolite repression or by alterations in culture pH. Activities responsible for oxidation of lignin and the ligninrelated phenol, 4-hydroxy-3-methoxyacetophenone, responded similarly to added nitrogen. Synthesis of a secondary metabolite, veratryl alcohol, like lignin oxidation, was suppressed quite sharply by glutamate and significantly by NH ₄ ⁺ . Results indicate that nitrogen metabolism affects ligninolytic activity as a part of secondary metabolism, and suggest a role for glutamate metabolism in regulating this phase of culture development.Non-Standard Abbreviations DMS 2,2-dimethylsuccinate - GLC gas-liquid chromatography - TCA trichloroacetic acid     

Metabolism of a phenylcoumaran substructure lignin model compound in ligninolytic cultures of Phanerochaete chrysosporium

The degradation of the phenylcoumaran substructure model compound methyl dehydrodiconiferyl alcohol by the white-rot wood decay fungus Phanerochaete chrysosporium was investigated using culture conditions optimized for lignin oxidation. Initial attack was in the cinnamyl alcohol side chain, which was oxidized to a glycerol structure. This was subsequently converted by loss of the two terminal carbon atoms, C and C, to yield a C-aldehyde structure, which was further oxidized to the C-acid compound. The next detected intermediate, a phenylcoumarone, was produced by double bond formation between C and C, and oxidation of the C-alcohol to an aldehyde group. Further oxidation of C to an acid yielded the next intermediate. The final identified degradation product was veratric acid. No products from the 5-substituted aromatic ring, and no phenolic products, were found. The initial glycerol-containing intermediate was a mixture of the threo and erythro forms, and no optical activity could be found, suggesting that its formation might have involved nonstereospecific C-C epoxidation followed by non-enzymatic hydrolysis of the epoxide.Abbreviations TLC thin layer chromatography - LDA lithium diisopropyl amide - DDQ 2,3-dichloro-5,6-dicyanobenzoquinone - MS mass spectrometry - UV ultraviolet spectroscopy     

Ligninolytic activity and levels of ammonia assimilating enzymes in Sporotrichum pulverulentum

Levels of ammonia-assimilating enzymes (glutamate dehydrogenase, glutamine synthetase, glutamate synthase) were determined in extracts of Sporotrichum pulverulentum grown under different conditions with respect to both nitrogen source and concentration. Evolution of ¹⁴CO₂ from ¹⁴C-synthetic lignin by fungal cultures grown under parallel conditions was also determined as a measure of lignin decomposition and the suppressive effect of nitrogen on ligninolysis confirmed. Under low nitrogen conditions, fungal extracts exhibited relatively high levels of NADP-dependent glutamate dehydrogenase and glutamine synthetase dehydrogenase. Conversely, in high nitrogen extracts, lower levels of NADP-dependent glutamate dehydrogenase and glutamine synthetase activity, and higher levels of NAD-dependent glutamate dehydrogenase, were recorded. Possible effects of enzyme activities on intracellular pool concentrations of glutamate/glutamine, and the implications for the regulation of lignin metabolism, are discussed.A preliminary report was presented at The Ekman Days 1981, International Symposium on Wood and Pulping Chemistry, Stockholm, Sweden, June 9–12, 1981.     

Influence of culture parameters on lignin metabolism byPhanerochaete chrysosporium

Culture parameters influencing metabolism of synthetic¹⁴C-lignins to¹⁴CO₂ in defined media have been studied in shallow batch cultures of the ligninolytic wood-destroying HymenomycetePhanerochaete chrysosporium Burds. Study of the effect of O₂ concentration in the gas phase above non-agitated cultures indicated essentially complete absence of attack on the lignin polymer at 5% O₂ in N₂, and a 2- to 3-fold enhancement by 100% O₂ as compared to air (21% O₂). Agitation of the cultures resulting in the formation of mycelial pellets greatly suppressed lignin decomposition. The optimum culture pH for lignin decomposition was 4 to 4.5, with marked suppression above 5.5 and below 3.5. The source of nutrient nitrogen (NO ₃ ^– , NH ₄ ⁺ , amino acids) had little influence on lignin decomposition, but the concentration of nitrogen was critical; decomposition at 24 mM was only 25–35% of that at 2.4 mM N. Thiamine was the only vitamin required for growth and lignin decomposition. Under the optimum conditions developed, decomposition of 5 mg of synthetic lignin was accompanied by utilization of approximately 100 mg of glucose. The influence of the various culture parameters was analogous for metabolism of synthetic lignin labeled in the ring-,side chain-, and methoxyl carbon atoms.     

Ligninolytic system of Phanerochaete chrysosporium: Inhibition by o-Phthalate

The degradation rate of [synthetic-¹⁴C]-lignin to ¹⁴CO₂ by Phanerochaete chrysosporium in cultures buffered with 0.01 M 2,2-dimethylsuccinate (DMS) was twice that in 0.01 M o-phthalate-buffered cultures. This difference could be totally accounted for by o-phthalate inhibition of the activity of the ligninolytic system. ¹⁴CO₂ production from ring-, sidechain-, and methoxyl-labeled lignins was inhibited, the degree of inhibition being dependent on o-phthalate concentration. Oxidations of ¹⁴C-glucose, ¹⁴C-acetovanillone, and ¹⁴C-apocynol were not inhibited; thus o-phthalate is not a general inhibitor, and might inhibit activities involved in attack of the lignin polymer. DMS is a suitable buffer for the ligninolytic system. Degradation rates of ring-labeled lignin to ¹⁴CO₂ of 10–15% in 24 h were obtained consistently over the pH range 3.6–4.5, with an optimum near pH 4.0.Non-Standard Abbreviations DMS dimethylsuccinate     

Biosynthesis of the secondary metabolite veratryl alcohol in relation to lignin degradation in Phanerochaete chrysosporium

The lignin-degrading basidiomycete Phanerochaete chrysosporium synthesizes veratryl alcohol (3,4-dimethoxybenzyl alcohol) via phenylalanine, 3,4-dimethoxycinnamyl alcohol and veratrylglycerol. Study of the conversion of 3,4-dimethoxycinnamyl alcohol to veratrylglycerol and veratryl alcohol showed is to be (a) catalyzed by a secondary metabolic system, (b) markedly suppressed by culture agitation, and (c) strongly inhibited by l-glutamate. The amount of veratryl alcohol synthesized de novo was positively correlated with the O₂ concentration after primary growth. Other work has shown that the cinnamyl alcohol terminal residue in a lignin substructure model compound is degraded via arylglycerol and benzyl alcohol structures in ligninolytic cultures of P. chrysosporium, and that the ligninolytic system exhibits traits (a)-(c) above. Ligninolytic activity is also strongly and positively correlated with O₂ concentration. The results here suggest, therefore, that the actual biosynthetic secondary metabolic product is 3,4-dimethoxycinnamyl alcohol, but that this is degraded by the ligninolytic system to veratryl alcohol via veratrylglycerol. Veratryl alcohol is only slowly metabolized by the fungus, and accumulates.Non-standard abbreviation tlc thin layer chromatography     

Fungal degradation of kraft lignin and lignin sulfonates prepared form synthetic 14C-lignins

Kraft lignins (KL), bleached kraft lignins (BKL), and lignin sulfonates (LS) were prepared from synthetic ¹⁴C-lignins labeled in the aromatic nuclei or in the propyl side chains. These and control lignins (CL) were incubated with the lignin-decomposing white-rot fungus, Phanerochaete chrysosporium Burds., in a defined culture medium containing cellulose as growth substrate. Decomposition was monitored by measuring the ¹⁴CO₂ evolved. Average percentages of the [ring-¹⁴C]- and [side chain-¹⁴C]-lignins, respectively, recovered as ¹⁴CO₂ at the cessation of ¹⁴CO₂ evolution were: KL, 41 and 31; BKL, 42 and 26; LS, 28 and 21; and CL, 26 and 24. Gel permeation chromatography of radiolabeled materials extracted from spent cultures showed that substantial degradation to nonvolatile products had occurred. The polymeric components in the extracts were further degraded in fresh cultures. These results indicate that industrial lignins are significantly bioalterable, and that under favorable conditions industrial lignins are substantially biodegradable.     

Inhibition of Glutamate Dehydrogenase in Brain Mitochondria and Synaptosomes by Mg2+ and Polyamines: A Possible Cause for Its Low In Vivo Activity

Abstract: Magnesium and the polyamines putrescine, spermidine, and spermine inhibited the activity of glutamate dehydrogenase in permeabilized rat brain mitochondria in a concentration-dependent manner. The inhibitory effect was observed on both the reductive amination of 2-oxoglutarate and oxidative deamination of glutamate, as well as in the presence and absence of ADP and leucine, the allosteric activators of the enzyme. Kinetic studies at various concentrations of substrates showed that inhibition by magnesium and spermine was very pronounced at 2-oxoglutarate concentrations less than 0.5 m M and NADH levels less than 0.08 m M . The presence of the former compounds also accentuated the inhibitory effect of high concentrations of 2-oxoglutarate (>2.0 m M ) and NADH (>0.32 m M ). Addition of magnesium and spermine to suspensions of synaptosomes decreased the amount of ammonia produced from glutamate. It is suggested that polyamines and magnesium, normal constituents of mammalian brain, are responsible, at least in part, for the low glutamate dehydrogenase activity in vivo.     

Wood stimulates the demethoxylation of [O14CH3]-labeled lignin model compounds by the white-rot fungi Phanerochaete chrysosporium and Phlebia radiata

Mineralization of polymeric wood lignin and its substructures is a result of complex reactions involving oxidizing and reducing enzymes and radicals. The degradation of methoxyl groups is an essential part of this process. The presence of wood greatly stimulates the demethoxylation of a non-phenolic lignin model compound (a [O¹⁴CH₃]-labeled β-O-4 dimer) by the lignin-degrading white-rot fungi Phlebia radiata and Phanerochaete chrysosporium. When grown on wood, both fungi produced up to 47 and 40% ¹⁴CO₂ of the applied ¹⁴C activity, respectively, under air and oxygen in 8 weeks. Without wood, the demethoxylation of the dimer by both fungi was lower, varying between 0.5 and 35%. Addition of nutrient nitrogen together with glucose decreased demethoxylation when the fungi were grown on spruce wood under air. Because the evolution of ¹⁴CO₂ in the absence of wood was poor, the fungi may have preferably used wood as a carbon and nitrogen source. The amount of fungal mycelium, as determined by the ergosterol assay, did not show connection to demethoxylation. P. radiata also showed a high demethoxylation of [O¹⁴CH₃]-labeled vanillic acid in the presence of birch wood. The degradation of lignin and lignin-related substances should be studied in the presence of wood, the natural substrate for white-rot fungi.     

Alteration of cyanobacterial glutamine synthetase activity in vivo in response to light and NH 4 +

Extractable glutamine synthetase activity of the cyanobacterium Anabaena cylindrica was reduced by approximately 50% when N₂-fixing cultures were treated with 10 mM NH ₄ ⁺ or were placed in darkness. The deactivated enzyme could be rapidly reactivated (within 5 min) by adding 40 mM 2-mercaptoethanol to the biosynthetic reaction mixture. The enzyme could also be reactivated in vivo by replacing the culture in light or by removing NH ₄ ⁺ . When the enzyme was deactivated by simultaneously adding NH ₄ ⁺ and placing the culture in darkness, reactivation occurred on reillumination and removal of NH ₄ ⁺ . The removal of NH ₄ ⁺ in darkness did not result in reactivation. On in vitro reactivation of glutamine synthetase from dark or NH ₄ ⁺ -treated cultures the maximum glutamine synthetase activity observed frequently exceeded that of glutamine synthetase extracted from untreated cultures. Anacystis nidulans showed a similar type of reversible dark deactivation to A. cylindrica but Plectonema boryanum and a Nostoc did not. With A. cylindrica, a direct positive correlation between the size of the intracellular pool of glutamate and biosynthetic glutamine synthetase activity occurred during light/dark shifts, and on treatment with NH ₄ ⁺ . The changes in activity of glutamine synthetase in A. cylindrica in response to light resemble in some respects the light modulation of enzymes of the oxidative and reductive pentose phosphate pathways noted in cyanobacteria by others.     

Organ-specific changes in the activity and subunit composition of glutamine-synthetase isoforms of barley (Hordeum vulgare L.) after growth on different levels of NH+ 4

One cytosolic glutamine synthetase (GS, EC 6.3.1.2) isoform (GS 1a) was active in the germinating seeds of barley (Hordeum vulgare L.). A second cytosolic GS isoform (GS 1b) was separated from the leaves as well as the roots of 10-d-old seedlings. The chloroplastic isoform (GS 2) was present and active only in the leaves. The three GS isoforms were active in N-supplied (NH⁺ ₄ or NO ₃ ^– ) as well as in N-free-grown seedlings. This indicates (i) that a supply of nitrogen to the germinating seeds was not necessary for the induction of the GS isoforms and (ii) that no nitrogen-specific isoforms appeared during growth of seedlings with different nitrogen sources. The activity of GS, however, depended on the seedlings' nitrogen source: the specific activity was much higher in the leaves and much lower in the roots of NH⁺ ₄-grown barley than in the respective organs of NO ₃ ^– -fed or N free-grown plants. With increasing concentrations of NH⁺ ₄ (supplied hydroponically during growth), the specific activity of GS 1b increased in the leaves, but decreased in the roots. The activity of GS 2 (leaf) also increased with increasing NH⁺ ₄ supply, whereas GS 1a activity (leaf and root) was not affected. The changes in the activities of GS 1b and GS 2 were correlated with changes in the subunit compositions of the active holoenzymes: growth at increased levels of external NH⁺ ₄ resulted in an increased abundance of one of the four GS subunits, and of two of the five GS 1b subunits in the leaves. In the roots, however, the abundance of these two GS 1b subunits was decreased under the same growth conditions, indicating an organ-specific difference either in the expression of the genes coding for the respective GS 1b subunits or in the assembly of the GS 1b holoenzymes. Furthermore, growth at different levels of NH⁺ ₄ resulted in changes in the substrate affinities of the isoforms GS 1b (root and leaf) and GS 2 (leaf), presumably due to the changes in the subunit compositions of the active holoenzymes.Abbreviations FPLC fast protein liquid chromatography - GHA -glutamyl hydroxamate - GS glutamine synthetase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.     

Leucine biosynthesis: effect of branched-chain amino acids and threonine on a-isopropylmalate synthase activity from aerobic and anaerobic microorganisms

$\text{a-}\text{Isopropylmalate}$ synthase activity was demonstrated in the Sephadex G 25 gel filtrated crude extracts of one yeast and 43 bacterial strains belonging to 14 families. The enzyme was inhibited by leucine from all strains Bacteroides fragilis, Clostridia and several phototropic bacteria. The enzyme was inhibited by leucine from all strains investigated. In crude extracts of 17 species (8 genera) the leucine-mediated inhibition could be relieved by the addition of valine or isoleucine , but not by the addition of threonine or alanine. The enzymes from 11 species (7 genera) were inhibited by 1 mM valine and isoleucine, whereas the enzyme activity from 5 bacteria (4genera) were not so affected. These results suggest that valine and isoleucine are specifically involved in the regulation of leucine biosynthesis in several bacteria. The affect of valine and isoleucine on the IPM-synthase activity from mycobacteria and Corynebacterium autotrophicum lends support to the reclassification of Mycobacterium flavum 301 to C. autotrophicum. The antagonism between 5′,5′,5′-trifluoroleucine and amino acids and $\text{a-}\text{ketoisovalerate}$ was $\text{a-}\text{isopropylmalate}$ synthase in the presence or abssence of leucine and the reversal of the 5′,5′,5′-trifluoroleucine-mediated growth inhibition by these amino acids.