Regulation of lysine biosynthesis in Brevibacterium flavum

L-lysine synthesis pathway enzyme activities: β-aspartate kinase (EC.2.7.2.4), diaminopimelate decarboxylase (EC.4.1.1.20) for two L-lysine producing strains Brevibacterium flavum 22LD and RC-115 were studied. It has been found that β-aspartate kinase and diaminopimelate decarboxylase in the Br. flavum RC-115 are less sensitive to feed-back inhibition by lysine and threonine. It is supposed that desensitized β-aspartate kinase in the Br. flavum RC-115 can be determined by genetical changes of the regulatory properties of the β-aspartate kinase. Auxotrophity in the locus of homoserine dehydrogenase was tested and no homoserine dehydrogenase (EC.1.1.1.3) activity was found in either strain. The combination of these both types of mutation supplemented by the lack of catabolic repression in the RC-115 strain makes it an active lysine producer in the medium with high carbohydrates content.     

Separation of the formation of γ-coniceine and aliphatic amines from got activity in Conium maculatum

L-Alanine:5-keto-octanal aminotransferase has been separated from GOT and GPT activities by DEAE-cellulose chromatography. Two transaminase peaks A and B were observed for γ-coniceine formation and assay showed that these peaks also contained the amino acid:aldehyde aminotransferase (AAT) responsible for aliphatic amine biosynthesis. Kinetic studies showed that γ-coniceine formation with transaminase A had K_{m for L-alanine and 5-keto-octanal of 27 mM and 1.6 mM respectively. The Vmaxwas 1.3 nkats/mg protein and the activation energy was 3.0 kJ/mol. For transaminase B the Kinm} for L-alanine and 5-keto-octanal are 55 mM and 0.14 mM respectively with a V_maxof 3.3 nkats/mg protein and an activation energy of 0.33 kJ/mol. Transaminase A and B had the same MW and have distinguishable pH_max. γ-Coniceine formation by transaminase A was inhibited uncompetitively by glyoxalate and competitively by pyruvate, whereas with transaminase B uncompetitive inhibition was also observed with glyoxalate; no inhibition was observed with pyruvate which at low concentration (0.25 M) showed slight stimulation of activity.     

Metabolism of L-beta-lysine in a Pseudomonas: conversion of 6-N-acetyl-L-beta-lysine to 3-keto-6-acetamidohexanoate and of 4-aminobutyrate to succinic semialdehyde by different transaminases.

Some kinetic properties of two new species of transaminase found in extracts of a β-lysine-utilizing Pseudomonas are reported. Transaminase A catalyzes transamination between 6-N-acetyl-l-β-lysine (3-amino-6-acetamidohexanoate) and α-ketoglutarate to form 3-keto-6-acetamidohexanoate and glutamate. Transaminase B catalyzes a reaction between 4-aminobutyrate and pyruvate to form succinic semialdehyde and alanine. The formation of both transaminases is induced by growth of the bacteria on l-β-lysine, although transaminase B is also produced in the absence of this substrate. Transaminase A requires pyridoxal phosphate for activity. The β-keto acid formed from acetyl-β-lysine by transaminase A has been purified and characterized by decarboxylation, conversion to a formazan, reduction to a stable β-hydroxy acid, and conversion of the latter to its methyl ester. Transaminase B, unlike previously reported transaminases utilizing 4-aminobutyrate, cannot use α-ketoglutarate as an amino group acceptor. This enzyme is not stimulated by addition of pyridoxal phosphate, but is inhibited by hydroxylamine or cyanide. Both transaminases appear to function in the main pathway of β-lysine degradation.     

The fungus Penicillium variabile Sopp 1912 isolated from permafrost deposits as a producer of rugulovasines

It has been established that relict fungi Penicillium variabile Sopp can synthesize clavine alkaloids, rugulovasines A and B, which are revealed in this species for the first time. Submerged cultivation of the strain-producer revealed several microcycles of conidia formation. The synthesis of alkaloids was also of a cyclic character. The synchronism of cyclic rugulovasine biosynthesis and conidia formation was revealed. Zinc ions stimulated fungal growth but had a negative effect on the biosynthesis of rugulovasines.     

Role of L-alanine: 4,5-dioxovalerate transaminase in chlorophyll synthesis in Bajra (Pennisetum typhoideum) seedlings

L-alanine:4,5-dioxovalerate transaminase activity and chlorophyll levels were estimated in lead and mercury treated Bajra seedlings. The enzyme activity increased with age upto 2nd day of germination and decreased on consequent days, where as the chlorophyll content increased with age upto 4th day and remained constant on day 5. Both the metals have no effect on L-alanine: 4,5-dioxovalerate transaminase activity but reduced chlorophyll levels. In vitro incubation of the enzyme with metal solutions showed that the enzyme activity was inhibited by mercury, while lead had no effect. Studies on sub-cellular localization of the L-alanine:4,5-dioxovalerate transaminase showed that it is present in all fractions. The non-correlation between L-alanine: 4,5-dioxovalerate transaminase activity and chlorophyll synthesis is evident from different activity profiles with age and response to heavy metal treatment in the seedlings. Hence, our results suggest the non-involvement of L-alanine:4,5-dioxovalerate transaminase in chlorophyll synthesis in bajra seedlings.     

Structural Analysis and Mutant Growth Properties Reveal Distinctive Enzymatic and Cellular Roles for the Three Major L-Alanine Transaminases of Escherichia coli

In order to maintain proper cellular function, the metabolism of the bacterial microbiota presents several mechanisms oriented to keep a correctly balanced amino acid pool. Central components of these mechanisms are enzymes with alanine transaminase activity, pyridoxal 5′-phosphate-dependent enzymes that interconvert alanine and pyruvate, thereby allowing the precise control of alanine and glutamate concentrations, two of the most abundant amino acids in the cellular amino acid pool. Here we report the 2.11-Å crystal structure of full-length AlaA from the model organism Escherichia coli, a major bacterial alanine aminotransferase, and compare its overall structure and active site composition with detailed atomic models of two other bacterial enzymes capable of catalyzing this reaction in vivo, AlaC and valine-pyruvate aminotransferase (AvtA). Apart from a narrow entry channel to the active site, a feature of this new crystal structure is the role of an active site loop that closes in upon binding of substrate-mimicking molecules, and which has only been previously reported in a plant enzyme. Comparison of the available structures indicates that beyond superficial differences, alanine aminotransferases of diverse phylogenetic origins share a universal reaction mechanism that depends on an array of highly conserved amino acid residues and is similarly regulated by various unrelated motifs. Despite this unifying mechanism and regulation, growth competition experiments demonstrate that AlaA, AlaC and AvtA are not freely exchangeable in vivo, suggesting that their functional repertoire is not completely redundant thus providing an explanation for their independent evolutionary conservation.     

Influence of glutamate dehydrogenase activity on L-proline synthesis

The influence of glutamate dehydrogenase activity on the production of L-proline in the producer strain Brevibacterium flavum AP111 was studied. Transformants of the Brevibacterium flavum strain AP111 were obtained with the use of a recombinant plasmid previously constructed by us, pVOG10. It includes the encoding sequence of the gdh gene, which is responsible for the synthesis of glutamate dehydrogenase. The production of L-proline in transformants was shown to have increased by more than 40%. The results can serve as the basis for the use of this approach to improve strains producing other amino acids of the glutamine family.     

Ammonia assimilation and glutamate incorporation in coenzyme F420 derivatives ofMethanosarcina barkeri

Methanosarcina barkeri was able to grow on L-alanine and L-glutamate as sole nitrogen sources. Cell yields were 0.5 g/l and 0.7 g/l (wet wt), respectively. The mechanism of ammonia assimilation inMethanosarcina barkeri strain MS was studied by analysis of enzyme activities. Activity levels of nitrogen-assimilating enzymes in extracts of cells grown on different nitrogen sources (ammonia, 0.05–100 mM; L-alanine, 10 mM; L-glutamate, 10 mM) were compared. Activities of glutamate dehydrogenase, glutamate synthase, glutamine synthetase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase could be measured in cells grown on these three nitrogen sources. Alanine dehydrogenase was not detected under the growth conditions used. None of the measured enzyme activities varied significantly in response to the NH₄ ⁺ concentration. The length of the poly--glutamyl side chain of F₄₂₀ derivatives turned out to be independent of the concentration of ammonia in the culture medium.Abbreviations ADH alanine dehydrogenase - FO 7,8-didemethyl-8-hydroxy-5-deazariboflavin - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - GS glutamine synthetase - H₄MPT tetrahydromethanopterin     

Biosynthesis and biological activity of β-(5-methylisoxazolin-3-on-2-yl)alanine in higher plants

Enzyme preparations from Leucaena seedlings catalysed the formation of β-(5-methylisoxazolin-3-on-2-yl)alanine (MIA) by using 3-hydroxy-5-methylisoxazole (HMI) and O-acetyl-L-serine. Some properties of this enzyme are described. The β-substituted alanine synthases from Pisum and Citrullus seedlings could not catalyse the synthesis of MIA. The phytotoxic effect of HMI on rice seedlings is reduced by alanylation.     

Affinity purification and properties of rat liver mitochondrial L-alanine:4,5-dioxovalerate transaminase and its inhibition by hemin

The present study describes a new rapid procedure for purification of L-alanine:4,5-dioxovalerate transaminase from rat liver mitochondria which was purified 243-fold with a 32% yield to apparent homogeneity. The purification procedure involved protamine sulfate treatment, followed by phenyl-Sepharose CL-4B column chromatography and alanine-Sepharose 4B affinity chromatography. The Km values for L-alanine and 4,5-dioxovalerate were 3.3 and 0.28 mM, respectively. The enzyme-bound pyridoxal phosphate content was estimated to be two molecules per enzyme molecule. The purified enzyme was inhibited by the reaction product pyruvic acid, substrate analog, methylglyoxal, and sulfhydryl inhibitors. Excess concentrations of 4,5-dioxovalerate was also found to inhibit the enzyme and our experiments failed to demonstrate reversibility of the reaction. Only hemin among the intermediate compounds of heme metabolism tested was shown to be an inhibitor of purified alanine:4,5-dioxovalerate transaminase. Hemin was further shown as an uncompetitive inhibitor of both alanine and dioxovalerate.     

Substrate specificity of human glutamine transaminase K as an aminotransferase and as a cysteine S-conjugate beta-lyase

Rat kidney glutamine transaminase K (GTK) exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. The β-lyase reaction products are pyruvate, ammonium and a sulfhydryl-containing fragment. We show here that recombinant human GTK (rhGTK) also exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. S-(1,1,2,2-Tetrafluoroethyl)-l-cysteine is an excellent aminotransferase and β-lyase substrate of rhGTK. Moderate aminotransferase and β-lyase activities occur with the chemopreventive agent Se-methyl-l-selenocysteine. l-3-(2-Naphthyl)alanine, l-3-(1-naphthyl)alanine, 5-S-l-cysteinyldopamine and 5-S-l-cysteinyl-l-DOPA are measurable aminotransferase substrates, indicating that the active site can accommodate large aromatic amino acids. The α-keto acids generated by transamination/l-amino acid oxidase activity of the two catechol cysteine S-conjugates are unstable. A slow rhGTK-catalyzed β-elimination reaction, as measured by pyruvate formation, was demonstrated with 5-S-l-cysteinyldopamine, but not with 5-S-l-cysteinyl-l-DOPA. The importance of transamination, oxidation and β-elimination reactions involving 5-S-l-cysteinyldopamine, 5-S-l-cysteinyl-l-DOPA and Se-methyl-l-selenocysteine in human tissues and their biological relevance are discussed.     

Enzymic synthesis of γ-coniceine in Conium maculatum chloroplasts and mitochondria

Further studies of the transaminase responsible for the first committed step in alkaloid formation in Conium maculatum have shown the L-alanine: 5-ketooctanal transaminase to occur in both the mitochondria and chloroplast. Experiments suggest that these enzymes are the isoenzymes Transaminase A and B respectively previously isolated by the author. It is suggested that the chloroplast enzyme is normally responsible for alkaloid production.     

Transfection ofBrevibacterium flavum with bacteriophage BFB10 DNA

The virulent bacteriophageBFB10 is infective toBrevibacterium flavum Its DNA (about 48 kilobase pairs) was used for optimization of the DNA transfer intoB. flavum cells treated with lysozyme. Efficiencies of transfection up to 60 transfectants per ng DNA were obtained The described procedure can also be used for transformation ofB. flavum with plasmid DNA.     

Biochemical, genetic, and regulatory studies of alanine catabolism in Escherichia coli K12.

Summary E. coli K12 was found to utilise both D-and L-stereoisomers of alanine as sole sources of carbon, nitrogen and energy for growth. This capability was absolutely dependent upon the possession of an active membrane-bound D-alanine dehydrogenase, and was lost by mutants in which the enzyme was defective. The Michaelis constant for the enzyme with D-alanine as substrate was 30 mM, and the pH optimum about 8.9. D-alanine was the most active substrate, L-alanine was inactive and several other D-amino acids were 10–50% as active as D-alanine. Oxidation of D-alanine was linked to oxygen via a cytochrome-containing respiratory chain. Synthesis of the dehydrogenase was induced 16 to 23-fold by incubation with D-or L-alanine, but only D-alanine was intrinsically active as an inducer. L-alanine was active either as a substrate or inducer only in the presence of an uninhibited alanine racemase which converted it to the D-isomer. The map-location of their structural genes between ara and leu, together with other similarities, indicate that D-alanine dehydrogenase and the alaninase of Wijsman (1972a) are the same enzyme. Both D-and L-alanine were intrinsically active as inducers of alanine racemase synthesis. The synthesis of both D-alanine dehydrogenase and alanine racemase was found to be regulated by catabolite repression.     

Excess generation of endogenous heme inhibits L-alanine:4,5-dioxovalerate transaminase in rat liver mitochondria

In the present study, we examined the possibility that the excess heme generation within mitochondria may provide a local concentration, sufficient to inhibit the activity of L-alanine:4,5-dioxovalerate transaminase, the enzyme proposed for an alternate route of delta-aminolevulinic acid biosynthesis in mammalian system. This was accomplished by assaying together L-alanine:4,5-dioxovalerate transaminase and heme synthetase activities in intact mitochondria isolated from rat liver. Endogenous heme in intact mitochondria has been generated in excess, by increasing the concentration of the substrate of heme synthetase. Our studies showed that the activity of L-alanine:4,5-dioxovalerate transaminase decreased as the rate of heme formation increased. In intact mitochondria, almost 50% inhibition of alanine:4,5-dioxovalerate transaminase was obtained with 4.0 mumole of heme generation. We conclude that end product inhibition of L-alanine:4,5-dioxovalerate transaminase by hemin, which was proposed in earlier report by us (FEBS Letter (1985), 189, 129), is an important physiological mechanism for the regulation of hepatic heme biosynthesis.     

Purification and properties of two forms of hepatic phenylalanine:pyruvate transaminase from glucagon treated rats.

Two forms of phenylalanine:pyruvate transaminase (EC 2.6.1. aminotransferases, the exact EC number has not been assigned) termed A and B were obtained from the liver supernatant fraction of glucagon-treated rats by DEAE-Sephadex A-50 column chromatography. Each of the two forms was further purified by hydroxylapatite, Sephadex G-100 chromatography, and preparative gel electrophoresis. Both the A and B forms have been purified to homogeneity as judged by analytical and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Moreover, histidine was found to be a competitive inhibitor of phenylalanine with both purified proteins. These findings conclusively support the view that phenylalanine:pyruvate transaminase and histidine:pyruvate transaminase reactions are catalyzed by the same protein. The overall purification was 710-fold for the A form and 1200-fold for the B form. The apparent molecular weight for both A and B are 74,000 ±6000 as determined by gel filtration. Sodium dodecyl sulfate gel electrophoresis revealed that the A form has two identical subunits of molecular weight 42,000, whereas the B form has two nonidentical subunits of molecular weight 42,000 and 44,000. The amino acid composition for the A and B forms of the enzyme are different. The major differences are in glycine, alanine and leucine. The isoelectric point for A was 7.8 and for B was 7.3. However, the A and B forms of the enzyme are of immunological identity. The substrate specificity determined for both the A and B form was phenylalanine >asparagine >alanine >leucine >histidine. The K_m for phenylalanine was 7.70 mm for the A form, 6.00 mm for the B form. For histidine, the K_m was 13.70 mm for the A form, 12.50 mm for the B form.     

Genetics and Regulation of the Major Enzymes of Alanine Synthesis in Escherichia coli

Genetic analysis of alanine synthesis in the model genetic organism Escherichia coli has implicated avtA, the still uncharacterized alaA and alaB genes, and probably other genes. We identified alaA as yfbQ. We then transferred mutations in several transaminase genes into a yfbQ mutant and isolated a mutant that required alanine for optimal growth. For cells grown with carbon sources other than pyruvate, the major alanine-synthesizing transaminases are AvtA, YfbQ (AlaA), and YfdZ (which we designate AlaC). Growth with pyruvate as the carbon source and multicopy suppression suggest that several other transaminases can contribute to alanine synthesis. Expression studies showed that alanine modestly repressed avtA and yfbQ but had no effect on yfdZ. The leucine-responsive regulatory protein (Lrp) mediated control by alanine. We purified YfbQ and YfdZ and showed that both are dimers with K_ms for pyruvate within the intracellular range of pyruvate concentration.The enzymes and pathway of alanine synthesis in the model organism Escherichia coli have not been well characterized (25). The most likely pathway is transamination of pyruvate by glutamate, catalyzed by glutamic-pyruvic transaminase (GPT). However, labeling studies have suggested some unanticipated complexities (7, 25, 26). Claire Berg and colleagues performed the only genetic analysis of alanine synthesis. They identified three genes that participate in alanine synthesis, namely, avtA, alaA, and alaB (1, 2, 36, 40). The activity of AvtA, also called transaminase C, was initially detected as an alanine-synthesizing enzyme with valine, not glutamate, as the nitrogen donor (27). Loss of either avtA or alaA did not affect growth and was apparent only in an ilvE background (2, 36, 40). An alaA mutant had normal AvtA and GPT activities, which suggested that AlaA was not a transaminase (1, 36). The alaA gene was physically mapped, but its product was not subsequently characterized (1). The alaB gene was identified from its partial suppression of the phenotype of an ilvE alaA strain (36). Multicopy alaB had elevated GPT activity, which suggested that alaB specifies a GPT (36). Except for a partial physical map of the alaB region, nothing else is known about alaB and its product (36).Our goal in this study was to determine the enzymes of alanine synthesis using current knowledge of known and potential transaminase genes. Our genetic analysis suggests that AvtA, YfbQ, and YfdZ are the major enzymes of alanine synthesis, but eight other transaminases can potentially synthesize alanine. To confirm these conclusions, we also analyzed the regulation of avtA, yfbQ, and yfdZ and purified and partially characterized YfbQ and YfdZ.     

Optimal conditions for induction of certain mutation types inBrevibacterium flavum by N-Methyl-N’-nitro-N-nitrosoguanidine

Conditions under which it is possible to induce auxotrophic mutants and DL-selenomethionine-resistant mutants inB. flavum by N-methyl-N’-nitro-N-nitrosoguanidine were determined. The yield of auxotrophic mutants was increased to 3% during mutagenesis in the first stage and to 1.5% in the second stage when using the enrichment-selective method with vancomycin. The optimal vancomycin concentration for inactivation of prototrophic cells growing in a minimal medium was 200 mg/L and the optimal time of treatment was 8 h. When testing the effect of three amino acid analogues (dl-ethionine,dl-selenomethionine and L-methionine sulfoximine) it was found thatB. flavum is sensitive todl-selenomethionine present in the minimal cultivation medium. Mutants resistant to 1 mg/mL of selenomethionine were isolated. Both isotope studies and measurement of growth indicate thatdl-ethionine also entersB. flavum cells, although its competition with endogenously synthesized methionine is not significant.     

The Role of Cryptochrome 1 and Phytochromes in the Control of Plant Photomorphogenetic Responses to Green Light

We studied the role of cryptochrome 1 (CRY1) and phytochromes in the photomorphogenetic responses of plants to the middle-wavelength region of photosynthetically active radiation. A comparison was performed of green light (GL) action on growth, GA activity and IAA and ABA contents during seedling deetiolation of two Arabidopsis thaliana (L.) Heynh lines of Landsnerg erecta ecotype (wild type Ler and mutant hy4) and of Phaseolus vulgaris L. It was shown that a growth responses of Ler hypocotyls to GL of 515 nm and Ler cotyledons to GL of 542 nm were weaker than those of the hy4 mutant defected in the CRY1 synthesis. Far-red light (730 nm) neutralized the effect of GL (533 nm) on the phytohormone balance in the primary kidney bean leaves. The data obtained permit a supposition that plants possess several photoregulatory systems functioning under GL of higher (515 nm) and lower emission energy (542–553 nm). A possible operation of GL receptors, other than cryptochrome 1 and phytochromes, is discussed.