Segmental differences in the stability of thetrp-repressor peptide backbone

Summary Exchange lifetimes of amide protons intrp-repressor with and without the corepressor,l-tryptophan, were studied by heteronuclear 2D NMR spectroscopy. The amide proton exchange times revealed pronounced differences in the stability of different regions of thetrp-repressor. The dimeric core of the molecule is relatively compact and homogeneous in terms of the measured parameters in both apo- and holorepressors. On the other hand the DNA-binding region appears less stable and more susceptible to the exchange of its backbone protons with the solvent. The NMR findings reported here are consistent with and amplify information on the stability of thetrp-repressor obtained by other methods.Dedicated to the memory of Professor V.F. Bystrov     

Amplification of the tryptophan operon gene in Escherichia coli chromosome to increase l-tryptophan biosynthesis

Classical mutagenesis could desensitize the feedback inhibition of l-tryptophan (l-Trp) biosynthesis. Among the mutants, a5-fluorotryptophan-resistant strain, Escherichia coli EMS4-C25 produced 3 g/l of l-Trp within 18 h. The feedback-resistant l-Trp operon gene (trp) prepared from E. coli EMS4-C25 was inserted into pUC19 and pHSG576 to generate pTC701 and pTC576, respectively. When pHSG576 and pTC701 were introduced into E. coli EMS4-C25, chromosomal integration occured through homologous recombination. By using Souther hybridization, we demostrated that the integrated plasmids existed as multicopies. The strains with integrated foreign trp operon gene had higher activities of anthranilate synthase and Trp synthase than those found for the host strain and produced 9.2 g/l of l-Trp with 13% conversion yield from d-glucose. The integration and implification of the trp-operon-beraing plasmid avoided the plasmid instability and increased l-TRp production. Correspondence to: E.-C. Chan     

Indolic constituents and indole-3-acetic acid biosynthesis in the wild-type and a tryptophan auxotroph mutant of Arabidopsis thaliana

 The tryptophan auxotroph mutant trp3-1 of Arabidopsis thaliana (L.) Heynh., despite having reduced levels of l-tryptophan, accumulates the tryptophan-derived glucosinolate, glucobrassicin and, thus, does not appear to be tryptophan-limited. However, due to the block in tryptophan synthase, the mutant hyperaccumulates the precursor indole-3-glycerophosphate (up to 10 mg per g FW). Instability of indole-3-glycerophosphate leads to release of indole-3-acetic acid (IAA) from this metabolite during standard workup of samples for determination of conjugated IAA. The apparent increase in “conjugated IAA” in trp3-1 mutant plants can be traced back entirely to indole-3-glycerophosphate degradation. Thus, the levels of neither free IAA nor conjugated IAA increase detectably in the trp3-1 mutant compared to wild-type plants. Precursor-feeding experiments to shoots of sterile-grown wild-type plants using [²H]₅-l-tryptophan have shown incorporation of label from this precursor into indole-3-acetonitrile and indole-3-acetic acid with very little isotope dilution. It is concluded that Arabidopsis thaliana shoots synthesize IAA from l-tryptophan and that the non-tryptophan pathway is probably an artifact. Received: 1 March 2000 / Accepted: 10 April 2000     

Interaction of the trp repressor with trp operator DNA fragments

The interaction of the trp repressor with several trp operator DNA fragments has been examined by DNA gel retardation assays and by circular dichroism, in the absence and presence of the corepressor l-tryptophan. The holorepressor binds stoichiometrically to both the trpO and aroH operators, forming 1:1 complexes. In the presence of excess protein, additional complexes are formed with these operator fragments. The relative electrophoretic mobilities of the 1:1 complexes differ significantly for trp and aroH operators, indicating that they differ substantially in gross structure. A mutant trp operator, trpO ^c, has low affinity for the holorepressor, and forms only complexes with stoichiometries of 2:1 (repressor: DNA) or higher, which have a very low electrophoretic mobility. Specific binding is also accompanied by a large increase in the intensity of the near ultraviolet circular dichroism, with only a small blue shift, which is consistent with significant changes in the conformation of the DNA. Large changes in the chemical shifts of three resonances in the ³¹P NMR spectrum of both the trp operator and the aroH operator occur on adding repressor only in the presence of L-tryptophan, consistent with localised changes in the backbone conformation of the DNA.Abbreviations CD circular dichroism - trpO, trpR aroH trp operator fragments - trpO ^c trpMH mutant trp operator fragments     

Isolation and Identification of a Sexual Hormone in Yeast

5-Fluorotryptophan (5FT), indolmycin (IM), 4-fluorotryptophan and 7-azatryptophan were found on screening to be tryptophan antagonists among various chemically synthesized and naturally occurring tryptophan analogues for the isolation of l-tryptophan (l-Trp) producing mutants of Bacillus subtilis K.

From among 5FT resistant mutants, potent l-Trp producers were obtained using an improved isolation medium. Growth of the isolated 5FT-resistant l-Trp producer, AJ 11709, was inhibited by IM. From among 5FT and IM resistant mutants, the best strain, AJ 11979, which produced 9.0 g/liter of l-Trp from 13% glucose on 120hr cultivation, was selected.     

Amino acid transport system y+L of human erythrocytes: Specificity and cation dependence of the translocation step

The transport specificity of system y⁺L of human erythrocytes was investigated and the carrier was found to accept a wide range of amino acids as substrates. Relative rates of entry for various amino acids were estimated from their trans-effects on the unidirectional efflux of l-[¹⁴C]-lysine. Some neutral amino acids, l-lysine and l-glutamic acid induced marked trans-acceleration of labeled lysine efflux; saturating concentrations of external l-leucine and l-lysine increased the rate by 5.3±0.63 and 6.2±0.54, respectively. The rate of translocation of the carrier-substrate complex is less dependent on the structure of the amino acid than binding. Translocation is slower for the bulkier analogues (l-tryptophan, l-phenylalanine); smaller amino acids, although weakly bound, are rapidly transported (l-alanine, l-serine). Half-saturation constants (±sem) calculated from this effect (l-lysine, 10.32±0.49 m and l-leucine, 11.50±0.50 m) agreed with those previously measured in cis-inhibition experiments. The degree of trans-acceleration caused by neutral amino acids did not differ significantly in Na⁺, Li⁺ or K⁺ medium, whereas the affinity for neutral amino acids was dramatically decreased if Na⁺ or Li⁺ were replaced by K⁺. The observation that specificity is principally expressed in substrate binding indicates that the carrier reorientation step is largely independent of the forces of interaction between the carrier and the transport site.We wish to thank Dr C.A.R. Boyd for helpful discussions and Prof. H.N. Christensen for sharing with us very relevant bibliographic material. We are grateful to FONDECYT (1282/91) and DTI (B 2674) (Chile) for financial assistance.     

The effect of selective deuteration on magnetization transfer in larger proteins

Summary The effects of selective deuteration on calculated NOESY intensities have been analyzed for the structure of theE. coli trp aporepressor, a 25 kDa protein. It is shown that selectively deuteratedtrp aporepressor proteins display larger calculated NOESY intensities than those for the same interproton distances in the natural abundance protein. The relatively larger magnetization transfer is demonstrated by a comparison of the NOE build-up curves for specific proton pairs, and for the calculated NOE intensities of short-range NOEs to backbone amide protons. This increase in intensity is especially pronounced for the NH¹–NH¹⁺¹ cross peaks in the -helical regions, and particularly for amide protons of two sequential deuterated residues. The effect is shown to be further intensified for longer mixing times. It is also shown that in all cases, each amide proton exhibits stronger NOEs to its own side chain, with an enhanced effect for deuterated derivatives. This theoretical analysis demonstrates that an evaluation of the relative NOE intensities for different selectively deuterated analogs may be an important tool in assigning NMR spectra of large proteins. These results also serve as a guide for the interpretation of NOEs in terms of distances for structure calculations based on data using selectively deuterated proteins.     

Genetic Changes in Regulation Occurring in the Tryptophan Biosynthetic Pathway of l-Tryptophan Producing Mutants Derived from Bacillus subtilis K

The regulatory mechanism for l-tryptophan (l-Trp) synthesis was compared between the wild type strain and l-Trp producing mutants of B. subtilis K. In the wild type strain, indolmycin (IM) repressed the synthesis of anthranilate synthetase (AS) more strongly than 5-fluorotryptophan ? (5FT), which repressed AS to the same extent as l-Trp did. 5FT inhibited the activity of AS as strongly as l-Trp did, while IM had no inhibitory effect. In the 5FT resistant strains, the syntheses of AS and tryptophan synthetase (TS-B) were markedly increased by genetic derepression, while AS remained still sensitive to the feedback inhibition by l-Trp. The facts that IM repressed the syntheses of AS and TS-B in the strain which was 5FT^r and IM^S, and did not repress those in the IM-resistant mutant suggested that IM acts as a co-repressor in a different way from 5FT.     

Hyper-production of l-trytophan via fermentation with crystallization

A stable and fast l-tryptophan producer, AGX1757, was isolated from Escherichia coli W3110 trpAE1 trpR tnaA, which carried pSC101-trpI15·14. Cells of AGX1757 did not lose the composite plasmid during fermentation. Whenever a fed-batch culture of AGX1757 attained an l-tryptophan concentration of about 30 g/l, indole began to appear in the broth. The emergence of indole was caused by inhibition of tryptophan synthase due to accumulated l-tryptophan. Hence, the production rate of l-tryptophan sharply decreased. A higher solubility of l-tryptophan in the supernatant of culture broth (about 32 g/l) than that in the initial medium (about 22 g/l) was attributed to some unknown interaction between l-tryptophan and certain macromolecular material(s) coming from the bacterial cells. An addition of non-ionic detergents into the supernatant was effective for decreasing the solubility of l-tryptophan, hence causing crystallization of l-tryptophan. Pluronic L-61 was supplied from outside to an extent of 0.5% in terms of wt% concentration at around 45 h of fermentation when the l-tryptophan accumulated reached about 25 g/l. This addition actually caused crystallization of l-tryptophan and, as a result, the inhibitory effect of tryptophan synthase by l-tryptophan accumulated in the broth could be alleviated. Thus far, further fermentation became possible. l-Tryptophan of more than 50 g/l was finally produced by feeding solutions of both glucose and anthranilic acid. Correspondence to: H. Tsunekawa     

Adsorption studies for the separation ofl-tryptophan froml-serine and indole in a bioconversion medium

l-tryptophan was produced froml-serine and indole by immobilized Escherichia coli cells in organic-aqueous systems. Selective adsorption was the method chosen to enable both product separation andl-serine reutilization. Amongst various adsorbents tested activated carbons and neutral polymeric resins (XAD-4 and XAD-7) showed good performance. The neutral resins could selectively concentrate thel-tryptophan from dilute aqueous solutions and adsorbed only 5% of the unconvertedl-serine. High separation factors (l-tryptophan/l-serine and indole/l-tryptophan) were obtained with these adsorbents. Despite a lower capacity, the XAD-7 resin had the advantage of desorbingl-tryptophan with basic or acidic solutions, while organic solvents were required to desorb, at the same concentration levels, this compound from XAD-4.In a packed bed column filled with XAD-4 resin or activated carbon, totall-tryptophan adsorption and recovery were achieved at linear velocities up to 5.0 cm/min and 3.2 cm/min respectively. Successive sorbent reutilization, following continuous sorption and elution steps, was carried out in packed bed columns with the neutral resins and activated carbon.Thel-form of tryptophan, after crystallization, was identified by HPTLC.List of Symbols HPLC High Performance Liquid Chromatography - HPTLC High Performance Thin Layer Chromatography - Trp tryptophan - Ser Serine - A amount of sorbent(g) - c equilibrium solute concentration in the aqueous phase (g/dm³) - c _i initial (before adding the sorbent) liquid phase concentration (g/dm³) - C _T tryptophan concentration in the inlet solution (g/dm³) - C _To tryptophan concentration in the outlet solution (g/dm³) - E _z axial dispersion coefficient (m²/s) - k experimental constant (Eq. 1, 2 and 3) - K ₁ rate constant of adsorption (min^–1) - L column length(m) - n experimental constant (eq. 1, 2 and 3) - q equilibrium solid phase concentration (g solute/g sorbent) - q _max maximum capacity of sorbent (g solute/g sorbent) - t time(s) - v liquid velocity (m/s) - V volume of liquid phase(dm³) - V _e eluted volume(dm³) - V _r volume needed to saturate the column (dm³)     

1H and 15N NMR resonance assignments and solution secondary structure of oxidized Desulfovibrio desulfuricans flavodoxin

Summary Sequence-specific ¹H and ¹⁵N resonance assignments have been made for 137 of the 146 nonprolyl residues in oxidized Desulfovibrio desulfuricans [Essex 6] flavodoxin. Assignments were obtained by a concerted analysis of the heteronuclear three-dimensional ¹H-¹⁵N NOESY-HMQC and TOCSY-HMQC data sets, recorded on uniformly ¹⁵N-enriched protein at 300 K. Numerous side-chain resonances have been partially or fully assigned. Residues with overlapping ¹H^N chemical shifts were resolved by a three-dimensional ¹H-¹⁵N HMQC-NOESY-HMQC spectrum. Medium-and long-range NOEs, ³J_NH coupling constants, and ¹H^N exchange data indicate a secondary structure consisting of five parallel -strands and four -helices with a topology similar to that of Desulfovibrio vulgaris [Hidenborough] flavodoxin. Prolines at positions 106 and 134, which are not conserved in D. vulgaris flavodoxin, contort the two C-terminal -helices.Abbreviations CSI chemical shift index - DQF-COSY double-quantum-filtered correlation spectroscopy - DIPSI decoupling in the presence of scalar interactions - FMN flavin mononucleotide - GARP globally optimized alternating phase rectangular pulse - HMQC heteronuclear multiple-quantum coherence - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser effect - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy - TPPI time-proportional phase increments - TSP 3-(trimethylsilyl)propionic-2,2,3,3-d ₄ acid, sodium salt     

Transport ofl-tryptophan inSaccharomyces cerevisiae

In addition to the general amino acid transport system (GAP) ofS. cerevisiae l-tryptophan is transported by another system with approximately 25% capacity of GAP, with aK _T of 0.41±0.08 mmol/L and with a similar specificity as GAP (lower inhibition by Met, Pro, Ser, Thr and 2-aminoisobutyric acid; greater inhibition by Glu and His). The pH optimum of this system is at 5.0–5.5, activation energy above the transition point (20°C) was 20 kJ/mol, below the transition point 55 kJ/mol. The transport by this system was virtually unidirectional, efflux amounting to at most 10% into a tryptophan-free medium. The transport itself was blocked by 2,4-dinitrophenol, antimycin A and uranyl nitrate. The system was synthesized de novo during preincubation with glucose=fructose>trehalose >ethanol within 30 min, and was degraded with a half-time of 15 min in the absence of further synthesis. The accumulation ratios ofl-tryptophan ingap1 mutants were concentration-dependent (200∶1 at 1 μmoll-Trp/L, 4∶1 at 2.5 mmoll-Trp/L) and decreased with increasing suspension density from 200∶1 to 5∶1 (for 10 μmoll-Trp/L). The involvement of hydrogen ions in the uptake was clearly demonstrated by the effect of D₂O even if it could not be established by either shifts of pH_out or membrane depolarization.     

Resonance assignment and structural analysis of acid denatured E. coli [U-15N]-glutaredoxin 3

Virtually complete sequence specific ¹H and ¹⁵N resonance assignments are presented for acid denatured reduced E. coli glutaredoxin 3. The sequential resonance assignments of the backbone rely on the combined use of 3D F₁-decoupled ROESY-¹⁵N-HSQC and 3D ¹⁵N-HSQC-(TOCSY-NOESY)-¹⁵N-HSQC using a single uniformly ¹⁵N labelled protein sample. The sidechain resonances were assigned from a 3D TOCSY-¹⁵N-HSQC and a homonouclear TOCSY spectrum. The presented assignment strategy works in the absence of chemical exchange peaks with signals from the native conformation and without ¹³C/¹⁵N double labelling. Chemical shifts, ³J(H, NH) coupling constants and NOEs indicate extensive conformational averaging of both backbone and side chains in agreement with a random coil conformation. The only secondary structure element persisting at pH 3.5 appears to be a short helical segment comprising residues 37 to 40.Abbreviations HSQC heteronuclear single quantum coherence - NMR nuclear magnetic resonance - NOE nuclear Overhauser effect - NOESY two-dimensional NOE spectroscopy - ROE nuclear Overhauser effect in the rotating frame - ROESY two-dimensional ROE spectroscopy - TOCSY total correlation spectroscopy - TPPI time proportional phase incrementation Correspondence to: G. Otting     

Regulation of the synthesis of enzymes of tryptophan dissimilation in Acinetobacter calcoaceticus

Summary Acinetobacter calcoaceticus dissimilates tryptophan via the -ketoadipate pathway. The first enzyme, tryptophan oxygenase (l-tryptophan: oxygen oxidoreductase; EC 1.13.1.12), is substrate-induced by tryptophan. The second two enzymes, formamidase (aryl-formylamine amidohydrolase; EC 3.5.1.9) and kynureninase (l-kynurenine hydrolase; EC 3.7.1.3), are induced by the next intermediate, kynurenine. The last enzyme specific to tryptophan dissimilation, anthranilate oxidase, is substrate induced. This inductive pattern is in marked contrast to the extensive coordinacy of enzyme synthesis characteristic of the remainder of the -ketoadipate pathway.     

Aromatic amino acid biosynthesis in Alcaligenes eutrophus H 16

Properties and regulation of anthranilate synthase from Alcaligenes eutrophus H 16 were investigated. Anthranilate synthase was partially purified from crude extracts by affinity chromatography on tryptophan-substituted Sepharose, and was used for kinetic measurements. During the purification procedure the enzyme was stabilized by 50 mM l-glutamine or during chromatography on DEAE-cellulose and Sephadex G-200 with 30% glycerol, respectively.The glutamine dependent activity of anthranilate synthase was examined; it showed little change between pH 8.4 and pH 9.1. The Arrhenius plot was broken and the activation energy, H, calculated therefrom amounted to 8.9 kcal/mole up to 30°C and 5.5 kcal/mole at higher temperatures. The molecular weight determined by gelfiltration on Sephadex G-200 and by sucrose density gradient centrifugation resulted in 158000 and 126000, respectively. The K _m -values for the two substrates chorismate and glutamine were found to be 5 M and 560 M, respectively.Anthranilate synthase was strongly inhibited by l-tryptophan; the only amino acid that affected enzyme activity. Homotropic interactions for chorismate (Hill coefficient n=1.4) were obtained in the presence of l-tryptophan. 50% inhibition were caused by 10 M l-tryptophan at 100 M chorismate. The inhibition with respect to l-glutamine was noncompetitive.Anthranilate synthase was not associated to phosphoribosyl transferase and easily separable from the latter by different chromatographic methods.Abbreviation TEA triethanolamine     

l-threonine production by l-aspartate- and l-homoserine-resistant mutant of Escherichia coli

Summary Growth and l-threonine productivity of l-threonine producer Escherichia coli H-4290 were inhibited by precursor amino acids, l-homoserine and l-aspartate. l-Threonine hyper-producers were isolated among the mutants resistant to l-homoserine and l-aspartate. Mutants H-4351 (Hom^r) and H-4578 (Hom^r, Asp^r) accumulated 22.2 g/l and 24.3 g/l of l-threonine in test tube cultures, while the parental strain H-4290 accumulated 18.2 g/l. The enzyme level of aspartokinase I (first enzyme of the threonine operon) was enhanced 2.3 times (H-4351) and 3 times (H-4578) that of H-4290. Mutant H-4578 accumulated 76 g/l of l-threonine in a 2-l jar fermentor after 75 h cultivation.Abbreviations DAP diaminopimeric acid - Met ^l poor growth in methionine-free medium - AHV -amino--hydroxyvaleric acid - Thr-N^- lack of ability to utilize l-threonine as a nitrogen source - Rif rifampicin - Lys+Met^r resistant to l-lysine and dl-methionine     

An extreme-halophile archaebacterium possesses the interlock type of prephenate dehydratase characteristic of the Gram-positive eubacteria

The focal point of phenylalanine biosynthesis is a dehydratase reaction which in different organisms may be prephenate dehydratase, arogenate dehydratase, or cyclohexadienyl dehydratase. Gram-positive, Gram-negative, and cyanobacterial divisions of the eubacterial kingdom exhibit different dehydratase patterns. A new extremehalophile isolate, which grows on defined medium and is tentatively designated as Halobacterium vallismortis CH-1, possesses the interlock type of prephenate dehydratase present in Gram-positive bacteria. In addition to the conventional sensitivity to feedback inhibition by l-phenylalanine, the phenomenon of metabolic interlock was exemplified by the sensitivity of prephenate dehydratase to allosteric effects produced by extra-pathway (remote) effectors. Thus, l-tryptophan inhibited activity while l-tyrosine, l-methionine, l-leucine, and l-isoleucine activated the enzyme. l-Isoleucine and l-phenylalanine were effective at M levels; other effectors operated at mM levels. A regulatory mutant selected for resistance to growth inhibition caused by -2-thienylalanine possessed an altered prephenate dehydratase in which a phenomenon of disproportionately low activity at low enzyme concentration was abolished. Inhibition by l-tryptophan was also lost, and activation by allosteric activators was diminished. Not only was sensitivity to feedback inhibition by l-phenylalanine lost, but the mutant enzyme was now activated by this amino acid (a mutation type previously observed in Bacillus subtilis). It remains to be seen whether this type of prephenate dehydratase will prove to be characteristic of all archaebacteria or of some archaebacterial subgroup cluster.     

Effects of combined administration ofl-tryptophan and tricyclic antidepressants on α2- and β-adrenoceptors and monoamine levels in rat brain

In order to test whether co-administration of a serotonin precursor with antidepressant drugs could potentiate the effects of the antidepressants on monoamines or adrenoceptors in rat brain,l-tryptophan (20 mg/kg) was administered to rats daily for 7 or 15 days, either alone or in combination with desipramine (10 mg/ kg) or amitriptyline (10 mg/kg). After treatment withl-tryptophan for 7 days, increases were observed in rat hypothalamic and frontal cortex 5-hydroxy-3-indoleacetic acid levels as well as in hypothalamic dopamine and nucleus accumbens 3,4-dihydroxyphenylacetic acid levels. After 15 days, hippocampal -adrenoceptor density was found to be decreased. There was no evidence of potentiation of desipramine or amitriptyline action whenl-tryptophan was administered in combination with the antidepressants. On the contrary, the antidepressants appeared to interact withl-tryptophan to reduce its effects.     

Substrate Oxidation by Indoleamine 2,3-Dioxygenase: EVIDENCE FOR A COMMON REACTION MECHANISM*

The kynurenine pathway is the major route of l-tryptophan (l-Trp) catabolism in biology, leading ultimately to the formation of NAD⁺. The initial and rate-limiting step of the kynurenine pathway involves oxidation of l-Trp to N-formylkynurenine. This is an O₂-dependent process and catalyzed by indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase. More than 60 years after these dioxygenase enzymes were first isolated (Kotake, Y., and Masayama, I. (1936) Z. Physiol. Chem. 243, 237–244), the mechanism of the reaction is not established. We examined the mechanism of substrate oxidation for a series of substituted tryptophan analogues by indoleamine 2,3-dioxygenase. We observed formation of a transient intermediate, assigned as a Compound II (ferryl) species, during oxidation of l-Trp, 1-methyl-l-Trp, and a number of other substrate analogues. The data are consistent with a common reaction mechanism for indoleamine 2,3-dioxygenase-catalyzed oxidation of tryptophan and other tryptophan analogues.     

StereoselectiveO-glycosylation oftrans-4-hydroxy-l-proline derivatives promoted by silver zeolite

Trans-4-hydroxy-l-proline has been converted to four imino- and carboxyl-blocked derivatives which are suitable for the synthesis of 4-O-glycosyl conjugates. Reaction of these derivatives with 2,3,5-tri-O-benzyl--l-arabinofuranosyl chloride in the presence of a silver zeolite promoter yielded the blocked -furanosyl amino-acid conjugates. Deprotection gavetrans-4-(-l-arabinofuranosyloxy)-l-proline which was characterised as its crystalline isopropyl ester.¹³C-NMR Data are presented for the compounds described.