Tryptophan Synthase Α-subunit Research Articles

Structures of wild-type and P28L/Y173F tryptophan synthase α-subunits from Escherichia coli

The α-subunit of tryptophan synthase (αTS) catalyzes the cleavage of indole-3-glycerol phosphate to glyceraldehyde-3-phosphate and indole, which is used to yield the amino acid tryptophan in tryptophan biosynthesis. Here, we report the first crystal structures of wild-type and double-mutant P28L/Y173F α-subunit of tryptophan synthase from Escherichia coli at 2.8 and 1.8 Å resolution, respectively. The structure of wild-type αTS from E. coli was similar to that of the α 2β 2 complex structure from Salmonella typhimurium. As compared with both structures, the conformational changes are mostly in the interface of α- and β-subunits, and the substrate binding region. Two sulfate ions and two glycerol molecules per asymmetric unit bind with the residues in the active sites of the wild-type structure. Contrarily, double-mutant P28L/Y173F structure is highly closed at the window for the substrate binding by the conformational changes. The P28L substitution induces the exposure of hydrophobic amino acids and decreases the secondary structure that causes the aggregation. The Y173F suppresses to transfer a signal from the α-subunit core to the α-subunit surface involved in interactions with the β-subunit and increases structural stability.

Fluorescence and folding properties of Tyr mutant tryptophan synthase α-subunits from Escherichia coli

The fluorescence of tyrosine has been used to monitor a folding process of tryptophan synthase α-subunit from Escherichia coli, because this protein has 7 tyrosines, but not tryptophan. Here to assess the contribution of each Tyr to fluorescence properties of this protein during folding, mutant proteins in which Tyr was replaced with Phe were analyzed. The result shows that a change of Tyr fluorescence occurring during folding of this protein is contributed to ∼40% each by Tyr 4 and Tyr 115, and to the remaining ∼20% by Tyr 173 and Tyr 175. Y173F and Y175F mutant proteins showed an increase in their fluorescence intensity by ∼40% and ∼10%, respectively. These increases appear to be due to multiple effects of increased hydrophobicity, quenching effect of nearby residue Glu 49, and/or energy transfer between Tyrs. Two data for Y173F α-subunit of urea-induced unfolding equilibrium monitored by UV and fluorescence were different. This result, together with ANS binding and far UV CD, shows that folding intermediate(s) of Y173F α-subunit, contrary to that of wild-type, may contain self-inconsistent properties such as more buried hydrophobicity, highly quenched fluorescence, and different dependencies on urea of UV absorbance, suggesting an ensemble of heterogeneous structures.

Homodimers of Mutant Tryptophan Synthase α-Subunits in Escherichia coli

Tryptophan synthase α-subunit from Escherichia coli functionally exists as a heterotetramer of α2β2 with β-subunit. While wild-type and mutant (F139W, T24M/F139W, and T24L/F139W) α-subunits were expressed as a monomer from recombinant plasmids in Escherichia coli, T24A/F139W, T24S/F139W, and T24K/F139W mutant α-subunits were abnormally expressed as soluble homodimers in addition to monomers. Monomers of dimer-forming mutant α-subunits retain high affinity to β-subunit, high activity in stimulating catalytic activities of β-subunit, and nearly intact content of secondary structure, indicating that the global structures of these monomers are identical to that of F139W α-subunit. However, fluorescence spectra of Trp139 and ANS binding indicate that significant perturbations occur in the mutant proteins. Interestingly, these defective properties of monomers caused by residue replacement were partially repaired by the dimer formation. As a result, it is suggested that dimers may be formed by domain or loop swapping, and that residue 24 may play important role in maintaining on-pathway of α-subunit folding.

Genetic differences in the Chlamydia trachomatis tryptophan synthase α-subunit can explain variations in serovar pathogenesis

The human pathogen Chlamydia trachomatis is an obligate intracellular bacterium, characterized by a developmental cycle that alternates between the infectious, extracellular elementary bodies and intracellular, metabolically active reticulate bodies. The cellular immune effector interferon gamma (IFN-γ) inhibits chlamydial multiplication in human epithelial cells by induction of the tryptophan degrading enzyme indoleamine 2,3 dioxygenase. IFN-γ causes persistent C. trachomatis serovar A infections with atypical reticulate bodies that are unable to redifferentiate into elementary bodies and show diminished expression of important immunogens, but not of GroEL. However, the sensitivity to IFN-γ varies among serovars of C. trachomatis. In our previous study significant IFN-γ-specific, but tryptophan reversible, induction of proteins in C. trachomatis A and L2 with molecular masses of approximately 30 and 40 kDa was observed on 2D-gels. The 30-kDa protein from C. trachomatis L2 migrated with a significantly lower molecular weight in C. trachomatis A. In this paper we include C. trachomatis B, C and D in our investigations and identify the proteins as alpha- and beta-subunits of the chlamydial tryptophan synthase using matrix-assisted laser desorption/ionization mass spectrometry. DNA sequencing of the trpA genes from C. trachomatis A and C shows that the TrpA in these serovars is a 7.7-kDa truncated version of C. trachomatis D and L2 TrpA. The truncation probably impairs the TrpA activity, thus elucidating a possible molecular mechanism behind variations in the pathogenesis of C. trachomatis serovars.

Time-resolved fluorescence anisotropy study of the refolding reaction of the alpha-subunit of tryptophan synthase reveals nonmonotonic behavior of the rotational correlation time.

Time-resolved fluorescence anisotropy of a bound extrinsic probe was studied in an effort to characterize dynamic properties of the transient partially folded forms that appear during the folding of the alpha-subunit of tryptophan synthase (alphaTS) from Escherichia coli. Previous studies have shown that alphaTS, a single structural domain, can be cleaved into autonomously folding amino- and carboxy-folding units comprising residues 1-188 and 189-268, respectively [Higgins, W., Fairwell, T., and Miles, E. W. (1979) Biochemistry 18, 4827-4835]. By use of a double-kinetic approach [Jones, B. E., Beechem, J. M., and Matthews, C. R. (1995) Biochemistry 34, 1867-1877], the rotational correlation time of 1-anilino-8-naphthalene sulfonate bound to nonpolar surfaces of folding intermediates was measured by time-correlated single photon counting at varying time delays following initiation of folding from the urea-denatured form by stopped-flow techniques. Comparison of the rotational correlation times for the full-length alphaTS and the amino-terminal fragment suggests that folding of the amino-terminal fragment and carboxy-terminal fragment is coordinated, not autonomous, on the milliseconds to seconds time scale. If a spherical shape is assumed, the apparent hydrodynamic radius of alphaTS after 5 ms is 26.8 A. The radius increases to 28.5 A by 1 s before decreasing to the radius for native alphaTS, 24.7 A, on a longer time scale (>25 s). Viewed within the context of the kinetic folding model of alphaTS [Bilsel, O., Zitzewitz, J. A., Bowers, K. E. , and Matthews, C. R. (1999) Biochemistry 38, 1018-1029], the initial collapse reflects the formation of an off-pathway burst-phase intermediate in which at least part of the carboxy folding unit interacts with the amino folding unit. The subsequent increase in rotational correlation time corresponds to the formation of an on-pathway intermediate that leads to the native conformation. The apparent increase in the radius for the on-pathway intermediate may reflect a change in the interaction of the two-folding units, thereby forming a direct precursor for the alpha/beta barrel structure.

Folding mechanism of the alpha-subunit of tryptophan synthase, an alpha/beta barrel protein: global analysis highlights the interconversion of multiple native, intermediate, and unfolded forms through parallel channels.

A variety of techniques have been used to investigate the urea-induced kinetic folding mechanism of the alpha-subunit of tryptophan synthase from Escherichia coli. A distinctive property of this 29 kDa alpha/beta barrel protein is the presence of two stable equilibrium intermediates, populated at approximately 3 and 5 M urea. The refolding process displays multiple kinetic phases whose lifetimes span the submillisecond to greater than 100 s time scale; unfolding studies yield two relaxation times on the order of 10-100 s. In an effort to understand the populations and structural properties of both the stable and transient intermediates, stopped-flow, manual-mixing, and equilibrium circular dichroism data were globally fit to various kinetic models. Refolding and unfolding experiments from various initial urea concentrations as well as forward and reverse double-jump experiments were critical for model discrimination. The simplest kinetic model that is consistent with all of the available data involves four slowly interconverting unfolded forms that collapse within 5 ms to a marginally stable intermediate with significant secondary structure. This early intermediate is an off-pathway species that must unfold to populate a set of four on-pathway intermediates that correspond to the 3 M urea equilibrium intermediate. Reequilibrations among these conformers act as rate-limiting steps in folding for a majority of the population. A fraction of the native conformation appears in less than 1 s at 25 degrees C, demonstrating that even large proteins can rapidly traverse a complex energy surface.

Cryo-crystallography of a True Substrate, Indole-3-glycerol Phosphate, Bound to a Mutant (αD60N) Tryptophan Synthase α2β2 Complex Reveals the Correct Orientation of Active Site αGlu49

The reversible cleavage of indole-3-glycerol by the α-subunit of tryptophan synthase has been proposed to be catalyzed by αGlu49 and αAsp60. Although previous x-ray crystallographic structures of the tryptophan synthase α2β2 complex showed an interaction between the carboxylate of αAsp60 and the bound inhibitor indole-3-propanol phosphate, the carboxylate of αGlu49 was too distant to play its proposed role. To clarify the structural and functional roles of αGlu49, we have determined crystal structures of a mutant (αD60N) α2β2 complex in the presence and absence of the true substrate, indole-3-glycerol phosphate. The enzyme in the crystal cleaves indole-3-glycerol phosphate very slowly at room temperature but not under cryo-conditions of 95 K. The structure of the complex with the true substrate obtained by cryo-crystallography reveals that indole-3-glycerol phosphate and indole-3-propanol phosphate have similar binding modes but different torsion angles. Most importantly, the side chain of αGlu49 interacts with 3-hydroxyl group of indole-3-glycerol phosphate as proposed. The movement of the side chain of αGlu49 into an extended conformation upon binding the true substrate provides evidence for an induced fit mechanism. Our results demonstrate how cryo-crystallography and mutagenesis can provide insight into enzyme mechanism.

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.

Scanning calorimetric study of tryptophan synthase α-subunits from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid

Denaturation temperatures (Td) and denaturation enthalpies (ΔH) for α-subunits from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid in which the C-terminal domain comes from E. coli and N-terminal domain comes from S. typhimurium were determined by scanning microcalorimetry (DASM4) at various pH values. The Td of the E. coli protein was the highest at each pH and the Td of the hybrid protein was close to that of S. typhimurium. ΔH values of the three proteins were similar to each other at the same temperature. The results suggest that the difference in the stabilities between E. coli and S. typhimurium proteins is caused by the difference of hydrophobicity with replacements of Val to Ile at positions 52 and 166, which are buried in the interior of the molecule.

Assignment of tyrosine resonances in the 1H‐NMR spectrum of tryptophan synthase α‐subunit

In order to monitor the conformational changes of tryptophan synthase alpha-subunit from Escherichia coli in solution resulting from amino acid substitutions, we have assigned the Tyr resonances in the aromatic region of the 1H-NMR spectrum to specific residues. In the spectrum of the alpha-subunit deuterated with [2,3,4,5,6-2H5]Phe and [3,5-2H2]Tyr, the C2 and C6 protons of Tyr gave completely isolated signals at acidic p2H. Some of the C3 and C5 proton resonances overlapped with each other at acidic p2H. By using a series of mutant alpha-subunits in which each Tyr was singly substituted with His or Phe, we can now assign each of seven Tyr resonances in the aromatic region to a specific residue. We have previously studied the conformational stability of a series of variant alpha-subunits at position 49 [Yutani et al. (1987) Proc. Natl Acad. Sci. USA 84, 4441-4444]. We now compare the 1H-NMR spectra in the aromatic region of the wild-type alpha-subunit and mutant alpha-subunits substituted with Phe or Gly in place of Glu-49. The results suggest that the major conformational effects of substitutions at position 49 are localized close to the position of substitution.

Correlation of surface properties with conformational stabilities of wild-type and six mutant tryptophan synthase alpha-subunits substituted at the same position.

The surface properties of wild-type and six mutant alpha-subunits of tryptophan synthase substituted at the same position, 49, which is buried in the interior, were measured by surface tension, foaming and emulsifying properties to correlate the surface properties with the stabilities. The conformational stabilities of the seven alpha-subunits differed dramatically depending on the characteristics of the substituting residues [Yutani et al. (1987) Proc. Natl. Acad. Sci., 84, 4441-4444]. The mutant proteins substituted by isoleucine and phenylalanine in place of glutamic acid at position 49 were more stable than the other proteins and showed higher surface tension and lower foaming and emulsifying properties than the wild-type and other mutant proteins. Good correlations were observed between these surface properties and values of the Gibbs free energy of unfolding in water, of the proteins. This indicates that the surface properties of the alpha-subunits of tryptophan synthase depend closely on the conformational stabilities.

Comparison of denaturation of tryptophan synthase α-subunits from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid

Guanidine hydrochloride-induced denaturation and thermal denaturation of three kinds of tryptophan synthase α subunit have been compared by circular dichroism measurements. The three α subunits are from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid in which the C-terminal domain comes from E. coli (α-2 domain) and the N-terminal domain comes from S. typhimurium (α-1 domain). Analysis of denaturation by guanidine hydrochloride at 25 °C showed that the α-2 domain of S. typhimurium was more stable than the α-2 domain of E. coli, but the α-1 domain of S. typhimurium was less stable than the α-1 domain of the E. coli protein; overall, the hybrid protein was slightly less stable than the two original proteins. It is concluded that the stability to guanidine hydrochloride denaturation of each of the domains of the interspecies hybrid is similar to the stability of the domain of the species from which it originated. The E. coli protein was more stable to thermal denaturation than the other proteins near the denaturation temperature, but the order of their thermal stability was reversed at 25 °C and coincided with that obtained from guanidine hydrochloride-induced denaturation.

NMR evidence for the stepwise unfolding of the two domains of tryptophan synthase α-subunit

1H-NMR signals of HisC-2 proton were employed as a probe to investigate the unfolding of the α-subunit of tryptophan synthase (EC 4.2.1.20) from Escherichia coli. Two proteolytic fragments of the α-subunit, α-1 (N-terminal 188 residues) and α-2 (C-terminal 80 residues), which correspond to the two domains of the α-subunit and contain two histidine residues, respectively, were prepared. Assignments of four HisC-2 peaks to the domains could be done by comparing the 1H-NMR spectra of the two fragments with that of the intact α-subunit. NMR spectra of the α-subunit were measured at various urea concentrations. The results support the idea that the unfolding of the α-subunit by denaturant proceeds by stepwise unfolding of the two domains.

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

The α subunit of the Escherichia coli tryptophan synthase catalyzes the reversible aldolytic reaction: Indole-3-glycerol phosphate ⇌ 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 α subunit treated at concentrations of 5-azidoindole less than or equal to 2 m m show a progressive 30–40% activation. A gradual inactivation occurs only in samples irradiated at concentrations in excess of 2 m m 5-azidoindole, and this inactivation is complete at 8–10 m m. A quantitatively similar activation occurs in the forward reaction (indole synthesis), however inactivation in this case is incomplete, with complexes treated at 8–12 m m 5-azidoindole retaining 30–40% relative activity in this reaction. When treated α subunits were assayed for their abilities to complement the β 2-subunit in the reactions indole + l-serine → l-tryptophan + H 2O and indole-3-glycerol phosphate + l-serine → l-tryptophan + glyceraldehyde 3-phosphate, quantitatively lesser amounts of activation followed by total inactivation are observed over a similar range of 5-azidoindole concentrations.

Effect of single amino acid substitutions at the same position on stability of a two-domain protein

In order to elucidate the role of individual amino acid residues on the conformational stability of a protein, the stabilities of the wild-type tryptophan synthase α-subunit from Escherichia coil and its five mutant proteins substituted by single amino acid residues at the same position 49 were compared. The five mutant proteins have glutamine, methionine, valine, serine, or tyrosine in place of glutamic acid of the wild-type protein at position 49. Denaturation of these proteins, which consist of two domains, by guanidine hydrochloride can be analyzed as a two-step process. We obtained the equilibrium constants between the native and the denatured forms and between the native and the stable intermediate forms for the above six proteins in the absence of denaturant at three pH values.

PH dependence of stability of the wild-type tryptophan synthase α-subunit and two mutant proteins (Glu49 → Met or Gln)

In order to elucidate the role of individual amino acid residues in the electrostatic interaction affecting the conformational stability of proteins, the pH dependence of stability of tryptophan synthase α-subunit of the wild-type and two mutant proteins, trpA33 (Glu49 → Met) and trpA11 (Glu49 → Gln), has been compared by means of circular dichroism measurements in the absence and presence of guanidine hydrochloride. The denaturation of the three proteins is discussed, assuming the existence of one stable intermediate. In the first denaturation step, i.e. the transition from the native to the intermediate state, the midpoint of the conformational transition in the acid region was pH 3.9 and pH 5.1 for the wild type and the trpA11 proteins, respectively; whereas, in the alkaline region the midpoint was pH 10.9 and pH 11.6 for the wild-type and the trpA11 proteins, respectively. The trpA33 protein belonged to the stronger group in stability in both the acid and alkaline regions. In the acid region in the presence of 0.4 m-guanidine hydrochloride, the trpA11 protein was more labile than the wild-type and trpA33 proteins; whereas, in the alkaline region in 0.8 m-guanidine hydrochloride, the order of stability among the three proteins was the trpA33, trpA11 and wild-type proteins at pH values between 7.5 and 9.8. In the second denaturation step, i.e. the transition from the intermediate to the completely denatured state, the order of stability among the three proteins was the trpA33, wild-type and trpA11 proteins. These results showed that a negatively charged group at position 49 served as a destabilizing factor in the alkaline region, and that the hydrophobicity of the residue at position 49 contributed to the stabilization of the conformation of the protein.

State of Tyr49 in a mutant tryptophan synthase alpha-subunit substituted at position 49.

The states of tyrosine residues in an alpha-subunit of wild-type tryptophan synthase from Escherichia coli and a mutant protein which has tyrosine in place of glutamic acid at position 49, were examined by absorption spectrum, spectrophotometric titration of phenolic hydroxyl ionization, and deuteration kinetics of phenolic hydrogen monitored by fluorescence measurement. The difference absorption spectrum of the mutant protein against the wild-type protein at pH 7.0 and 25 degrees C had peaks at 289, 282, and 276.5 nm. These positions corresponded to those in the absorption spectrum of L-tyrosine derivatives in a non-aqueous solvent at 77 K and these bands were well-resolved even at 25 degrees C as if the tyrosine residue were fixed at lower temperature. The titration curve of the mutant protein at 3 degrees C differed from that of the wild-type protein only above pH 12.7, where the difference molar extinction coefficient at 295 nm reached a plateau, indicating that ionization of Tyr49 took place at an abnormally high pH. These results suggest that Tyr49 is buried in the hydrophobic interior and fixed in a certain orientation. The deuterium exchanges of phenolic hydrogen at pH 7.0 and 13 degrees C in the wild-type and mutant proteins consisted of a single and two first order processes, respectively, all three having smaller rate constants than that of free tyrosine, indicating that these tyrosine residues are buried. It is concluded that Tyr49 in the mutant protein is not on the surface of the molecule.

Amino-terminally formylated tryptophan synthase alpha-subunit produced by the trp operon cloned in a plasmid vector.

The trp operon of Escherichia coli cloned in a multicopy plasmid produced an amino-terminally formylated tryptophan synthase alpha-subunit, as well as the normal deformylated one.

Comparison of Denaturation by Guanidine Hydrochloride of the Wild Type Tryptophan Synthase α-Subunit of Escherichia coli and Two Mutant Proteins (Glu 49—Met or Gln)

In order to elucidate the roles of individual amino acid residues in the conformational stability of proteins, the denaturation by guanidine hydrochloride of the wild-type trytophan synthase alpha-subunit of Escherichia coli and two mutant proteins, trpA33 (Glu 49 leads to Met) and trpA11 (Glu 49 leads to Gln), has been compared by means of CD measurements at pH 7.0 and various temperatures. CD spectra of the two mutant proteins were similar to that of the wild-type protein. The trpA33 and the trpA11 proteins were more and less resistant, respectively, to guanidine hydrochloride than the wild-type protein at 9.7 to 49.6 degrees C. The free energy change of unfolding in water delta delta Gnd H2O, was evaluated assuming a three state denaturation, since the denaturation curves of three proteins suggested the presence of one stable intermediate. The values of delta Gnd H2O for the trpA33, the wild-type, and the trpA11 proteins at 25.8 degrees C and pH 7.0 were 13.4,8.8, and 6.3 kcal/mol, respectively. The delta Gnd H2O of the trpA11 protein was almost independent of temperature, though that of the trpA33 protein was remarkably dependent on temperature. The conformation stabilities of the three proteins were correlated with the hydrophobicities of the substituted amino acid residues.

Substrate interactions with the α-subunit of the Escherichia coli tryptophan synthase : A study of the activity of mutant α-subunits

Extracts of 19 trpA mutant strains of Escherichia coli were examined for their relative activity in the reversible aldolytic reaction catalyzed by the trpA gene product, the α-subunit of tryptophan synthase, in combination with the β-subunit of this enzyme. The specific activities in this reaction, indoleglycerol-P (InGP) ⇌ indole + glyceraldehyde-3-P, were determined for both the forward reaction (InGP to indole) and the reverse reaction (indole to InGP). The majority of the mutant α-subunits had <10% of the wild-type activity in the forward reaction, as expected since these mutant strains were selected for defects in this reaction. In contrast, the majority of these mutant enzymes had >50% of the wild-type activity in the reverse reaction. Several had 5 to 15% of wild-type specific activity in the forward reaction but 60 to 100% of wild-type specific activity in the reverse reaction. Spontaneous revertant strains, selected for their increased ability to catalyze the forward reaction effectively, contained α-subunits with the expected higher specific activities in the forward reaction but without parallel changes in the reverse reaction activity.