Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase was studied. The tryptophan synthase (EC 4.2.1.20) of Escherichia coli catalyzed beta-substitution reaction of L-serine into L-tryptophan and the amino acid racemase (EC 5.1.1.10) of Pseudomonas putida catalyzed the racemization of D-serine simultaneously in one reactor. Under optimal conditions established for L-tryptophan production, a large-scale production of L-tryptophan was carried out in a 200-liter reactor using intact cells of E. coli and P. putida. After 24 h of incubation with intermittent indole feeding, 110 g liter-1 of L-tryptophan was formed in molar yields of 91 and 100% for added DL-serine and indole, respectively. Continuous production of L-tryptophan was also carried out using immobilized cells of E. coli and P. putida. The maximum concentration of L-tryptophan formed was 5.2 g liter-1 (99% molar yield for indole), and the concentration decreased to 4.2 g liter-1 after continuous operation for 20 days.     

Red-edge-excitation fluorescence spectroscopy of indole and tryptophan

Studies on the dependence of indole and tryptophan fluorescence emission spectra on excitation wavelength, ^ex, show that the emission shifts to longer wavelengths for red-edge excitation in different solid and viscous solvents. In solid systems the spectral shifts for excitation in the range from 290 to 310 nm can reach tens of nm, and they are more significant than changes of ^ex. In a viscous medium the magnitude of this effect is shown to be directly related to the dipole-reorientational relaxation of solvent molecules in the environment of the chromophore, which allows the relaxation times to be estimated. The method involves simple steady-state measurements of fluorescence spectra at the maximum and at the red edge of the absorption band. Since it is not necessary to obtain information on the fluorescence spectra of completely relaxed states, this method for the estimation of relaxation times may have advantages in studies of proteins compared with the conventional relaxation shift method, and may produce complementary information to that obtained by nanosecond time-resolved spectroscopy.     

Pigment production from tryptophan by an Achromobacter species

Duerre, John A. (University of North Dakota, Grand Forks), and Patrick J. Buckley. Pigment production from tryptophan by an Achromobacter species. J. Bacteriol. 90:1686-1691. 1965.-A microorganism was isolated from the soil near the University of North Dakota. Biochemical and morphological characteristics indicated that this organism would best be classified as a member of the family Achromobacteraceae, genus Achromobacter, species unknown. The organism produced a red pigment when grown in a medium containing yeast extract and tryptophan. The pH optimum for pigment production was about 8.0 and the optimal temperature was 25 C. During a study of the nutritional requirements for growth and pigment production, it was found that the organism would grow and produce pigment in a medium containing tryptophan and nucleosides, but the rate of both growth and pigment formation in this medium was slower than that observed with tryptophan and yeast extract. The organism grew well in the presence of acid-hydrolyzed casein and nucleosides without producing pigment, indicating that the pigment is not necessary for growth. Resting-cell experiments definitely established tryptophan as the sole exogenous requirement for pigment production. The pigment was extracted from yeast extract-tryptophan medium with chloroform. Thin layer chromatographic analysis of the crude pigment extracted from this medium revealed the presence of two other pigments in addition to the major red pigment. One of these was a highly fluorescent orange pigment and the other a pink pigment. Only the red pigment was produced by resting cells in the presence of tryptophan alone. This pigment served as an electron acceptor when coupled with formic dehydrogenase, indicating its possible function as an oxidation-reduction pigment. The oxidized pigment had absorption peaks at 506 and 304 mmu. The peak at 506 mmu disappeared upon reduction with sodium sulfite. Shaking the reduced pigment in air proved to be an unsatisfactory method for returning the reduced pigment to the oxidized, colored state.     

Beta-elimination of indole from L-tryptophan catalyzed by bacterial tryptophan synthase: a comparison between reactions catalyzed by tryptophanase and tryptophan synthase

Although tryptophan synthase catalyzes a number of pyridoxal phosphate dependent beta-elimination and beta-replacement reactions that are also catalyzed by tryptophanase, a principal and puzzling difference between the two enzymes lies in the apparent inability of tryptophan synthase to catalyze beta-elimination of indole from L-tryptophan. We now demonstrate for the first time that the beta 2 subunit and the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli and from Salmonella typhimurium do catalyze a slow beta-elimination reaction with L-tryptophan to produce indole, pyruvate, and ammonia. The rate of the reaction is about 10-fold higher in the presence of the alpha subunit. The rate of indole production is increased about 4-fold when the aminoacrylate produced is converted to S-(hydroxyethyl)-L-cysteine by a coupled beta-replacement reaction with beta-mercaptoethanol. The rate of L-tryptophan cleavage is also increased when the indole produced is removed by extraction with toluene or by condensation with D-glyceraldehyde 3-phosphate to form indole-3-glycerol phosphate in a reaction catalyzed by the alpha subunit of tryptophan synthase. The amount of L-tryptophan cleavage is greatest in the presence of both beta-mercaptoethanol and D-glyceraldehyde 3-phosphate, which cause the removal of both products of cleavage. The cleavage reaction is not due to contaminating tryptophanase since the activity is not inhibited by (3R)-2,3-dihydro-L-tryptophan, a specific inhibitor of tryptophanase, but is inhibited by (3S)-2,3-dihydro-L-tryptophan, a specific inhibitor of tryptophan synthase. The cleavage reaction is also inhibited by D-tryptophan, the product of a slow racemization reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of indole chemistry to label tryptophan residues in protein for determination of tryptophan surface accessibility

Solvent accessibility can be used to evaluate protein structural models, identify binding sites, and characterize protein conformational changes. The differential modification of amino acids at specific sites enables the accessible surface residues to be identified by mass spectrometry. Tryptophan residues within proteins can be differentially labeled with halocompounds by a photochemical reaction. In this study, tryptophan residues of carbonic anhydrase are reacted with chloroform, 2,2,2-trichloroethanol (TCE), 2,2,2-trichloroacetate (TCA), or 3-bromo-1-propanol (BP) under UV irradiation at 280 nm. The light-driven reactions with chloroform, TCE, TCA, and BP attach a formyl, hydroxyethanone, carboxylic acid, and propanol group, respectively, onto the indole ring of tryptophan. Trypsin and chymotrypsin digests of the modified carbonic anhydrase are used to map accessible tryptophan residues using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Tryptophan reactivity is determined by identifying peptides with tryptophan residues modified with the appropriate label. The reactivity is calculated from the frequency that the modification is identified and a semiquantitative measure of the amount of products formed. Both of these measures of tryptophan reactivity correlate significantly with the accessible surface area of tryptophan residues in carbonic anhydrase determined from the X-ray crystal structure. Therefore the photochemical reaction of halocompounds with tryptophan residues in carbonic anhydrase indicates the degree of solvent accessibility of these residues.     

Peroxide oxidation of indole to oxindole by chloroperoxidase catalysis.

In the presence of chloroperoxidase, indole was oxidized by H2O2 to give oxindole as the major product. Under most conditions oxindole was the only product formed, and under optimal conditions the conversion was quantitative. This reaction displayed maximal activity at pH 4.6, although appreciable activity was observed throughout the entire pH range investigated, namely pH 2.5-6.0. Enzyme saturation by indole could not be demonstrated, up to the limit of indole solubility in the buffer. The oxidation kinetics were first-order with respect to indole up to 8 mM, which was the highest concentration of indole that could be investigated. On the other hand, 2-methylindole was not affected by H2O2 and chloroperoxidase, but was a strong inhibitor of indole oxidation. The isomer 1-methylindole was a poor substrate for chloroperoxidase oxidation, and a weak inhibitor of indole oxidation. These results suggest the possibility that chloroperoxidase oxidation of the carbon atom adjacent to the nitrogen atom in part results from hydrogen-bonding of the substrate N-H group to the enzyme active site.     

Inhibition of tumor necrosis factor-induced cell killing by tryptophan and indole

Cells sensitive to the cytocidal effect of tumor necrosis factor (TNF) were protected against this effect when growth in the presence of elevated concentrations of tryptophan. Several other indole derivatives also provided protection against TNF cytotoxicity. Most effective were indole itself and its monomethyl derivatives, providing a degree of protection greatly exceeding that observed with tryptophan. Protection was also observed against the cytocidal effect of TNF applied in the presence of a protein synthesis inhibitor. The protective effect of tryptophan was largely dependent on preexposure of the cells, for several hours, to a high concentration of this amino acid. On the other hand, indole was protective also when applied to cells together with TNF, or even two hours after TNF application. The inhibition of the cytotoxicity of TNF by tryptophan and other indole derivatives may serve as a useful experimental tool in exploring the mechanisms and the physiological implications of TNF cytotoxicity.     

The use of 6-(difluoromethyl)indole to study the activation of indole by tryptophan synthase

6-(Difluoromethyl)indole has been characterized and developed as a probe for the turnover of indole by the bifunctional enzyme, tryptophan synthase (alpha 2 beta 2). The neutral form of the indolyl species undergoes a slow and spontaneous hydrolysis to produce 6-formylindole with a rate constant (k1) of 0.0089 +/- 0.0001 min-1. The overall rate is independent of pH in the range of 3.5-10.5. Above pH 10.5, the observed rate increases are due to the high reactivity of the anionic form of the indole; deprotonation at N-1 accelerates hydrolysis by 10(4)-fold (k2, 97 +/- 2 min-1). The magnitude of this effect provides a technique for detecting the formation or stabilization of the anionic form of indole. 6-(Difluoromethyl)indole is recognized and processed by the beta subunit of tryptophan synthase. Selective inactivation of the beta subunit prevents enzymatic processing of 6-(difluoromethyl)indole. Chromatographic isolation and mass spectral analysis has identified 6-(difluoromethyl)tryptophan as the sole turnover product of the indolyl substrate. The lack of enzyme-promoted dehalogenation does not exclude the formation of an indole anion during turnover but rather the data suggest that rapid carbon-carbon bond formation (greater than 5300 min-1) prevents the accumulation of this anion.     

Crystalline hemoprotein from Pseudomonas that catalyzes oxidation of side chain of tryptophan and other indole derivatives.

A new enzyme which catalyzes the oxidation of the side chain of tryptophan and other indole derivatives, has been purified to apparent homogeneity from Pseudomonas and crystallized. The overall purification was about 25-fold with a yield of 4.5%. The purified enzyme was apparently homogeneous as judged by polyacrylamide gel electrophoresis. The molecular weight estimated by gel filtration was approximately 280,000 and sedimentation coefficient (S20,w) was 11 by sucrose density gradient ultracentrifugation. The absorption spectra indicated that the enzyme was a hemoprotein. The purified enzyme was shown to catalyze the reaction in which 1 mol each of NH3 and CO2 was formed at the expense of 1 mol each of L-tryptophan and molecular oxygen. Neither peroxidase nor catalase activity was detected in the purified enzyme and no formation of H2O2 was observed during the enzyme reaction. The product(s) of the reaction was unstable but was converted to and was identified as its stable quinoxaline derivative, 2-(3-indolyl)quinoxaline, in the presence of o-phenylenediamine. These results indicate that the product of the reaction was 3-indolylglycoaldehyde or 3-indolylglyoxal. A variety of other indole derivatives such as D-tryptophan, 5-hydroxyl-L-tryptophan, tryptamine, serotonin, melatonin, N-acetyl-L-tryptophan, N-acetyl-L-tryptophanamide, 3-indoleacetamide, 3-indolelactic acid, 3-indolepropionic acid, 3-indoleethanol, and skatole were also substrates.     

Metal ions in biological catalysis: from enzyme databases to general principles

We analysed the roles and distribution of metal ions in enzymatic catalysis using available public databases and our new resource Metal-MACiE (). In Metal-MACiE, a database of metal-based reaction mechanisms, 116 entries covering 21% of the metal-dependent enzymes and 70% of the types of enzyme-catalysed chemical transformations are annotated according to metal function. We used Metal-MACiE to assess the functions performed by metals in biological catalysis and the relative frequencies of different metals in different roles, which can be related to their individual chemical properties and availability in the environment. The overall picture emerging from the overview of Metal-MACiE is that redox-inert metal ions are used in enzymes to stabilize negative charges and to activate substrates by virtue of their Lewis acid properties, whereas redox-active metal ions can be used both as Lewis acids and as redox centres. Magnesium and zinc are by far the most common ions of the first type, while calcium is relatively less used. Magnesium, however, is most often bound to phosphate groups of substrates and interacts with the enzyme only transiently, whereas the other metals are stably bound to the enzyme. The most common metal of the second type is iron, which is prevalent in the catalysis of redox reactions, followed by manganese, cobalt, molybdenum, copper and nickel. The control of the reactivity of redox-active metal ions may involve their association with organic cofactors to form stable units. This occurs sometimes for iron and nickel, and quite often for cobalt and molybdenum. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Production of indole alkaloids by metabolic engineering of the tryptophan pathway in rice

Tryptophan decarboxylase (TDC) converts tryptophan (Trp) into tryptamine, consequently increasing the metabolic flow of tryptophan derivatives into the production of secondary metabolites such as indole alkaloids. We inserted an expression cassette containing OsTDC, a putative tryptophan decarboxylase gene from rice, into an expression plasmid vector containing OASA1D, the feedback‐resistant anthranilate synthase alpha‐subunit mutant (OASA1D). Overexpression of OASA1D has been reported to significantly increase Trp levels in rice. The co‐expression of OsTDC and OASA1D in rice calli led to almost complete depletion of the Trp pool and a consequent increase in the tryptamine pool. This indicates that TDC inactivity is a contributory factor for the accumulation of Trp in rice transgenics overexpressing OASA1D. Metabolic profiling of the calli expressing OsTDC and OASA1D revealed the accumulation of serotonin and serotonin‐derived indole compounds (potentially pharmacoactive β‐carbolines) that have not been reported from rice. Rice calli overexpressing OASA1D:OASA1D is a novel system for the production of significant amounts of pharmacologically useful indole alkaloids in rice.     

Biodiesel production via enzymatic catalysis

A review of current work in biodiesel production via enzymatic catalysis has been done. The parameters of the process as determined by laboratories are represented and analyzed. The main factors affecting interesterification are considered. The major types of oils and alcohols used in biodiesel synthesis are listed. The means of lipase enzyme immobilization, including exposure on the cell surface, are discussed.     

Indole-3-acetonitrile production from indole glucosinolates deters oviposition by Pieris rapae

Like many crucifer-specialist herbivores, Pieris rapae uses the presence of glucosinolates as a signal for oviposition and larval feeding. Arabidopsis thaliana glucosinolate-related mutants provide a unique resource for studying the in vivo role of these compounds in affecting P. rapae oviposition. Low indole glucosinolate cyp79B2 cyp79B3 mutants received fewer eggs than wild type, confirming prior research showing that indole glucosinolates are an important oviposition cue. Transgenic plants overexpressing epithiospecifier protein, which shifts glucosinolate breakdown toward nitrile formation, are less attractive to ovipositing P. rapae females. Exogenous application of indol-3-ylmethylglucosinolate breakdown products to cyp79B2 cyp79B3 mutants showed that oviposition was increased by indole-3-carbinol and decreased by indole-3-acetonitrile (IAN). P. rapae larvae tolerate a cruciferous diet by using a gut enzyme to redirect glucosinolate breakdown toward less toxic nitriles, including IAN, rather than isothiocyanates. The presence of IAN in larval regurgitant contributes to reduced oviposition by adult females on larvae-infested plants. Therefore, production of nitriles via epithiospecifier protein in cruciferous plants, which makes the plants more sensitive to generalist herbivores, may be a counter-adaptive mechanism for reducing oviposition by P. rapae and perhaps other crucifer-specialist insects.     

Metal ions in ribozyme folding and catalysis

Current research is reshaping basic theories regarding the roles of metal ions in ribozyme function. No longer viewed as strict metalloenzymes, some ribozymes can access alternative catalytic mechanisms depending on the identity and availability of metal ions. Similarly, reaction conditions can allow different folding pathways to predominate, with divalent cations sometimes playing opposing roles.     

Photoaffinity labeling of the indole sites on the Escherichia coli tryptophan synthase alpha-subunit

The alpha subunit of the Escherichia coli tryptophan synthase catalyzes the reversible aldolytic reaction: Indole-3-glycerol phosphate in equilibrium indole + glyceraldehyde 3-phosphate. The use of 5-azidoindole as a photoaffinity label has made the generation of a number of enzyme-substrate complexes possible, each with a given degree of saturation of the two postulated indole sites. When assayed in the reverse reaction (indole-3-glycerol phosphate synthesis), samples of alpha subunit treated at concentrations of 5-azidoindole less than or equal to 2 mM show a progressive 30-40% activation. A gradual inactivation occurs only in samples irradiated at concentrations in excess of 2 mM 5-azidoindole, and this inactivation is complete at 8-10 mM. A quantitatively similar activation occurs in the forward reaction (indole synthesis), however inactivation in this case is incomplete, with complexes treated at 8-12 mM 5-azidoindole retaining 30-40% relative activity in this reaction. When treated alpha subunits were assayed for their abilities to complement the beta 2-subunit in the reactions indole + L-serine leads to L-tryptophan + H2O and indole-3-glycerol phosphate + L-serine leads to L-tryptophan + glyceraldehyde 3-phosphate, quantitatively lesser amounts of activation followed by total inactivation are observed over a similar range of 5-azidoindole concentrations.