Aspartate kinase and homoserine dehydrogenase ofCandida utilis

Aspartate kinase and homoserine dehydrogenase activity were assayed in a dialyzed cell-free extract ofCandida utilis. Aspartate kinase was partly inhibited by ATP-Mg and by Mg²⁺ alone. There appear to be two isoenzymes of aspartate kinase in the yeast, one heatlabile, the other relatively heat-stable. The first is subject to feedback inhibition by threonine, the other is threonine-resistant. Neither aspartate kinase nor homoserine dehydrogenase is the rate-limiting enzyme in methionine biosynthesis. Homoserine dehydrogenase measured in the forward direction showed an activity five times higher than aspartate kinase. No regulatory interaction could be demonstrated for this enzyme. No repression of aspartate kinase and homoserine dehydrogenase synthesis by threonine, methionine or both amino acids was observed.     

DoesStreptomyces aureofaciens contain two alanine dehydrogenase isozymes ?

Summary Two NAD-dependent dehydrogenases exhibiting activity with L-alanine were detected in crude extracts ofStreptomyces aureofaciens. The dominant enzyme was found to be an alanine dehydrogenase with 6 or 4 subunits, each ofM, 45,000. The minor enzyme exhibited high activities with branched-chain amino acids, suggesting it is a valine dehydrogenase. The results demonstrate thatS. aureofaciens has only one alanine dehydrogenase, which is catalytically active in a six- or four-subunit structure.     

Biochemical and phylogenetic characterization of isocitrate dehydrogenase from a hyperthermophilic archaeon, Archaeoglobus fulgidus

A thermostable homodimeric isocitrate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was purified and characterized. The mol. mass of the isocitrate dehydrogenase subunit was 42 kDa as determined by SDS-PAGE. Following separation by SDS-PAGE, A. fulgidus isocitrate dehydrogenase could be renatured and detected in situ by activity staining. The enzyme showed dual coenzyme specificity with a high preference for NADP⁺. Optimal temperature for activity was 90° C or above, and a half-life of 22 min was found for the enzyme when incubated at 90° C in a 50 mM Tricine-KOH buffer (pH 8.0). Based on the N-terminal amino acid sequence, the gene encoding the isocitrate dehydrogenase was cloned. DNA sequencing identified the icd gene as an open reading frame encoding a protein of 412 amino acids with a molecular mass corresponding to that determined for the purified enzyme. The deduced amino acid sequence closely resembled that of the isocitrate dehydrogenase from the archaeon Caldococcus noboribetus (59% identity) and bacterial isocitrate dehydrogenases, with 57% identity with isocitrate dehydrogenase from Escherichia coli. All the amino acid residues directly contacting substrate and coenzyme (except Ile-320) in E. coli isocitrate dehydrogenase are conserved in the enzyme from A. fulgidus. The primary structure of A. fulgidus isocitrate dehydrogenase confirmes the presence of Bacteria-type isocitrate dehydrogenases among Archaea. Multiple alignment of all the available amino acid sequences of di- and multimeric isocitrate dehydrogenases from the three domains of life shows that they can be divided into three distinct phylogenetic groups. Received: 6 February 1997 / Accepted: 12 June 1997     

Alteration of substrate specificity of valine dehydrogenase from Streptomyces albus

The catabolism of branched chain amino acids, especially valine, appears to play an important role in furnishing building blocks for macrolide and polyether antibiotic biosyntheses. To determine the active site residues of ValDH, we previously cloned, partially characterized, and identified the active site (lysine) of Streptomyces albus ValDH. Here we report further characterization of S. albus ValDH. The molecular weight of S. albus ValDH was determined to be 38 kDa by SDS-PAGE and 67 kDa by gel filtration chromatography indicating that the enzyme is composed of two identical subunits. Optimal pHs were 10.5 and 8.0 for dehydrogenase activity with valine and for reductive amination activity with -ketoisovaleric acid, respectively. Several chemical reagents, which modify amino-acid side chains, inhibited the enzyme activity. To examine the role played by the residue for enzyme specificity, we constructed mutant ValDH by substituting alanine for glycine at position 124 by site-directed mutagenesis. This residue was chosen because it has been considered to be important for substrate discrimination by phenylalanine dehydrogenase (PheDH) and leucine dehydrogenase (LeuDH). The Ala-124–Gly mutant enzyme displayed lower activities toward aliphatic amino acids, but higher activities toward L-phenylalanine, L-tyrosine, and L-methionine compared to the wild type enzyme suggesting that Ala-124 is involved in substrate binding in S. albus ValDH.     

Regulation of aspartate-derived amino acid biosynthesis in the yeastSaccharomyces cerevisiae

The activity of three enzymes, aspartokinase, homoserine dehydrogenase, and homoserine kinase, has been studied in the industrial strainSaccharomyces cerevisiae IFI256 and in the mutants derived from it that are able to overproduce methionine and/or threonine. Most of the mutants showed alteration of the kinetic properties of the enzymes aspartokinase, which was less inhibited by threonine and increased its affinity for aspartate, and homoserine dehydrogenase and homoserine kinase, which both lost affinity for homoserine. Furthermore, they showed in vitro specific activities for aspartokinase and homoserine kinase that were higher than those of the wild type, resulting in accumulation of aspartate, homoserine, threonine, and/or methionine/S-adenosyl-methionine (Ado-Met). Together with an increase in the specific activity of both aspartokinase and homoserine kinase, there was a considerable and parallel increase in methionine and threonine concentration in the mutants. Those which produced the maximal concentration of these amino acids underwent minimal aspartokinase inhibition by threonine. This supports previous data that identify aspartokinase as the main agent in the regulation of the biosynthetic pathway of these amino acids. The homoserine kinase in the mutants showed inhibition by methionine together with a lack or a reduction of the inhibition by threonine that the wild type undergoes, which finding suggests an important role for this enzyme in methionine and threonine regulation. Finally, homoserine dehydrogenase displayed very similar specific activity in the mutants and the wild type in spite of the changes observed in amino acid concentrations; this points to a minor role for this enzyme in amino acid regulation.     

Overproduction of amino acids and the activity of methanol dehydrogenase of protaminobacter thiaminophagus mutants

The activity of methanol dehydrogenase of Protaminobacter thiaminophagus ATCC 21371 and its 10 mutants which where able to overproduce amino acids from methanol was studied. It was found that the activity of methanol dehydrogenase depended on the used strain varied from 56.1 to 100.6 mU/mg d. w. of cells. Specific production of amino acids was between 4.3 × 10^?7 and 13.0 × 10^?7 μg/CFU. Statistical analysis confirmed expected high positive correlation (r = 0.93) between activity of methanol dehydrogenase and specific production of amino acids. Based on the measurement of methanol dehydrogenase activity a rapid method of estimation of amino acid production ability of Protaminobacter thiaminophagus mutants was developed.     

Some aspects of relation between nitrogen metabolism inPisum sativum and mineral nutrition

Summary Plants ofPisum sativum grown in water culture were subjected to deficiencies in the macronutrients N, P, K, S, Ca, Mg, substitution of ammonium for nitrate and change in pH to pH 3 or 9. Free amino acids, soluble nitrogen, protein and glutamate dehydrogenase activity of shoots were measured for all nutrient types. Changes in amino acid concentrations correlated well with changes in glutamate dehydrogenase. It is considered whether this enzyme is indicative of the nutritional state of plants.     

Analysis of maize alcohol dehydrogenase by native-SDS two dimensional electrophoresis and autoradiography

Summary Alcohol dehydrogenase isozyme proteins were characterized by visualization on two dimensional polyacrylamide gels. Native first dimension electrophoresis separates isozymes by size and charge, while preserving enzyme activity and subunit interactions. SDS electrophoresis in the second dimension breaks subunit interactions and separates polypeptides primarily by molecular weight. Results revealed that ADH2 monomers are larger in molecular weight than ADH1 monomers. An EMS induced Adhl mutant was found to produce ADH1 monomers of reduced molecular weight. Autoradiography revealed that only a few proteins (five or six) including ADH1 and ADH2 actively incorporate labelled amino acids after prolonged anaerobiosis.     

Studies on Aspartic β-Semialdehyde Dehydrogenase in Pea Seedlings

Highly purified aspartic β-semialdehyde dehydrogenase was prepared from pea seedlings. The optimum pH and Km values for the substrates were determined, and the substrate and coenzyme specificities of this enzyme were examined.

Effects of various compounds on aspartic β-semialdehyde dehydrogenase prepared from pea seedlings were examined. From the behaviours toward thioglycolate and PCMB, this enzyme was concluded to be a -SH enzyme. The inhibitory effects of aldehydes and amino acids were also studied.     

The anaerobic fungusPiromyces sp. strain E2: nitrogen requirement and enzymes involved in primary nitrogen metabolism

The anaerobic fungusPiromyces sp. strain E2 appeared restricted in nitrogen utilization. Growth was only supported by ammonium as source of nitrogen. Glutamine also resulted in growth, but this was due to release of ammonia rather than to uptake and utilization of the amino acid. The fungus was not able to grow on other amino acids, albumin, urea, allantoin, or nitrate. Assimilation of ammonium is very likely to be mediated by NADP-linked glutamate dehydrogenase (NADP-GDH) and glutamine synthetase (GS). One transaminating activity, glutamate-oxaloacetate transaminase (GOT), was demonstrated. Glutamate synthase (GOGAT), NAD-dependent glutamate dehydrogenase (NAD-GDH), and the transaminating activity glutamate-pyruvate transaminase (GPT) were not detected in cell-free extracts ofPiromyces sp. strain E2. Specific enzyme activities of both NADP-GDH and GS increased four-to sixfold under nitrogen-limiting conditions.Abbreviations GDH Glutamate dehydrogenase - GOGAT Glutamate synthase - GOT Glutamate-oxaloacetate transaminase - GPT Glutamate-pyruvate transaminase - GS Glutamine synthetase     

Proline dehydrogenase from Pseudomonas fluorescens: Gene cloning, purification, characterization and homology modeling

The gene encoding proline dehydrogenase (ProDH) from Pseudomonas fluorescens was isolated using PCR amplification and cloned into pET23a expression vector. The expression of the recombinant target enzyme was induced by addition of IPTG. The produced His-fusion enzyme was purified and its kinetic properties were studied. The 3D structure modeling was also performed to identify key amino acids involved in FAD-binding and catalysis. The PCR product contained a 1033 bp open reading frame encoding 345 amino acid residue polypeptide chain. SDS-PAGE analysis revealed a MW of 40 kDa, whereas the native enzyme exhibited a MW of 40 kDa suggesting a monomeric protein. The K _m and V _max values of the P. fluorescens ProDH were estimated to be 35 mM and 116 μmol/min, respectively. ProDH activity was stable at alkaline pH and the highest activity was observed at 30°C and pH 8.5. The modeling analysis of the three dimensional structure elucidated that Lys-173 and Asp-202, which were oriented near the hydroxyl group of the substrate, were essential residues for the ProDH activity. This study, to our knowledge, is the first data on the cloning and biochemical and structural properties of P. fluorescens ProDH.     

The 2-oxoacid dehydrogenase multienzyme complex of Haloferax volcanii

Those aerobic archaea whose genomes have been sequenced possess four adjacent genes that, by sequence comparisons with bacteria and eukarya, appear to encode the component enzymes of a 2-oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the archaea. In Thermoplasma acidophilum, evidence has been presented that the heterologously expressed recombinant enzyme possesses activity with the branched chain 2-oxoacids and, to a lesser extent, with pyruvate. In the current paper, we demonstrate that in Haloferax volcanii the four genes are transcribed as an operon in vivo. However, no functional complex or individual enzyme, except for the dihydrolipoamide dehydrogenase component, could be detected in this halophile grown on a variety of carbon sources. Dihydrolipoamide dehydrogenase is present at low catalytic activities, the level of which is increased three to fourfold when Haloferax volcanii is grown on the branched-chain amino acids valine, leucine and isoleucine.     

Some properties of glutamine synthetase and glutamate synthase from Chlorobium vibrioforme f. thiosulfatophilum

The phototrophic green sulphur bacterium Chlorobium vibrioforme f. thiosulfatophilum assimilated ammonia via glutamine synthetase and glutamate synthase when grown with ammonia up to 30 mM, but above this level glutamate dehydrogenase was the key enzyme. Glutamine synthetase purified 42-fold was found to be adenylylated. The -glutamyltransferase activity of the enzyme was markedly inhibited by alanine, glycine, serine and lysine, and these amino acids in various combinations showed cumulative inhibition. Adenine nucleotides also inhibited enzyme activity, especially ATP. Glutamate synthase purified 222-fold had a maximum absorption at 440 nm which was reduced by sodium dithionite, and the enzyme was inhibited by atebrin indicating the presence of a flavin component. The enzyme had specific requirements for NADH, -ketoglutarate and l-glutamine, the K _m values for these were 13.5, 270 and 769 M respectively. Glutamate synthase was sensitive to feedback inhibition by amino acids, adenine nucleotides and other metabolites and the combined effects of these inhibitors was cumulative.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamic dehydrogenase     

Isoenzyme pattern and de novo synthesis of phosphodiesterase during differentiation (spherulation) in Physarum polycephalum

Summary Under conditions of CsCl-equilibrium sedimentation, phosphodiesterase in extracts made from growing Physarum microplasmodia forms two bands with buoyant densities of 1.3572 g/ml (Phosphodiesterase I) and 1.2937 g/ml (Phosphodiesterase II). In spherulating cultures induced by starvation, only phosphodiesterase I is present and true de novo synthesis of this enzyme during this differentiation was demonstrated by density labeling with deuterated amino acids. The synthesis is inhibited by cycloheximide, whereas only the total activity but not the density of the enzyme was influenced by actinomycin-C.In spherulating cultures induced by mannitol both isoenzymes are present as in the growing cultures.     

Glutathione-dependent formaldehyde dehydrogenase homolog from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain R5 is a propanol-preferring alcohol dehydrogenase

Genome search of Bacillus subtilis revealed the presence of an open reading frame annotated as glutathione-dependent formaldehyde dehydrogenase/alcohol dehydrogenase. The open reading frame consists of 1137 nucleotides corresponding to a polypeptide of 378 amino acids. To examine whether the encoded protein is glutathione-dependent formaldehyde dehydrogenase or alcohol dehydrogenase, we cloned and characterized the gene product. Enzyme activity assays revealed that the enzyme exhibits a metal ion-dependent alcohol dehydrogenase activity but no glutathione-dependent formaldehyde dehydrogenase or aldehyde dismutase activity. Although the protein is of mesophilic origin, optimal temperature for the enzyme activity is 60°C. Thermostability analysis by circular dichroism spectroscopy revealed that the protein is stable up to 60°C. Presence or absence of metal ions in the reaction mixture did not affect the enzyme activity. However, metal ions were necessary at the time of protein production and folding. There was a marked difference in the enzyme activity and CD spectra of the proteins produced in the presence and absence of metal ions. The experimental results obtained in this study demonstrate that the enzyme is a bona-fide alcohol dehydrogenase and not a glutathionedependent formaldehyde dehydrogenase.     

Metabolism of aspartate in Mycobacterium smegmatis

Mycobacterium smegmatis grows best on L-asparagine as a sole nitrogen source; this was confirmed. [14C]Aspartate was taken up rapidly (46 nmol.mg dry cells-1.h-1 from 1 mM L-asparagine) and metabolised to CO2 as well as to amino acids synthesised through the aspartate pathway. Proportionately more radioactivity appeared in the amino acids in bacteria grown in medium containing low nitrogen. Activities of aspartokinase and homoserine dehydrogenase, the initial enzymes of the aspartate pathway, were carried by separate proteins. Aspartokinase was purified as three isoenzymes and represented up to 8% of the soluble protein of M. smegmatis. All three isoenzymes contained molecular mass subunits of 50 kDa and 11 kDa which showed no activity individually; full enzyme activity was recovered on pooling the subunits. Km values for aspartate were: aspartokinases I and III, 2.4 mM; aspartokinase II, 6.4 mM. Aspartokinase I was inhibited by threonine and homoserine and aspartokinase III by lysine, but aspartokinase II was not inhibited by any amino acids. Aspartokinase activity was repressed by methionine and lysine with a small residue of activity attributable to unrepressed aspartokinase I. Homoserine dehydrogenase activity was 96% inhibited by 2 mM threonine; isoleucine, cysteine and valine had lesser effects and in combination gave additive inhibition. Homoserine dehydrogenase was repressed by threonine and leucine. Only amino acids synthesised through the aspartate pathway were tested for inhibition and repression. Of these, only one, meso-diaminopimilate, had no discernable effect on either enzyme activity.     

Mild reductions in cytosolic NADP-dependent isocitrate dehydrogenase activity result in lower amino acid contents and pigmentation without impacting growth

Transgenic tomato (Solanum lycopersicum) plants were generated targeting the cytosolic NADP-dependent isocitrate dehydrogenase gene (SlICDH1) via the RNA interference approach. The resultant transformants displayed a relatively mild reduction in the expression and activity of the target enzyme in the leaves. However, biochemical analyses revealed that the transgenic lines displayed a considerable shift in metabolism, being characterized by decreases in the levels of the TCA cycle intermediates, total amino acids, photosynthetic pigments, starch and NAD(P)H. The plants showed little change in photosynthesis with the exception of a minor decrease in maximum photosynthetic efficiency (F _v/F _m), and a small decrease in growth compared to the wild type. These results reveal that even small changes in cytosolic NADP-dependent isocitrate dehydrogenase activity lead to noticeable alterations in the activities of enzymes involved in primary nitrate assimilation and in the synthesis of 2-oxoglutarate derived amino acids. These data are discussed within the context of current models for the role of the various isoforms of isocitrate dehydrogenase within plant amino acid metabolism.     

Molecular cloning and characterization of NAD(+)-dependent xylitol dehydrogenase from Candida tropicalis ATCC 20913

Induction of xylitol dehydrogenase of Candida tropicalis ATCC 20913 by various carbon sources was investigated. The enzyme activity was induced when the yeast was grown on l-arabinose and d-xylose. A novel gene encoding the enzyme was cloned and characterized. The 1,095-bp coding sequence of the gene encodes a polypeptide of 364 amino acids, with a molecular mass of 39.4 kDa. Sequence analysis of the putative protein showed it to be a member of the zinc-containing alcohol dehydrogenase family and to have homology to xylitol dehydrogenase genes from other yeasts and fungi. The recombinant xylitol dehydrogenase expressed in Escherichia coli oxidized polyols such as xylitol and d-sorbitol and reduced ketoses such as d-xylulose and d-fructose. It required exclusively NAD or NADH as a cofactor.     

Rotihibin A,a Novel Plant Growth Regulator,from Streptomyces sp.

A thermophilic Thermoactinomyces sp. E79 producing a highly thermostable alkaline protease was isolated from soil. The protease, produced extracellularly by Thermoactinomyces sp. E79, was purified by DEAE-Sepharose CL-6B and Butyl-Toyopearl 650M column chromatography. The relative molecular mass was estimated to be 31,000 by SDS–polyacrylamide gel electrophoresis. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, suggesting the enzyme to be a serine protease. The optimum temperature for the enzyme activity was 85°C, and about 50% of the original activity remained after incubation at 90°C for 10 min in the presence of Ca^{2 +} . The optimum pH for the enzyme activity was 11.0 and the enzyme was fairly stable from pH 5.0 to 12.0. The gene for this thermostable alkaline protease was cloned in Escherichia coli and the expressed intracellular enzyme was activated by heat treatment. Sequence analysis showed an open reading frame of 1,152 base pairs, coding for a poiypeptide of 384 amino acids. The polypeptide was composed of a signal sequence (25 amino acids), a prosequence (81 amino acids), and a mature protein of 278 amino acids. The deduced amino acid sequence of the mature protease had high similarity with thermitase, a serine protease from Thermoactinomyces vulgaris, and the extent of sequence identity was 76%.