Nitrogen assimilation in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 assimilated ammonia via a constitutive glutamine synthetase/glutamate synthase enzyme system.Glutamine synthetase had a K _m for NH ₄ ⁺ of 0.38 mM whilst the nicotinamide adenine dinucleotide linked glutamate synthase had a K _m for glutamine of 0.55 mM. R. acidophila utilized only a limited range of amino acids as sole nitrogen sources: l-alanine, glutamine and asparagine. The bacterium did not grow on glutamate as sole nitrogen source and lacked glutamate dehydrogenase. When R. acidophila was grown on l-alanine as the sole nitrogen source in the absence of N₂ low levels of a nicotinamide adenine dinucleotide linked l-alanine dehydrogenase were produced. It is concluded, therefore, that this reaction was not a significant route of ammonia assimilation in this bacterium except when glutamine synthetase was inhibited by methionine sulphoximine. In l-alanine grown cells the presence of an active alanine-glyoxylate aminotransferase and, on occasions, low levels of an alanine-oxaloacetate aminotransferase were detected. Alanine-2-oxo-glutarate aminotransferase could not be demonstrated in this bacterium.Abreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulphoximine     

Metabolism of l-alanine in Desulfotomaculum ruminis and two marine Desulfovibrio strains

The degradation of l-alanine by three strains of sulfate-reducing bacteria that can grow with l-alanine as an energy source was investigated. In Desulfotomaculum ruminis and most likely also in two marine Desulfovibrio strains alanine is converted to pyruvate via an NAD-dependent alanine dehydrogenase. D. ruminis contained high activities of soluble NADH and NADPH dehydrogenases. In the marine strains the activities were much lower and the NADH dehydrogenase was partly associated with the membrane fraction.     

Morpholine-induced formation of l-alanine dehydrogenase activity in Mycobacterium strain HE5

An NAD-dependent, morpholine-stimulated l-alanine dehydrogenase activity was detected in crude extracts from morpholine-, pyrrolidine-, and piperidine-grown cells of Mycobacterium strain HE5. Addition of morpholine to the assay mixture resulted in an up to 4.6-fold increase of l-alanine dehydrogenase activity when l-alanine was supplied at suboptimal concentration. l-Alanine dehydrogenase was purified to near homogeneity using a four-step purification procedure. The native enzyme had a molecular mass of 160 kDa and contained one type of subunit with a molecular mass of 41 kDa, indicating a tetrameric structure. The sequence of 30 N-terminal amino acids was determined and showed a similarity of up to 81% to that of various alanine dehydrogenases. The pH optimum for the oxidative deamination of l-alanine, the only amino acid converted by the enzyme, was determined to be pH 10.1, and apparent K _m values for l-alanine and NAD were 1.0 and 0.2 mM, respectively. K _m values of 0.6, 0.02, and 72 mM for pyruvate, NADH, and NH₄ ⁺, respectively, were estimated at pH 8.7 for the reductive amination reaction. Received: 25 September 1998 / Accepted: 11 March 1999     

d-Alanine production from dl-alanine by Candida maltosa with asymmetric degrading activity

Summary To develop a practical process for d-alanine production from dl-alanine, we screened 107 yeasts for their asymmetric degrading activity against dl-alanine. Candida maltosa JCM1504 degraded the l-isomer ten times more rapidly than the d-isomer. The cells of this strain were used as a biocatalyst for eliminating the l-isomer. However, when the degradation reaction was conducted in the presence of a high concentration of dl-alanine, the pH of the reaction mixture was rapidly increased by the liberation of ammonia from l-alanine, and consequently the reaction stopped. This hindrance was overcome by controlling the pH value at 6.0 with H₂SO₄ during the reaction. Additionally, we found that the maximum rate of l-isomer degradation was obtained at 30° C and pH 6.0 under conditions of high aeration (1.0 vvm) and agitation (1200 rpm). Under the optimal conditions, the l-isomer of 200 g dl-alanine/l was completely degraded within 40 h and 90 g d-alanine/l remained in the reaction mixture. d-Alanine was easily isolated from the reaction mixture. The chemical and optical purity of the d-isomer product so obtained was 99.0% and 99.9% enantiomeric excess, respectively.Offprint requests to: I. Umemura     

Altered control of glutamate dehydrogenases in ornithine utilization mutants of Pseudomonas aeruginosa

Two classes of ornithine-nonutilizing (oru) mutants of Pseudomonas aeruginosa PAO were investigated. Strains carrying the oru-310 mutation were entirely unable to grow on l-ornithine as the only carbon and nitrogen source and were affected in the assimilation of a variety of nitrogen sources (e.g., amino acids, nitrate). The oru-310 mutation caused changes in the regulation of the catabolic NAD-dependent glutamate dehydrogenase; this enzyme was no longer inducible by glutamate but instead could be induced by ammonia. The oru-310 locus was cotransducible with car-9 and tolA in the 10 min region of the chromosome. An oru-314 mutant was severely handicapped in ornithine medium but could grow when a good carbon source was added; the mutant also showed pleiotropic growth effects related to nitrogen metabolism. The oru-314 mutation affected the regulation of the anabolic NADP-dependent glutamate dehydrogenase, which was no longer repressed by glutamate but showed normal derepression in the presence of ammonia. The oru-314 locus was mapped by transduction near met-9011 at 55 min. Both oru mutants could grow on l-glutamate, l-proline, or l-ornithine amended with 2-oxoglutarate, albeit slowly. We speculate that insufficient 2-oxoglutarate concentrations might account, at least in part, for the Oru^- phenotype of the mutants.     

Involvement of the glutamine synthetase/glutamate synthase pathway in ammonia assimilation by the wood-rotting fungusPleurotus ostreatus

The mycelium of the wood-rotting fungus,P. ostreatus, contains NAD-dependent glutamate synthase inhibited by azaserine.l-Glutamine andl-glutamate are the most important free amino acids in the mycelium. Feeding of the mycelium with nitrogenous substrates showed thatl-glutamate,l-aspartate andl-alanine are interconnected by way of transaminases. After the inhibition of glutamine synthetase by methionine-S-sulfoximine the synthesis ofl-glutamate was inhibited and the level of all free amino acids decreased. The¹⁵N-NMR spectra of mycelia after the addition of¹⁵NH₄Cl confirmed that the GS/GOGAT is the only pathway of ammonia assimilation inP. ostreatus and NAD-glutamate dehydrogenase should be the deaminating enzyme.     

Regulation of reduced nitrogen assimilation in Rhodobacter capsulatus E1F1

The phototrophic bacterium Rhodobacter capsulatus E1F1 assimilates ammonia and other forms of reduced nitrogen either through the GS/GOGAT pathway or by the concerted action of l-alanine dehydrogenase and aminotransferases. These routes are light-independent and very responsive to the carbon and nitrogen sources used for cell growth. GS was most active in cells grown on nitrate or l-glutamate as nitrogen sources, whereas it was heavily adenylylated and siginificantly repressed by ammonium, glycine, l-alanine, l-aspartate, l-asparagine and l-glutamine, under which conditions specific aminotransferases were induced. GOGAT activity was kept at constitutive levels in cells grown on l-amino acids as nitrogen sources except on l-glutamine where it was significantly induced during the early phase of growth. In vitro, GOGAT activity was strongly inhibited by l-tyrosine and NADPH. In cells using l-asparagine or l-aspartate as nitrogen source, a concerted induction of l-aspartate aminotransferase and l-asparaginase was observed. Enzyme level enhancements in response to nitrogen source variation involved de novo protein synthesis and strongly correlated with the cell growth phase.Abbreviations ADH l-alanine dehydrogenase - AOAT l-alanine:2-oxoglutarate aminotransferase - Asnase l-asparaginase - GOAT Glycine: oxaloacetate aminotransferase - GOGAT Glutamate synthase - GOT l-aspartate: 2-oxoglutarate aminotransferase - GS Glutamine synthetase - HPLC High-Pressure Liquid Chromatography - MOPS 2-(N-morpholino)propanesulfonic acid - MSX l-methionine-d,l-sulfoximine     

Construction of an arginine-producing strain of Serratia marcescens

Summary In Serratia marcescens Sr41, l-canavanine was demonstrated to be a weak cell growth inhibitor in minimal medium containing glucose as the sole carbon source. The inhibition of cell growth was enhanced by changing the carbon source from glucose to l-glutamic acid. An arginine regulatory mutant (i.e., argR mutant) in which formation of l-arginine biosynthetic enzymes was genetically derepressed was isolated by selecting for l-canavanine resistance on the glutamate medium. Furthermore, an l-arginine-producing strain was constructed by introducing the mutation leading to feedback-resistant N-acetylglutamate synthase into the argR mutant. The resulting transductant produced about 40 g/l of l-arginine, whereas the wild strain produced no l-arginine and the argR mutant only 3 g/l.     

Engineering of sugar metabolism of Corynebacterium glutamicum for production of amino acid l-alanine under oxygen deprivation

Corynebacterium glutamicum was genetically engineered to produce l-alanine from sugar under oxygen deprivation. The genes associated with production of organic acids in C. glutamicum were inactivated and the alanine dehydrogenase gene (alaD) from Lysinibacillus sphaericus was overexpressed to direct carbon flux from organic acids to alanine. Although the alaD-expressing strain produced alanine from glucose under oxygen deprivation, its productivity was relatively low due to retarded glucose consumption. Homologous overexpression of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the alaD-expressing strain stimulated glucose consumption and consequently improved alanine productivity. In contrast gapA overexpression did not affect glucose consumption under aerobic conditions, indicating that oxygen deprivation engendered inefficient regeneration of NAD⁺ resulting in impaired GAPDH activity and reduced glucose consumption in the alanine-producing strains. Inactivation of the alanine racemase gene allowed production of l-alanine with optical purity greater than 99.5%. The resulting strain produced 98 g l⁻¹ of l-alanine after 32 h in mineral salts medium. Our results show promise for amino acid production under oxygen deprivation.     

Utilization of d-amino acids by dadR mutants of Salmonella typhimurium

Utilization of d-amino acids being substrates of d-amino acid dehydrogenase of Salmonella typhimurium was examined. The experiments were done with wild type strains and the mutants dadA missing the enzyme activity and dadR in which its synthesis is released from catabolite repression. Growth on d-tryptophan, d-histidine and d-methionine used as precursors of the l-amino acids was faster when the respective auxotrophs carried dadR mutations. The dadR mutants grew faster when d-or l-alanine was present as a sole source of nitrogen. Experiments with d-amino acid dehydrogenase in vitro provided evidence that d-tryptophan is its substrate with a very low affinity to the dehydrogenase.     

Phenylalanine and tyrosine metabolism in the facultative methylotroph Nocardia sp. 239

Nocardia sp. 239 is able to use l-tyrosine and both d- and l-phenylalanine as carbon-, energy- and nitrogen sources for growth. The catabolism of these compounds is by way of (4-hydroxy)phenylpyruvate and (4-hydroxy)-phenylacetate as intermediates and the pathways merge at the level of homogentisate. The conversion of the amino acids into (4-hydroxy)phenylpyruvate is catalyzed by an inducible NAD-dependent phenylalanine dehydrogenase and l-tyrosine aminotransferase, respectively. Incubation of the organism in media with l-phenylalanine plus phenyl-pyruvate resulted in diauxic growth, with phenylpyruvate used first. Phenylalanine dehydrogenase activity cold only be detected after depletion of phenylpyruvate, in the ensuing second growth phase on l-phenylalanine. During growth on phenylalanine plus methanol, low levels of phenylalanine dehydrogenase were detected and this resulted in simultaneous utilization of the two substrates. Following diepoxyoctane treatment, mutants of Nocardia sp. 239 affected in phenylalanine and phenylpyruvate degradation were isolated. Double mutants blocked in both phenylalanine dehydrogenase and phenylpyruvate decarboxylase completely failed to catabolize phenylalanine. The absence of these enzymes did not affect growth on tyrosine.Abbreviations RuMP ribulose monophosphate - EMS ethylmethanesulphonate - NTG N-methyl-N-nitro-N-nitrosoguanidine     

d-Alanine dehydrogenase

Pseudomonas aeruginosa PA01 was found to utilise both thed- andl-isomers of -alanine and also -alanine as sole sources of carbon and energy for growth. Enzymological studies of wild-type cultures and comparison with mutants deficient in growth upon one or more isomers of alanine led to the following conclusions: (i) utilisation ofd-alanine involved its direct oxidation by an inducible, membrane-bound, cytochrome-linked dehydrogenase; (ii) utilisation ofl-alanine required its conversion to the directly oxidisabled-form by a soluble racemase; (iii) utilisation of -alanine, likel-alanine, involves both the racemase andd-alanine dehydrogenase enzymes, but in addition must involve other enzymes the identity, of which is still speculative; (iv)P. aeruginosa, likeEscherichia coli, appears to take upd-alanine andl-alanine by means of two specific permeases.Abbreviation DCPIP 2,6-dichlorophenol-indophenol     

A mutant of Saccharomyces cerevisiae lacking catabolic NAD-specific glutamate dehydrogenase Growth characteristics of the mutant and regulation of enzyme synthesis in the wild-type strain

NAD-specific glutamate dehydrogenase (GDH-B)¹ was induced in a wild-type strain derived of - 1278b by -amino acids, the nitrogen of which according to known degradative pathways is transferred to 2-oxoglutarate. A recessive mutant (gdhB) devoid of GDH-B activity grew more slowly than the wild type if one of these amino acids was the sole source of nitrogen. Addition of ammonium chloride, glutamine, asparagine or serine to growth media with inducing -amino acids as the main nitrogen source increased the growth rate of the gdhB mutant to the wild-type level and repressed GDH-B synthesis in the wild type. Arginine, urea and allantoin similarly increased the growth rate of the gdhB mutant and repressed GDH-B synthesis in the presence of glutamate, but not in the presence of aspartate, alanine or proline as the main nitrogen source. These observations are consistent with the view that GDH-B in vivo deaminates glutamate. Ammonium ions are required for the biosynthesis of glutamine, asparagine, arginine, histidine and purine and pyrimidine bases. Aspartate and alanine apparently are more potent inducers of GDH-B than glutamate.Anabolic NADP-specific glutamate dehydrogenase (GDH-A) can not fulfil the function of GDH-B in the gdhB mutant. This is concluded from the equal growth rates in glutamate, aspartate and proline media as observed with a gdhB mutant and with a gdhA, gdhB double mutant in which both glutamate dehydrogenases are lacking. The double mutant showed an anomalous growth behaviour, growth rates on several nitrogen sources being unexpectedly low.The following abbreviations and symbols are used GDH-A NADP-specific glutamate dehydrogenase [l-glutamate - NADP⁺ oxido-reductase (deaminating), EC 1.4.1.4] - gdhA genotype associated with GDH-A deficiency - GDH-B NAD-specific glutamate dehydrogenase, [L-glutamate NAD⁺ oxido-reductase (deaminating), EC 1.4.1.2] - gdhB genotype associated with GDH-B deficiency - gdhCR genotype associated with derepressed GDH-B synthesis - specific growth rate (h^-1) - x cell density - t time (h)     

Observations on enzymes of ammonia assimilation in two different strains of Cyanidium caldarium

Two strains of Cyanidium caldarium, one able to utilize nitrate as a substrate, and the other not, were tested for the presence of enzymes of ammonia assimilation. The nitrate-assimilating strain exhibits glutamate dehydrogenase activity. By contrast, the other strain lacks glutamate dehydrogenase; it possesses high alanine dehydrogenase and l-alanine aminotransferase activities which suggest that this strain may incorporate ammonia through reductive amination of pyruvate and may form glutamate from 2-ketoglutarate by a transamination reaction with alanine. Neither strain reveals glutamate synthase activity. Both strains contain similar levels of glutamine synthetase.     

Biochemical and genetic characterization of a mutant of Salmonella typhimurium defective in a locus for glutamate dehydrogenase activity

Summary By two consecutive treatments with N-methyl-N-nitro-N-nitrosoguanidine we obtained mutant SM151 of Salmonella typhimurium which differs from the parental LT2 strain in: a) is able to use l-glutamate as carbon source (first mutation), and b) requires that amino acid for growth (second mutation). It was found that the requirement of mutant SM151 for glutamate was due to a very low activity of glutamate dehydrogenase. Both glutamate-oxaloacetate transaminase and aspartase activities were present at normal levels. Glutamate dehydrogenase activity was strongly repressed by glutamate; aspartase activity was under severe catabolite (glucose) repression, while glutamate-oxaloacetate transaminase was partially repressed by glutamate. By conjugation and transduction the locus gdh, responsible for the low activity of the glutamate dehydrogenase of mutant SM151, was located at about minute 128 of the bacterial chromosome and found to be linked to the argC, argF, and metB loci.     

Expression and regulation of the Escherichia coli glutamate dehydrogenase gene (gdh) in Rhizobium japonicum

The glutamate dehydrogenase (gdh) gene of Escherichia coli was transferred into an ammonium assimilation deficient mutant (Asm^-) of Rhizobium japonicum (CJ9) using plasmid pRP301, a broad host range derivative of RP₄. Exconjugants capable of growth on ammonia as sole N-source occurred at a frequency of 6.8×10^-6. Assimilatory GDH (NADP⁺) activity was detected in the strain carrying the E. coli gdh gene and the pattern of ammonia assimilation via GDH was similar to that of the Asm⁺ wild type strain. However, GDH mediated ammonia assimilation was not subject to regulation by l-glutamate. Nitrogenase activity was expressed ex planta in R. japonicum CJ9 harbouring the gdh gene, however, the presence of the gdh gene did not restore symbiotic effectiveness to the CJ9 Asm^- strain in nodules. The gdh plasmid was maintained in approximately 90% of the isolates recovered from soybean nodules.Abbreviations gdh glutamate dehydrogenase - Asm^- mutant ammonia assimilation deficient mutant     

Altered control of chorismate mutase leads to tryptophan auxotrophy of Pichia guilliermondii

We have isolated the tryptophan auxotrophic mutant strain, PK101, of Pichia guilliermondii. This strain is not defective in any of the tryptophan biosynthetic enzymes, but its chrismate mutase, an enzyme of the phenylalanine-tyrosine biosynthesis, is changed. In comparison with the wild type chorismate mutase, the enzyme of PK101 is characterized by a complete loss of sensitivity to l-phenylalanine inhibition and to a considerable loss of sensitivity to l-tryptophan activation. Furthermore, the chorismate mutase activity of the mutant is more than 7-fold higher in the absence of l-tryptophan than in the wild type. The PK101 enzyme is also changed in the pH optimum and in some kinetic constants. We found an increased intracellular pool of both phenylalanine and tyrosine and a reduced contents of tryptophan in the mutant cells. Our genetic data indicate that the mutant phenotype is dominant over the wild type.     

Threonine metabolism byPseudomonas aeruginosa

83 strains ofPseudomonas aeruginosa were unable to utilizel-threonine as carbon-energy source, although this compound served as sole nitrogen source. Auxotrophs ofP. aeruginosa 9-D2 that requiredl-serine or glycine for growth could grow in the presence ofl-threonine. Extracts ofP. aeruginosa 9-D2 grown in the presence ofl-threonine contained threonine dehydrogenase and alpha-amino beta-ketobutyrate: CoA ligase activities; threonine aldolase was not detectable. Cells grown in the absence ofl-threonine produced no detectable threonine dehydrogenase.l-Leucine neither stimulated nor repressed threonine dehydrogenase levels. Glycine, and to a lesser extentl-serine, repressedl-threonine-mediated threonine dehydrogenase synthesis. A mutant of strain 9-D2 was isolated that could utilizel-threonine as sole carbon-energy source. This strain produced elevated levels of threonine dehydrogenase, but only slightly higher levels of alpha-amino beta-ketobutyrate: CoA ligase activities.     

Utilization of l-alanine by Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 can satisfy all its nitrogen and carbon requirements from l-alanine. Addition of 100 M methionine sulfoximine to alanine grown cultures had no effect on growth rate indicating that deamination of alanine via alanine dehydrogenase and re-assimilation of the released NH ₄ ⁺ by glutamine synthetase/glutamate synthase was an insignificant route of nitrogen transfer in this bacterium. Determination of aminotransferase activities in cell-free extracts failed to demonstrate the presence of direct routes from alanine to either aspartate or glutamate. The only active aminotransferase involving l-alanine was the alanine-glyoxylate enzyme (114–167 nmol·min^–1·mg^–1 protein) which produced glycine as end-product. The amino group of glycine was further transaminated to yield aspartate via a glycineoxaloacetate aminotransferase (117–136 nmol·min^–1 ·mg^–1 protein). No activity was observed when 2-oxoglutarate was substituted for oxaloacetate. The formation of glutamate from aspartate was catalysed by aspartate-2-oxoglutarate aminotransferase (85–107 nmol·min^–1·mg^–1 protein). Determinations of free intracellular amino acid pools in alanine and alanine+100 M methionine sulfoximine grown cells showed the predominance of glutamate, glycine and aspartate, providing further evidence that in alanine grown cultures R. acidophila satisfies its nitrogen requirements for balanced growth by transamination.Abbreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulfoximine - GOT glutamate-oxaloacetate aminotransferase - GPT glutamate-pyruvate amino-transferase - AGAT alanine-glyoxylate aminotransferase - GOAT glycine-oxaloacetate aminotransferase - GOTAT glycine-2-oxoglutarate aminotransferase - AOAT alanine-oxaloacetate aminotransferase     

Utilization of l-alanine as carbon and nitrogen source by Deusulfovibrio HL21

Desulfovibrio HL21 is unable to grow with amino acids as energy substrates. Alanine, serine, aspartate and to some extent glutamate were used as carbon and nitrogen sources in the presence of hydrogen as the energy substrate. Dense cell suspensions converted alanine stoichiometrically to acetate, NH ₄ ⁺ and presumably HCO ₃ ^- , but at a very low rate. Desulfovibrio HL21 cells grown with alanine as carbon and nitrogen source contained increased levels of NAD(P)-dependent l-alanine dehydrogenase as compared to cells grown with NH₄Cl as nitrogen source. Unfavourable kinetic properties of this alanine dehydrogenase, repression of the synthesis of the enzyme by NH ₄ ⁺ and a low rate of NADH oxidation all have a negative effect on the rate of degradation of alanine and may partly explain the inability of the strain to grow with alanine as an energy substrate.