Tryptophan Synthase Activity Research Articles

Enzymatic Production of L-tryptophan by a Thermophilic Strain of Bacillus licheniformis Isolated from a Local Hot Spring of Paniphala, Asansol Area of West Bengal

A thermophilic bacterial strain having the ability to produce L-tryptophan enzymatically was isolated and identified from a less explored hot spring of West Bengal. The isolate was identified using polyphasic taxonomic approach as a strain of Bacillus licheniformis. Initially, the 16S rRNA gene and later the whole genome of the isolate was sequenced and submitted to the NCBI Gene Bank for future reference. The isolate showed considerable tryptophan synthase activity and may be a potential candidate for mass production of L-tryptophan by enzymatic means.

Directed Evolution Improves the Enzymatic Synthesis of L-5-Hydroxytryptophan by an Engineered Tryptophan Synthase.

L-5-Hydroxytryptophan is an important amino acid that is widely used in food and medicine. In this study, L-5-hydroxytryptophan was synthesized by a modified tryptophan synthase. A direct evolution strategy was applied to engineer tryptophan synthase from Escherichia coli to improve the efficiency of L-5-hydroxytryptophan synthesis. Tryptophan synthase was modified by error-prone PCR. A high-activity mutant enzyme (V231A/K382G) was obtained by a high-throughput screening method. The activity of mutant enzyme (V231A/K382G) is 3.79 times higher than that of its parent, and kcat/Km of the mutant enzyme (V231A/K382G) is 4.36 mM-1∙s-1. The mutant enzyme (V231A/K382G) reaction conditions for the production of L-5-hydroxytryptophan were 100 mmol/L L-serine at pH 8.5 and 35°C for 15 h, reaching a yield of L-5-hydroxytryptophan of 86.7%. Directed evolution is an effective strategy to increase the activity of tryptophan synthase.

Changes in tryptophan synthase activity in the wild type and auxin-sensitive mutants of the moss Funaria hygrometrica Hedw.,accompanying protonema differentation

Changes in tryptophan synthase activity in the wild type and auxin-sensitive mutants of the moss Funaria hygrometrica Hedw.,accompanying protonema differentation

Engineering Improves Enzymatic Synthesis of L-Tryptophan by Tryptophan Synthase from Escherichia coli.

To improve the thermostability of tryptophan synthase, the molecular modification of tryptophan synthase was carried out by rational molecular engineering. First, B-FITTER software was used to analyze the temperature factor (B-factor) of each amino acid residue in the crystal structure of tryptophan synthase. A key amino acid residue, G395, which adversely affected the thermal stability of the enzyme, was identified, and then, a mutant library was constructed by site-specific saturation mutation. A mutant (G395S) enzyme with significantly improved thermal stability was screened from the saturated mutant library. Error-prone PCR was used to conduct a directed evolution of the mutant enzyme (G395S). Compared with the parent, the mutant enzyme (G395S /A191T) had a Km of 0.21 mM and a catalytic efficiency kcat/Km of 5.38 mM−1∙s−1, which was 4.8 times higher than that of the wild-type strain. The conditions for L-tryptophan synthesis by the mutated enzyme were a L-serine concentration of 50 mmol/L, a reaction temperature of 40 °C, pH of 8, a reaction time of 12 h, and an L-tryptophan yield of 81%. The thermal stability of the enzyme can be improved by using an appropriate rational design strategy to modify the correct site. The catalytic activity of tryptophan synthase was increased by directed evolution.

A surface‐exposed, network substitution results in a gain‐of‐function in tryptophan synthase6.5.0

Tryptophan synthase (TS) is a α2β2 dimeric enzyme that catalyzes the final two reactions in the production of tryptophan. The product of the alpha subunit (αTS), indole, is channeled to the beta subunit (βTS) where it is then condensed with serine to form tryptophan. TS is a valuable model system for understanding enzyme allostery, and specifically enzyme‐enzyme communication. We previously identified amino acid interaction networks in αTS using NMR‐based methods. We proposed that these network residues play an important role in catalysis and communication between the two subunits. Based on previous studies, αTS residue Ala198 was identified as a residue of interest. Ala198 is a surface‐exposed, network residue that is dynamic on the ms‐μs timescale, according to our previous NMR studies. To better understand the networks, we characterized the A198W variant by performing cell‐based assays, kinetic assays, molecular dynamics (MD) simulations, and NMR experiments. We found that E.coli cells expressing the A198W variant had a faster growth rate in minimal medium compared to cells expressing the wild‐type αTS. Our kinetic studies on purified TS indicated that the A198W variant had little effect on αTS activity, but increased the activity of the TS complex (i.e. from initial indole‐3‐glycerol phosphate substrate to final tryptophan product), perhaps due to an enhancement of indole channeling. MD simulations indicated that the A198W substitution led to structural dynamic changes that propagated throughout αTS, including several key residues at the α/β binding interface. The MD simulations were consistent with the indole channel being more open, more often, which could help explain the increase in TS activity in vitro and in vivo. NMR experiments also indicated that the A198W substitution induced structural dynamic changes throughout αTS, consistent with the MD simulations. Despite Ala198 being distal from both the active site of αTS (27Å) and the α/β binding interface (30Å), substitutions at this position result in changes to catalytic efficiency and the TS complex dynamics. These findings underscore the importance of network residues on enzyme dynamics, allostery, and subunit communication. Engineering of surface‐exposed network residues represents one way of controlling enzyme activity.6.5.0Support or Funding Information6.5.06.5.0

Enzymatic Synthesis of L-2-Methyltryptophan Catalyzed by Recombinant Escherichia Coli with Tryptophan Synthase Activity

Enzymatic Synthesis of L-2-Methyltryptophan Catalyzed by Recombinant Escherichia Coli with Tryptophan Synthase Activity

Optimization of L-Tryptophan Biosynthesis From L-Serine of Processed Iranian Beet and Cane Molasses and Indole by Induced Escherichia coli ATCC 11303 Cells.

Background:L-tryptophan is an important ingredient in medicines, especially in neuromedicines such as antidepressants. Many commercial processes employ various microorganisms with high tryptophan synthase activity to produce L-tryptophan from indole and L-serine, but these processes are very costly due to the costs of precursors, especially L-serine.Objectives:For this reason, we studied the ability to use processed Iranian cane and beet molasses as L-serine sources for L-tryptophan production, which enables us to reach a cost-effective process.Materials and Methods:Whole cells of Escherichia coli ATCC 11303 were induced for L-tryptophan synthase by addition of indole to the growth medium and bacterial cells harvested from the growth medium were used as biocatalysts in the production medium. Conditions of the production medium were optimized and Iranian cane and beet molasses were processed by solvent extraction with ethanol and n-butanol and used as L-serine sources of the production medium. Amount of L-tryptophan and theoretical yield of L-tryptophan production were determined by High Performance Liquid Chromatography and by a colorimetrical method on the basis of the remaining indole assay, respectively.Results:L-tryptophan production increased by 15 folds, when indole was used as an inducer. L-tryptophan was produced from processed Iranian beet molasses in satisfactory amounts (0.53 mM) and no exogenous pyridoxal phosphate was required as a cofactor under our experimental conditions.Conclusions:The obtained results proved that Iranian beet molasses include significant amounts of L-serine that makes them a suitable substitution for L-serine. Findings of the present study give impetus to use of Iranian beet molasses for cost-effective L-Trp production in the amino acid industry.

Effects of Polyhydroxy Compounds on Enzymatic Synthesis of L-Tryptophan Catalyzed by Tryptophan Synthase

The effects of polyethylene glycol (PEG) of different molecular (1000, 2000), glycerol, ethylene glycol on the catalytic activity of tryptophan synthase were studied. The results indicated that the addition of PEG 2000 increased the enzymatic activity of tryptophan synthase. The enzymatic activity of tryptophan synthase was enhanced 25.2% by 10 g L−1 PEG 2000. Reaction conditions were optimized by using 10 g L−1 PEG 2000 at pH 9 and 40 °C. L-Serine conversion rate reach 89.9% under the optimal conditions. The kinetic parameters indicated the specificity of TSase to substrate was improved. The mechanism of reaction catalyzed by tryptophan synthase

High cell density cultivation of recombinant E. coli with a pressurized culture

An air-pressurized culture was used to enhance the oxygen transfer rate in the high cell density cultivation (HCDC) of non-recombinant and recombinant (harbors pBR322trpAB) Escherichia coli K-12. Carbon dioxide generated by the cells under increased pressure was inhibitory and as a result, cellular growth stopped in the air-pressurized culture at a constant mass flow rate. Increasing the flow rate along with the pressure in the reactor enabled the cells to grow to 130 (non-recombinant) and 104 (recombinant) g/l due to the release of the CO 2. In addition, the specific activity of tryptophan synthase was increased. This study showed that an air-pressurized culture is able to meet the high demand for oxygen in the HCDC of E. coli.

Identification of the Geometric Requirements for Allosteric Communication between the α- and β-Subunits of Tryptophan Synthase

The pyridoxal 5'-phosphate-dependent tryptophan synthase alpha2beta2 complex is a paradigmatic protein for substrate channeling and allosteric regulation. The enzymatic activity is modulated by a ligand-mediated equilibrium between open (inactive) and closed (active) conformations of the alpha- and beta-subunit, predominantly involving the mobile alpha loop 6 and the beta-COMM domain that contains beta helix 6. The alpha ligand-triggered intersubunit communication seems to rely on a single hydrogen bond formed between the carbonyl oxygen of betaSer-178 of beta helix 6 and the NH group of alphaGly-181 of alpha loop 6. We investigated whether and to what extent mutations of alphaGly-181 and betaSer-178 affect allosteric regulation by the replacement of betaSer-178 with Pro or Ala and of alphaGly-181 with either Pro to remove the amidic proton that forms the hydrogen bond or Ala, Val, and Phe to analyze the dependence on steric hindrance of the open-closed conformational transition. The alpha and beta activity assays and the equilibrium distribution of beta-subunit catalytic intermediates indicate that mutations do not significantly influence the intersubunit catalytic activation but completely abolish ligand-induced alpha-to beta-subunit signaling, demonstrating distinct pathways for alpha-beta-site communication. Limited proteolysis experiments indicate that the removal of the interaction between betaSer-178 and alphaGly-181 strongly favors the more trypsin-accessible open conformation of the alpha-active site. When the hydrogen bond cannot be formed, the alpha-subunit is unable to attain the closed conformation, and consequently, the allosteric signal is aborted at the subunit interface.

Isolation of a cDNA for tryptophan synthase beta from rice and studies of its expression in a 5-methyltryptophan-resistant mutant of rice.

A cDNA clone encoding rice tryptophan synthase beta (TSB) was isolated and its transcript level was examined in TR-No. 73, a derivative of a previously isolated rice mutant (TR-1) that is resistant to 5-methyltryptophan. The cDNA sequence of the rice gene for TSB was very similar to that of genes for TSB from other plants. Northern blotting analysis revealed that the steady-state level of TSB mRNA in the 5MT-resistant mutant TR-No. 73 was 1.3 times higher than the level of TSB mRNA in control rice plants under standard conditions. The level of TSB mRNA in control rice plants decreased after treatment of plants with 5MT. Similarly, the level of TSB mRNA in TR-No. 73 initially decreased, although less so than in control rice. However, after 12 h of treatment with 5MT, the level of the transcript in TR-No. 73 returned to the level under standard conditions. The activity of tryptophan synthase (TS) in seedlings of TR-No. 73 was about 2.3 times higher than that in seedlings of control rice under standard conditions.

Characterization of Anthranilate Synthetase and Tryptophan Synthase in a 5-Methyltryptophan Resistant Mutant (MR 1) of Zea mays L.

Anthranilate synthetase (AS) and tryptophan synthase (TS) enzymes were analyzed for characterization of 5-methyltryptophan (5MT) resistant mutant plants (MR 1) in maize. There were no significant differences in the level of AS activity between MR I and control seedlings when grown on MS medium without 5MT. However, the level of AS was 2 times higher in MR I seedlings grown on MS medium with 25 ppm 5MT than in control seedlings. The activity of AS from control seedlings was 50 o/o inhibited by 5 pprn tryptophan added to extract, while the enzyme from MR I seedlings was 50 o/o inhibited by 4 times higher concentrations (20 ppm) of tryptophan. The level of TS activity in MR I was 4 times higher than that in control seedlings when cultured on MS medium. For a more detailed study on TS, the gene encoding the tryptophan synthase B subunit (TSB) was isolated from maize and sequenced. The gene showed high homology to TSB isolated from other organisms. Northern hybridization analysis indicated that the gene expressed in MR I seedlings grown on MS medium at a higher level than in control seedlings. From the results, it was found that MR I plants showed differeces in the character of AS, as reported previously in cell lines resistant to 5MT, and also notable diversity regarding TS activity when compared to control plants.

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.) G. Don immobilized by adhesion to glass fibres.

Suspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In-vitro assays for the specific activity of tryptophan decarboxylase (TDC) and tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension-cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension-cultured cells only after the addition of exogenous secologanin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of indole alkaloid biosynthetic enzymes in our system beyond, and including, strictosidine synthase.

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene in Escherichia coli.

The application of an inducible regulation system using the tryptophanase operon promoter (TPase promoter; Ptna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of Ptna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, L-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-Ptna TS was increased 500-fold compared to that of the cultured host strain. About 1 mol L-tryptophan/l reaction mixture was formed from indole and L-serine at 37 degrees C for 3.5 h.

Improved method for measuring the catalytic activity of tryptophan synthase α subunit in cell extracts

An improved method has been developed for measuring the catalytic activity of tryptophan synthase α-subunit in cell extracts using the indole-3-glycerol phosphate (InGP) → tryptophan reaction. The method involves the chemical and enzymatic synthesis of the substrate InGP immediately before use and avoids the preparation of salt-free hydroxylamine. The method is more convenient, safer and more reliable than the traditional method employing the InGP → indole reaction.

Production of S-(carboxymethyl)- l-cysteine from l-serine with tryptophan synthase

Synthesis of S-carboxymethyl)- l-cysteine ( l-CMC) from L-serine with tryptophan synthase was studied. In the β-replacement reaction l-serine catalyzed by tryptophan synthase of Escherichia coli, thioglycolic acid was a poor substrate but alkylthioglycolates were good substrates. S-(2-Ethylhexyloxycarbonylmethyl)- l-cysteine was synthesized from l-serine and 2-ethylhexylthioglycolate using intact cells of recombinant E. coli with high tryptophan synthase activity, and subsequently hydrolyzed to l-CMC with alkali in one reactor. Under optimum conditions established for l-CMC synthesis, 159 g. l −1 of l-CMC was formed at a molar yield of 94% based on when both l-serine and 2-ethylhexylthioglycolate were added. Synthesized l-CMC was confirmed to be l-form after isolation and identification of the product.

Conformational effects of ligand binding on the beta 2 subunit of Escherichia coli tryptophan synthase analyzed with monoclonal antibodies.

Five monoclonal antibodies recognizing five different epitopes of the native beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.1.2.20) were used to analyze the conformational changes occurring upon ligand binding or chemical modifications of the enzyme. For this purpose, the affinities of each antibody for the different forms of the enzyme were determined by using an enzyme-linked immunosorbent assay which allows measurement of the dissociation constant of antigen-antibody equilibrium in solution. The fixation of the coenzyme pyridoxal 5'-phosphate and the substrate L-serine modifies the affinity constants of most of the antibodies for the enzyme, thus showing the existence of extended conformational rearrangements of the protein. The association of the alpha subunit with the beta 2 subunit, which brings about an increase of the tryptophan synthase activity and abolishes the serine deaminase activity of beta 2, is accompanied by an important conformational change of the N-terminal domain of beta 2 (F1) since none of the anti-F1 monoclonal antibodies can bind to alpha 2 beta 2. Similarly, chemical modifications of beta 2 which are known to produce significant effects on the enzymatic activities of beta 2 result in changes of the affinities of the monoclonal antibodies which can be interpreted as the acquisition of different conformational states of the enzyme.

Factors affecting the biosynthesis of L-tryptophan by genetically modified strains of Escherichia coli.

Derivatives of Escherichia coli strain W3110 with increased tryptophan synthase (TS) activity were constructed. The biosynthesis of serine was shown to limit tryptophan production in minimal medium with indole as precursor. In the presence of serine and indole we obtained a correlation between the specific activity of TS and the specific productivity (qp) of tryptophan. Supplementation of the growth medium with glycine enhanced qp two-fold. In a strain with high serine hydroxymethyltransferase (SHMT) activity no such increase of tryptophan productivity was observed, although crude extracts from these cells were shown to produce tryptophan with indole, one-carbon units and glycine as precursors. Growth of the strain with high SHMT activity was inhibited in a medium with high glycine concentration. This inhibition could not be released by addition of isoleucine and valine. In a buffer system with permeabilized cells high in specific TS and SHMT activities we did not obtain any tryptophan production in presence of indole, glycine, one-carbon units and cofactors. On the other hand, in a buffer system with indole and serine as precursors we obtained high qp of tryptophan [13.3 g tryptophan (g dry wt cells)-1 h-1], which was correlated to the TS specific activity.

Entrapment of l-tryptophan producing Escherichia coli in different matrices: activity of immobilized cells

Cells of Escherichia coli induced for l-tryptophan synthase [ l-serine hydro-lyase (adding indole-glycerol-phosphate), EC 4.2.1.20] have been assayed in DMF and DMSO aqueous solvents as reaction medium. Up to 20% DMF/water, cells retained 90% of their tryptophan synthase activity. Concentrations of 20 mM indole, which did not inhibit this reactivity, could be reached with 5% DMF/water. Four matrices were compared for cell immobilization: polyacrylamide, foam particles of bovine seum albumin, alginate and κ-carrageenan. The best activity was retained with the latter matrix, and the preparations thus obtained allowed high productivity of l-tryptophan. Various systems of production of l-tryptophan with κ-carrageenan and DMF/water were studied .

Structural and functional influence of enzyme-antibody interactions: effects of eight different monoclonal antibodies on the enzymatic activity of Escherichia coli tryptophan synthase.

Twelve monoclonal antibodies directed against the beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.2.1.20) were produced from hybridoma clones. These monoclonal antibodies are found to recognize at least eight different epitopes on beta 2, and eight classes of monoclonal antibodies are thus defined. The effects of these monoclonal antibodies on the enzymatic activities of beta 2 are studied. The monoclonal antibodies from three classes rapidly inhibit the serine deaminase activity catalyzed by the beta 2 subunit alone; two of them lead to an inhibition plateau under stoichiometric conditions, and their inhibitory effects are cumulative. With the antibodies from two of these three classes, the tryptophan synthase activity of the alpha 2 beta 2 complex is recovered, through a competition between the alpha subunit and the monoclonal antibody. On the contrary, the antibody from the third class is inhibitory even in the presence of an excess of alpha subunit. The antibodies from the five other classes, though binding easily to the coated antigen in the enzyme-linked immunosorbent assay, react only very slowly with beta 2 in solution and, only after a long time of incubation, inhibit the enzymatic activity at different levels.