Most mesophilic bacteria have a multimeric tryptophan synthase complex (EC 4.2.1.20) that forms L-tryptophan from L-serine and 1-C-(indol-3-yl)glycerol 3-phosphate via an indole intermediate. This intermediate, which is formed by the alpha subunits, is transferred in an internal tunnel to the beta units, which convert it to tryptophan. In thermophilic organisms the high temperature enhances diffusion and causes the loss of indole. This enzyme, which does not combine with the alpha unit to form a complex, salvages the lost indole back to L-tryptophan. It has a much lower Km for indole than the beta subunit of EC 4.2.1.20.
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The enzyme appears in viruses and cellular organisms
Most mesophilic bacteria have a multimeric tryptophan synthase complex (EC 4.2.1.20) that forms L-tryptophan from L-serine and 1-C-(indol-3-yl)glycerol 3-phosphate via an indole intermediate. This intermediate, which is formed by the alpha subunits, is transferred in an internal tunnel to the beta units, which convert it to tryptophan. In thermophilic organisms the high temperature enhances diffusion and causes the loss of indole. This enzyme, which does not combine with the alpha unit to form a complex, salvages the lost indole back to L-tryptophan. It has a much lower Km for indole than the beta subunit of EC 4.2.1.20.
the enzyme is involved in L-tryptophan biosynthesis. TrpB2 does not interact with TrpA as in the case of TrpB1. TrpB2 provides an alternate route to generate Trp from serine and free indole (indole salvage)
only 17% of the indole that is released from the alpha-subunit goes on to form beta-MeTrp, demonstrating that the release of indole is decoupled from product formation. Thr binds non-covalently to the isolated beta-subunit, indicating that the beta-methyl group hinders entry into the catalytic cycle. The beta-methyl of Thr causes a steric clash that destabilizes the E(Aex1) intermediate
enzymatic production of psilocybin formation from 4-hydroxyindole and L-serine and of 7-phosphoryloxytryptamine (isonorbaeocystin), a non-natural congener of the Psilocybe alkaloid norbaeocystin (4-phosphoryloxytryptamine), and of serotonin (5-hydroxytryptamine) by means of the same in vitro approach, overview
the rate of Thr deamination by PfTrpS is 8.5fold faster than with Ser, competitive with the rate of beta-substitution. Substrate differentiation mechanism of the enzyme, molecular dynamics simulations analysis, overview
beta-substitution occurs in vitro with a 3.4fold higher catalytic efficiency for Ser over Thr using saturating indole, despite over 82000fold preference for Ser in direct competition using IGP. When the reaction is conducted with a 1000fold molar excess of Thr over Ser, only Trp is observed, with no trace of beta-MeTrp. Atypical mechanism of specificity: Thr binds efficiently but decreases the affinity for indole and disrupts the allosteric signaling that regulates the catalytic cycle
activity and isolated yields of L-beta-methyl-Trp are low compared to the wild-type reaction with l-Ser, which gives over 90% of L-Trp under the same conditions. Preperative scale synthesis (2S,3S)-L-beta-methyl tryptophan and derivatives
the rate of Thr deamination by PfTrpS is 8.5fold faster than with Ser, competitive with the rate of beta-substitution. Substrate differentiation mechanism of the enzyme, molecular dynamics simulations analysis, overview
the enzyme is involved in L-tryptophan biosynthesis. TrpB2 does not interact with TrpA as in the case of TrpB1. TrpB2 provides an alternate route to generate Trp from serine and free indole (indole salvage)
only 17% of the indole that is released from the alpha-subunit goes on to form beta-MeTrp, demonstrating that the release of indole is decoupled from product formation. Thr binds non-covalently to the isolated beta-subunit, indicating that the beta-methyl group hinders entry into the catalytic cycle. The beta-methyl of Thr causes a steric clash that destabilizes the E(Aex1) intermediate
the double-deletion mutant (DELTAtrpB1DELTAtrpB2) displays Trp auxotrophy, whereas individual single mutants (DELTAtrpB1 and DELTAtrpB2 strains) does not
double-deletion mutant (DELTAtrpB1DELTAtrpB2) displays Trp auxotrophy, whereas individual single mutants (DELTAtrpB1 and DELTAtrpB2 strains) do not. To examine the capacity of TrpB1 and TrpB2 in Trp synthesis via indole salvage, DtrpEB1 and DtrpEB2 mutant strains are constructed using strain KUW1 (DELTApyrFDtrpE) as a host, eliminating the route for endogenous indole synthesis. Indole complements the Trp auxotrophies of DELTAtrpEB1 (DELTApyrFDELTAtrpEDELTAtrpB1) and DELTAtrpEB2 (DELTApyrFDELTAtrpEDELTAtrpB2) to similar levels. The results indicate that TrpB1 and TrpB2 both contribute to Trp biosynthesis in Thermococcus kodakarensis and can utilize free indole, and that indolesalvage does not necessarily rely on TrpB2 to a greater extent
the last two steps of L-tryptophan (Trp) biosynthesis are catalyzed by Trp synthase, a heterotetramer composed of TrpA and TrpB. TrpB catalyzes the condensation of indole, synthesized by TrpA, and serine to Trp. TrpB2 catalyzes the TrpB reaction but does not interact with TrpA as in the case of TrpB1. TrpB1 and TrpB2 both contribute to Trp biosynthesis in Thermococcus kodakarensis and can utilize free indole, and indole salvage does not necessarily rely on TrpB2 to a greater extent
TrpB2 acts as an indole rescue protein, which prevents the escape of this costly hydrophobic metabolite from the cell at the high growth temperatures of hyperthermophiles
the enzyme is part of the tryptophan synthase complex. Indole formation is catalyzed by the alpha-subunit (TrpBalpha), L-tryptophan production is catalyzed by the beta-subunit (TrpBbeta)
tryptophan synthase (TrpS) catalyzes the final steps in the biosynthesis of L-tryptophan from L-serine (Ser) and indole-3-glycerol phosphate (IGP). Native TrpS can also catalyze a productive reaction with L-threonine (Thr), leading to (2S,3S)-beta-methyltryptophan
site-directed mutagenesis of the beta-subunit, the mutant shows altered substrate specificity compared to the wild-type enzyme, single-step enzymatic synthesis of beta-methyl-Trp derivatives, overview. Although 2-, 4-, 6- or 7-substituted indoles are accepted by StTrpS beta-L66V, along with L-Thr, 5-subsituted indoles prove to be very poor substrates for the enzyme. Variant beta-L166V can better accommodate L-Thr as a substrate
site-directed mutagenesis of the beta-subunit, the mutant shows altered substrate specificity compared to the wild-type enzyme, single-step enzymatic synthesis of beta-methyl-Trp derivatives, overview. Although 2-, 4-, 6- or 7-substituted indoles are accepted by StTrpS beta-L66V, along with L-Thr, 5-subsituted indoles prove to be very poor substrates for the enzyme. Variant beta-L166V can better accommodate L-Thr as a substrate
biocatalytic production of psilocybin and derivatives in tryptophan synthase-enhanced reactions, in vitro reconstituted indole alkaloid synthesis pathway including enzyme PsiD, PsiK and PsiM, and ATP and S-adenosyl-L-methionine, method, overview. Assays run only with TrpB and PsiD result in identical chromatograms
Pyrococcus furiosus enzyme engineering by directed evolution of selected TrpB mutant PfTrpB4D11 results in an modified improved enzyme, L-threonine may substitute for L-serine in the beta-substitution reaction of the engineered subunit of tryptophan synthase yielding (2S,3S)-beta-methyltryptophan (beta-MeTrp) in a single step. The trace activity of the wild-type beta-subunit on this substrate is enhanced more than 1000fold by directed evolution. Structural and spectroscopic data indicate that this increase is correlated with stabilization of the electrophilic aminoacrylate intermediate. The engineered biocatalyst also reacts with a variety of indole analogues and thiophenol for diastereoselective C-C, C-N, and C-S bond forming reactions. The new activity circumvents the 3-enzyme pathway that produces beta-MeTrp in nature and offers a simple and expandable route to preparing derivatives of this valuable building block
usage of directed evolution to engineer TrpB from Pyrococcus furiosus (PfTrpB) to retain activity in the absence of its TrpA partner. Further engineering of this stand-alone enzyme achieves tha catalysis the of efficient beta-substitution of L-threonine (Thr), yielding (2S,3S)-beta-methyltryptophan (beta-MeTrp) in a single step
an engineered variant of tryptophan synthase from Salmonella enterica (StTrpS) can catalyse the efficient condensation of L-threonine and various indoles to generate bmTrp and derivatives in a single step. Although L-serine is the natural substrate for TrpS, targeted mutagenesis of the StTrpS active site provides a variant (bL166V) that can better accommodate L-Thr as a substrate. The condensation of L-Thr and indole proceeds with retention of configuration at both alpha- and beta-positions to give (2S,3S)-beta-methyl-Trp
an engineered variant of tryptophan synthase from Salmonella enterica (StTrpS) can catalyse the efficient condensation of L-threonine and various indoles to generate bmTrp and derivatives in a single step. Although L-serine is the natural substrate for TrpS, targeted mutagenesis of the StTrpS active site provides a variant (bL166V) that can better accommodate L-Thr as a substrate. The condensation of L-Thr and indole proceeds with retention of configuration at both alpha- and beta-positions to give (2S,3S)-beta-methyl-Trp
recombinant C-terminally His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21-A1(DE3) by nickel affinity chromatography, ultrafiltration, and desalting gel filtration
gene trpB1, phylogenetic tree, expression in Escherichia coli strain BL21, transcript levels of trpB1 in the host, DELTAtrpB1 strains by quantitative RT-PCR expression analysis
gene trpB2, phylogenetic tree, expression in Escherichia coli strain BL21, transcript levels of trpB2 in the host, DELTAtrpB2 strains by quantitative RT-PCR expression analysis
the enzyme participates in psilocybin formation from 4-hydroxyindole and L-serine, which are less cost-intensive substrates, compared to the previous method. The pharmaceutical interest in this psychotropic natural product as a future medication to treat depression and anxiety is strongly reemerging. Enzymatic production of 7-phosphoryloxytryptamine (isonorbaeocystin), a non-natural congener of the Psilocybe alkaloid norbaeocystin (4-phosphoryloxytryptamine), and of serotonin (5-hydroxytryptamine) by means of the same in vitro approach
A novel tryptophan synthase beta-subunit from the hyperthermophile Thermotoga maritima. Quaternary structure, steady-state kinetics, and putative physiological role
The tryptophan synthase beta-subunit paralogs TrpB1 and TrpB2 in Thermococcus kodakarensis are both involved in tryptophan biosynthesis and indole salvage
The tryptophan synthase beta-subunit paralogs TrpB1 and TrpB2 in Thermococcus kodakarensis are both involved in tryptophan biosynthesis and indole salvage