Trp Synthase Research Articles

Structural Basis of the Stereochemistry of Inhibition of Tryptophan Synthase by Tryptophan and Derivatives.

We have examined the reaction of Salmonella enterica serovar typhimurium tryptophan (Trp) synthase α2β2 complex with l-Trp, d-Trp, oxindolyl-l-alanine (OIA), and dioxindolyl-l-alanine (DOA) in the presence of disodium (dl)-α-glycerol phosphate (GP), using stopped-flow spectrophotometry and X-ray crystallography. All structures contained the d-isomer of GP bound at the α-active site. (3S)-OIA reacts with the pyridoxal-5'-phosphate (PLP) of Trp synthase to form a mixture of external aldimine and quinonoid complexes. The α-carboxylate of OIA rotates about 90° to become planar with the PLP when the quinonoid complex is formed, resulting in a conformational change in the loop of residues 110-115. The COMM domain of the Trp synthase-OIA complex is found as a mixture of two conformations. The (3R)-diastereomer of DOA binds about 5-fold more tightly than (3S)-OIA and also forms a mixture of aldimine and quinonoid complexes. DOA forms an additional H-bond between the 3-OH of DOA and βLys-87. l-Trp does not form a covalent complex with the PLP of Trp synthase. However, d-Trp forms a mixture of two external aldimine complexes which differ in the orientation of the α-carboxylate. In one conformation, the α-carboxylate is in the plane of the PLP, while in the other conformation, the α-carboxylate is perpendicular to the PLP plane. These results confirm that the stereochemistry of the transient indolenine quinonoid intermediate in the mechanism of Trp synthase is (3S) and demonstrate the linkage between aldimine and quinonoid reaction intermediates in the β-active site and allosteric communications with the α-active site.

Sequence and Misfolding in the Ancient TIM Barrel Motif

Protein folding energy landscapes have been driven by evolution to minimize the energetic and topological frustration experienced during the folding reaction. Small 2 state folders, whose folding rates correlate with their topologies, are good examples of evolution at work. However, larger proteins are often found to have partially-folded intermediates on their energy landscape, some of which can be misfolded. (βα)8 TIM barrel proteins, one of the most common motifs in biology, are one such class of proteins. Previous studies on several diverse TIM barrels have shown that the structures of their misfolded intermediates are not conserved. We hypothesize that large, local in sequence clusters of isoleucine, leucine, and valine (ILV) residues drive the formation of the misfolded intermediate in TIM barrels. These clusters are not conserved in the structures, but do correlate with strong protection against hydrogen exchange in folding intermediates. To test the generality of this hypothesis, we expanded our studies to the indole-3-glycerol phosphate synthase from S. solfataricus (sIGPS). sIGPS has two ILV clusters in different locations than those found in the E. coli α-subunit of trp synthase. The common fold but sequence variations allows a test of the importance of ILV clusters in the early misfolding events. Continuous-flow trFRET has been used to measure pair-wise distance distributions for several βα segments in the misfolded intermediate that appears after a few 10's of microseconds. To complement the pair-wise measurements, SAXS experiments provide global dimensions of the earliest folding events. The insights obtained will explore the link between sequence, ILV clusters and misfolding for the TIM barrel architecture. NIH GM23303.

Inhibition of Escherichia coli tryptophan indole-lyase by tryptophan homologues

We have designed, synthesized and evaluated homotryptophan analogues as possible mechanism-based inhibitors for Escherichia coli tryptophan indole-lyase (tryptophanase, TIL, E.C. 4.1.99.1). As a quinonoid structure is an intermediate in the reaction mechanism of TIL, we anticipated that homologation of the physiological substrate, l-Trp would provide analogues resembling the transition state for β-elimination, and potentially inhibit TIL. Our results demonstrate that l-homotryptophan (1a) is a moderate competitive inhibitor of TIL, with Ki=67μM, whereas l-bishomotryptophan (1b) displays more potent inhibition, with Ki=4.7μM. Pre-steady-state kinetics indicated the formation of an external aldimine and quinonoid with 1a, but only the formation of an external aldimine for 1b, suggesting differences in the inhibition mechanism. These results demonstrate that formation of a quinonoid complex is not required for strong inhibition. In addition, the Trp analogues were evaluated as inhibitors of Salmonella typhimurium Trp synthase. Our results indicate that compound 1b is at least 25-fold more selective toward TIL than Trp synthase. We report that compound 1b is comparable to the most potent inhibitor previously reported, while displaying high selectivity for TIL. Thus, 1b is a potential lead for the development of novel antibacterials.

The tryptophan synthase β‐subunit paralogs TrpB1 and TrpB2 in Thermococcus kodakarensis are both involved in tryptophan biosynthesis and indole salvage

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. In the hyperthermophilic archaeon Thermococcus kodakarensis, trpA and trpB (trpB1) are located adjacently in the trpCDEGFB1A operon. Interestingly, several organisms possess a second trpB gene (trpB2) encoding TrpB2, located outside of the trp operon in T. kodakarensis. Until now, the physiological function of trpB2 has not been examined genetically. In the present study, we report the biochemical and physiological analyses of TrpB2 from T. kodakarensis. Kinetic analysis indicated that TrpB2 catalyzed the TrpB reaction but did not interact with TrpA as in the case of TrpB1. When growth phenotypes were examined for gene disruption strains, the double-deletion mutant (ΔtrpB1ΔtrpB2) displayed Trp auxotrophy, whereas individual single mutants (ΔtrpB1 and ΔtrpB2 strains) did not. It has been proposed previously that, in Thermotoga maritima, TrpB2 provides an alternate route to generate Trp from serine and free indole (indole salvage). To accurately examine the capacity of TrpB1 and TrpB2 in Trp synthesis via indole salvage, we constructed ΔtrpEB1 and ΔtrpEB2 strains using strain KUW1 (ΔpyrFΔtrpE) as a host, eliminating the route for endogenous indole synthesis. Indole complemented the Trp auxotrophies of ΔtrpEB1 (ΔpyrFΔtrpEΔtrpB1) and ΔtrpEB2 (ΔpyrFΔtrpEΔtrpB2) to similar levels. The results indicate that TrpB1 and TrpB2 both contribute to Trp biosynthesis in T. kodakarensis and can utilize free indole, and that indole salvage does not necessarily rely on TrpB2 to a greater extent.

Effects of Pressure and Osmolytes on the Allosteric Equilibria of Salmonella typhimurium Tryptophan Synthase

Osmolytes are common constituents of bacteria that may be produced or accumulate at high concentrations, up to 1 M, when cells are subjected to stresses like ionic strength and temperature. However, the effects of osmolytes on the allosteric properties of bacterial enzymes have rarely been examined. We have studied the effects of osmolytes and hydrostatic pressure on the allosteric equilibria of Salmonella typhimurium tryptophan (Trp) synthase. Trp synthase is a well-studied multienzyme complex with activity tightly regulated by allosteric interactions between the α- and β-subunits. Trp synthase activity is affected by a wide range of physical parameters, including monovalent cations, pH, ligands, solvents, and hydrostatic pressure. Osmolytes, including betaine, taurine, sucrose, and polyethylene glycol, activate Trp synthase 2-3-fold in the absence of monovalent cations, indicating that osmolytes can stabilize the active closed conformation. However, in the presence of monovalent cations, osmolytes have only minor effects on activity and allosteric equilibria, but 1 M betaine stabilizes the Trp synthase-Ser-indoline complex against apparent pressure-induced subunit dissociation. Na(+) and K(+) are more effective at shifting the α-aminoacrylate-indoline quinonoid equilibrium toward the quinonoid side, with a K(Q) of 8-10, than NH(4)(+)(K(Q) ~ 2). Furthermore, pressure-jump experiments show that the mechanism of indoline reaction to form a quinonoid complex may be different for the NH(4)(+) enzyme than the Na(+) and K(+) forms. These results show that osmolytes have subtle but significant effects on the allosteric properties of Trp synthase, and these effects may be important in vivo.

Effects of hydrostatic pressure on the conformational equilibrium of tryptophan synthase from Salmonella typhimurium

A wide range of parameters influence allosteric communications between the alpha- and beta-subunits of the Trp synthase alpha(2)beta(2) multienzyme complex with L-Ser, including monovalent cations, pH, temperature, ligands, organic solvents, and hydrostatic pressure. The conformational change from closed to open can be monitored either by absorbance at 423 nm or fluorescence at 495 nm from the pyridoxal-5'-phosphate-L-Ser complex. Pressure perturbation was used to quantify the effects of monovalent cations, ligands, and mutations on the conformational equilibrium of Trp synthase. P-jump kinetics in the presence of Na(+), NH(4) (+), and Na(+) together with benzimidazole were also examined. The plots of lnk versus P are nonlinear and require a compressibility (beta(double dagger) (o)) term to obtain a good fit. beta(double dagger) (o) is positive for the Na(+) enzyme but negative for NH(4) (+) and Na(+) with benzimidazole. These results suggest that there is a large contribution of solvation to the kinetics of the conformational change of Trp synthase. The relaxation kinetics are also different if the P-jumps are made by increasing or decreasing pressure, suggesting that the enzyme conformations are ensembles of microstates.

Tryptophan deficiency affects organ growth by retarding cell expansion in Arabidopsis

Tryptophan (Trp) is an essential amino acid required not only for protein synthesis but also for the production of many plant metabolites, including the hormone auxin. Mutations that disrupt Trp biosynthesis result in various developmental defects in plant organs, but how Trp affects organ growth and development remains unclear. Here, we identify an Arabidopsis mutant, small organ1 (smo1/trp2-301), which exhibits a reduction in the size of its aerial organs as a result of the retardation of growth by cell expansion, rather than by the retardation of growth by cell proliferation. smo1/trp2-301 contains a lesion in TSB1 that encodes a predominantly expressed Trp synthase beta-subunit, and is allelic with trp2 mutants. Further analyses show that in trp2 leaf cells, the nuclear endoreduplication is impaired and chloroplast development is delayed. Furthermore, cell expansion and leaf growth in trp2 can be restored by the exogenous application of Trp, but not by auxin, and the general protein synthesis is not apparently affected in trp2 mutants. Our findings suggest that the deficiency in Trp or its derivatives is a growth-limiting factor for cell expansion during plant organogenesis.

Pressure and Temperature Jump Relaxation Kinetics of the Conformational Change in Salmonella typhimurium Tryptophan Synthase l-Serine Complex: Large Activation Compressibility and Heat Capacity Changes Demonstrate the Contribution of Solvation

Tryptophan synthase is an alpha2beta2 multienzyme complex that exhibits coupling of the alpha- and beta-subunit reactions by tightly controlled allosteric interactions. A wide range of parameters can affect the allosteric interactions, including monovalent cations, pH, alpha-site and beta-site ligands, temperature, and pressure. Rapid changes in hydrostatic pressure (P-jump) and temperature (T-jump) were used to examine the effects of pressure and temperature on the rates of the interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex. The intense fluorescence emission of the Tryptophan synthase L-Ser external aldimine complex at 495 nm, with 420 nm excitation, provides a probe of the conformational state of Trp synthase. P-jump measurements allowed the determination of rate constants for the reactions in the presence of Na(+), Na(+) with benzimidazole (BZI), and NH4(+). The data require a compressibility term, beta(o)(double dagger), to obtain good fits, especially for the NH4(+) and BZI/Na(+) data. The compressibility changes are consistent with changes in solvation in the transition state. The transition state for the relaxation is more similar in volume to the closed aminoacrylate complex in the presence of Na(+), while it is more similar to the open external aldimine in the presence of NH4(+). Differences between the relaxations for positive and negative P-jumps may arise from changing relative populations of microstates with pressure. T-jump experiments of the Na(+) form of the tryptophan synthase-L-Ser complex show large changes in rate and amplitude over the temperature range from 7 to 45 degrees C. The Arrhenius plots show strong curvature, and hence require a heat capacity term, DeltaC(p)(double dagger), to obtain good fits. The values of DeltaC(p)(double dagger) are very large and negative (-3.6 to -4.4 kJ mol(-1) K(-1)). These changes are also consistent with large changes in solvation in the transition state for interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex.

Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein

The earliest kinetic folding events for (betaalpha)(8) barrels reflect the appearance of off-pathway intermediates. Continuous-flow microchannel mixing methods interfaced to small-angle x-ray scattering (SAXS), circular dichroism (CD), time-resolved Förster resonant energy transfer (trFRET), and time-resolved fluorescence anisotropy (trFLAN) have been used to directly monitor global and specific dimensional properties of the partially folded state in the microsecond time range for a representative (betaalpha)(8) barrel protein. Within 150 micros, the alpha-subunit of Trp synthase (alphaTS) experiences a global collapse and the partial formation of secondary structure. The time resolution of the folding reaction was enhanced with trFRET and trFLAN to show that, within 30 micros, a distinct and autonomous partially collapsed structure has already formed in the N-terminal and central regions but not in the C-terminal region. A distance distribution analysis of the trFRET data confirmed the presence of a heterogeneous ensemble that persists for several hundreds of microseconds. Ready access to locally folded, stable substructures may be a hallmark of repeat-module proteins and the source of early kinetic traps in these very common motifs. Their folding free-energy landscapes should be elaborated to capture this source of frustration.

Arabidopsis Indole Synthase, a Homolog of Tryptophan Synthase Alpha, is an Enzyme Involved in the Trp‐independent Indole‐containing Metabolite Biosynthesis

The plant tryptophan (Trp) biosynthetic pathway produces many secondary metabolites with diverse functions. Indole-3-acetic acid (IAA), proposed as a derivative from Trp or its precursors, plays an essential role in plant growth and development. Although the Trp-dependant and Trp-independent IAA biosynthetic pathways have been proposed, the enzymes, reactions and regulatory mechanisms are largely unknown. In Arabidopsis, indole-3-glycerol phosphate (IGP) is suggested to serve as a branchpoint component in the Trp-independent IAA biosynthesis. To address whether other enzymes in addition to Trp synthase alpha (TSA1) catalyze IGP cleavage, we identified and characterized an indole synthase (INS) gene, a homolog of TSA1 in Arabidopsis. INS exhibits different subcellular localization from TSA1 owing to the lack of chloroplast transit peptide (cTP). In silico data show that the expression levels of INS and TSA1 in all examined organs are quite different. Histochemical staining of INS promoter-GUS transgenic lines indicates that INS is expressed in vascular tissue of cotyledons, hypocotyls, roots and rosette leaves as well as in flowers and siliques. INS is capable of complementing the Trp auxotrophy of Escherichia coliDeltatrpA strain, which is defective in Trp synthesis due to the deletion of TSA. This implies that INS catalyzes the conversion of IGP to indole and may be involved in the biosynthesis of Trp-independent IAA or other secondary metabolites in Arabidopsis.

Quantitative effects of allosteric ligands and mutations on conformational equilibria in Salmonella typhimurium tryptophan synthase

Allosteric communications are important in coordination of the reactions in the tryptophan (Trp) synthase α 2β 2 multienzyme complex. We have measured the conformational equilibria of l-Ser and l-Trp complexes, using absorption and fluorescence spectrophotometry with hydrostatic pressure equilibrium perturbation. The effects of monovalent cations, disodium α-glycerophosphate (Na 2GP), indoleacetylglycine (IAG), and benzimidazole (BZI), as well as of βE109D and βD305A mutations, on K eq for the conformational equilibria were determined. The l-Ser external aldimine–aminoacrylate equilibrium ( K eq = [external aldimine]/[aminoacrylate]) has the largest value with Na + (0.12), followed by K + (0.04), Li + (7.6 × 10 −4), Rb + (4.3 × 10 −4), NH 4 + (2.3 × 10 −4), no cation (2.0 × 10 −4) and Cs + (1.6 × 10 −5). α-Site ligands, Na 2GP and IAG, have modest 3- to 40-fold effects on K eq in the direction of aminoacrylate, but BZI in the presence of Na + gives a low value of K eq comparable to that obtained with Cs +. There is no additivity of free energy for Na 2GP and BZI, suggesting a common pathway for allosteric communications for both ligands. The values of Δ V o range from −126 mL/mol for the Na + complex to −204 mL/mol for the Na + complex with BZI. The βD305A mutation changes the K eq by a factor of at least 10 5 (26.7 kJ/mol) and nearly abolishes allosteric communications. There are also dramatic decreases in the magnitude of both Δ V o and Δ S for the l-Ser external aldimine–aminoacrylate equilibrium for βD305A Trp synthase, consistent with a large decrease in solvation accompanying the conformational change in βD305A Trp synthase relative to wild-type Trp synthase. The βE109D mutation has more modest but significant effects on K eq, which differ with the ligand, ranging from 40-fold for GP to 2200-fold for BZI, even though βGlu-109 is not directly involved in allosteric communications. The effect of GP on the external aldimine–quinonoid intermediate equilibrium of the Trp synthase- l-Trp complex is similar to that of GP on the Trp synthase- l-Ser external aldimine–aminoacrylate equilibrium. These results have allowed a quantitative comparison of the allosteric effects of ligand and mutations in Trp synthase. These allosteric effects are finely tuned to control the synthesis of l-Trp without resulting in substrate or product inhibition.

DEFINING SUBSTRATE SPECIFICITY IN TRYPTOPHAN SYNTHASE BETA‐SUBUNIT HOMOLOGS

Pyrococcus furiosus and many other Archaea, hyperthermophilic bacteria, and some plants, possess two trp synthase β subunit genes which encode two separate yet closely related enzymes; TrpB1 and TrpB2. TrpB1s are considered the canonical β-subunit of the trp synthase α2β2 quaternary complex, whereas TrpB2s are a newly discovered homolog. In P. furiosus, TrpB2 is located outside of a full set of trp operon genes, but several Archaea and hyperthermophilic bacteria possess only TrpB2s and no other trp operon genes. The only TrpB2 that has been characterized is that from T. maritima which was found to catalyze a canonical TrpB1 reaction using L-Ser and indole as substrates. We have found that the P. furiosus, T. maritima, and A. aeolicus TrpB2s, as well as the S. typhimirium TrpB1, can utilize L-Ser and L-Cys as substrates in the enzymatic biosynthesis of L-Trp. The P. furiosus TrpB2 appears to follow a biphasic kinetic pattern with two Km’s and two Vmax’s with both substrates. We have applied LC-MS to define the substrate specificity between L-Ser and L-Cys in a competition experiment using β-[13C]-L-Ser. These experiments have revealed that Ser/Cys substrate specificity in the S. typhimirium α2β2 complex is imparted to the β-subunit by the α-subunit, yet in other organisms, like the protozoan Cryptosporidium parvum, which possesses a TrpB1 but no TrpA, the α-subunit is not necessary to discriminate between L-Ser and L-Cys.

Rapid Kinetics of Na+ Binding to Thrombin

The kinetic mechanism of Na(+) binding to thrombin was resolved by stopped-flow measurements of intrinsic fluorescence. Na(+) binds to thrombin in a two-step mechanism with a rapid phase occurring within the dead time of the spectrometer (<0.5 ms) followed by a single-exponential slow phase whose k(obs) decreases hyperbolically with increasing [Na(+)]. The rapid phase is due to Na(+) binding to the enzyme E to generate the E:Na(+) form. The slow phase is due to the interconversion between E(*) and E, where E(*) is a form that cannot bind Na(+). Temperature studies in the range from 5 to 35 degrees C show significant enthalpy, entropy, and heat capacity changes associated with both Na(+) binding and the E to E(*) transition. As a result, under conditions of physiologic temperature and salt concentrations, the E(*) form is negligibly populated (<1%) and thrombin is almost equally partitioned between the E (40%) and E:Na(+) (60%) forms. Single-site Phe mutations of all nine Trp residues of thrombin enabled assignment of the fluorescence changes induced by Na(+) binding mainly to Trp-141 and Trp-215, and to a lesser extent to Trp-148, Trp-207, and Trp-237. However, the fast phase of fluorescence increase is influenced to different extents by all Trp residues. The distribution of these residues over the entire thrombin surface demonstrates that Na(+) binding induces long-range effects on the structure of the enzyme as a whole, contrary to the conclusions drawn from recent structural studies. These findings elucidate the mechanism of Na(+) binding to thrombin and are relevant to other clotting factors and enzymes allosterically activated by monovalent cations.

Hydrostatic Pressure as a Structural and Mechanistic Probe of Tryptophan Synthase and Tryptophan Indole‐lyase

The effects of hydrostatic pressure on the conformation and activity of tryptophan (Trp) synthase α2β2 complex from Salmonella typhimurium and tryptophan indole-lyase (Trpase) from Escherichia coli have been examined. The data demonstrate that the open conformation of Trp synthase is favored by higher pressure. The ΔVo and Ko for the equilibrium between the closed and open conformations of the Trp synthase-L-Ser complex are −126 mL/mol and 0.12 for the Na+form and −171 mL/mol and 2.3 x 10−4 for the NH4+ form. Pressure-jump experiments of the H463F mutant Trpase L-Trp and L-Met complexes show relaxations at 500 nm after rapid pressure changes of 100–400 bar for the complexes with both L-Trp and L-Met. However, the apparent rate constants for relaxation of L-Trp, but not L-Met, show a significant increase with pressure. These results suggest that there may be a contribution to the deprotonation reaction of Trpase either from quantum mechanical tunneling or from a mechanical coupling of protein motion and proton transfer for the reaction of L-Trp, but not with L-Met.

Hydrostatic Pressure Affects the Conformational Equilibrium of Salmonella typhimurium Tryptophan Synthase

The effect of hydrostatic pressure on the tryptophan (Trp) synthase alpha2beta2 complex from Salmonella typhimurium has been investigated. Trp synthase has been shown previously to exhibit low-activity (open) and high-activity (closed) conformations. The equilibrium between the open and closed conformations of Trp synthase has been found to be affected by a wide range of variables, including alpha-subunit ligands, monovalent cations, organic solvents, pH, and temperature. The absorption spectrum of the Trp synthase-L-Ser complex shows an increase in absorption of the 423 nm band of the external aldimine, which is a characteristic of the open conformation, as hydrostatic pressure is increased from 1 to 2000 bar. The deltaV(o) and K(o) for the equilibrium between the closed and open conformations of the Trp synthase-L-Ser complex are -126 mL/mol and 0.12 for the Na+ form and -171 mL/mol and 2.3 x 10(-4) for the NH4+ form. When the Trp synthase-L-Ser complex is subjected to pressure jumps of 100-400 bar, relaxations are observed, exhibiting an increase in fluorescence emission at wavelengths greater than 455 nm, with 405 nm excitation. The relaxation to the new equilibrium position requires two exponentials to fit the data in the presence of 0.1 M Na+ and three exponentials to obtain a reasonable fit in the absence of cations and with 0.1 M NH4+. Fluorescence emission at 325 nm, with excitation at 280 nm, also increases when the Trp synthase-L-Ser complex is subjected to pressure jump. These data demonstrate that the open conformation of Trp synthase is favored by higher pressure. Thus, the open conformation has a smaller apparent net system volume than the closed conformation. We estimate that there are 35-47 more waters in the solvation shell of the open conformation than in that of the closed conformation.

Detection of Open and Closed Conformations of Tryptophan Synthase by 15N-Heteronuclear Single-Quantum Coherence Nuclear Magnetic Resonance of Bound 1-15N-L-Tryptophan

1-15N-L-Tryptophan (1-15N-L-Trp) was synthesized from 15N-aniline by a Sandmeyer reaction, followed by cyclization to isatin, reduction to indole with LiAlH4, and condensation of the 15N-indole with L-serine, catalyzed by tryptophan synthase. 1-15N-L-Trp was complexed with wild-type tryptophan synthase and beta-subunit mutants, betaK87T, betaD305A, and betaE109D, in the absence or presence of the allosteric ligands sodium chloride and disodium alpha-glycerophosphate. The enzyme complexes were observed by 15N-heteronuclear single-quantum coherence nuclear magnetic resonance (15N-HSQC NMR) spectroscopy for the presence of 1-15N-L-Trp bound to the beta-active site. No 15N-HSQC signal was detected for 1-15N-L-Trp in 10 mm triethanolamine hydrochloride buffer at pH 8. 1-15N-L-Trp in the presence of wild-type tryptophan synthase in the absence or presence of 50 mm sodium chloride showed a cross peak at 10.25 ppm on the 1H axis and 129 ppm on the 15N axis as a result of reduced solvent exchange for the bound 1-15N-L-Trp, consistent with formation of a closed conformation of the active site. The addition of disodium alpha-glycerophosphate produced a signal twice as intense, suggesting that the equilibrium favors the closed conformation. 15N-HSQC NMR spectra of betaK87T and betaE109D mutant Trp synthase with 1-15N-L-Trp showed a similar cross peak either in the presence or absence of disodium alpha-glycerophosphate, indicating the preference for a closed conformation for these mutant proteins. In contrast, the betaD305A Trp synthase mutant only showed a 15N-HSQC signal in the presence of disodium alpha-glycerophosphate. Thus, this mutant Trp synthase favored an open conformation in the absence of disodium alpha-glycerophosphate but was able to form a closed conformation in the presence of disodium alpha-glycerophosphate. Our results demonstrate that the 15N-HSQC NMR spectra of 1-15N-L-Trp bound to Trp synthase can be used to determine the conformational state of mutant forms in solution rapidly. In contrast, UV-visible spectra of wild-type and mutant Trp synthase in the presence of L-Trp with NaCl and/or disodium alpha-glycerophosphate are more difficult to interpret in terms of altered conformational equilibria.

Proline Replacements and the Simplification of the Complex, Parallel Channel Folding Mechanism for the Alpha Subunit of Trp Synthase, a TIM Barrel Protein

The kinetic folding mechanism for the alpha subunit of tryptophan synthase (αTS) from Escherichia coli involves four parallel channels whose inter-conversions are controlled by three cis/ trans prolyl isomerization reactions (τ 1, τ 2 and τ 3). A previous mutational analysis of all 19 proline positions, including the unique cis Asp27–Pro28 peptide bond, revealed that the G 3P28G, P78A or P96A mutations selectively eliminated the fast, τ 1 (ten seconds), folding phase, while the P217M and P261A mutations eliminated the medium, τ 2 (40 seconds) and the slow, τ 3 (∼300 seconds) folding phases, respectively. To further elucidate the role of these proline residues and to simplify the folding mechanism, a series of double and triple mutants were constructed at these critical positions, and comprehensive kinetic and thermodynamic experiments were performed. Although it was not possible to construct a stable system that was free of proline isomerization constraints, a double mutant variant, G 3P28G/P217M, in which the refolding of more than 90% of the unfolded protein is not limited by proline isomerization reactions was identified. Further, long-range interactions between several of these residues appear to be a crucial part of the cooperative network of structure that stabilizes the TIM barrel motif for αTS.

Parallel channels and rate-limiting steps in complex protein folding reactions: prolyl isomerization and the alpha subunit of Trp synthase, a TIM barrel protein.

A kinetic folding mechanism for the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, involving four parallel channels with multiple native, intermediate and unfolded forms, has recently been proposed. The hypothesis that cis/trans isomerization of several Xaa-Pro peptide bonds is the source of the multiple folding channels was tested by measuring the sensitivity of the three rate-limiting phases (tau(1), tau(2), tau(3)) to catalysis by cyclophilin, a peptidyl-prolyl isomerase. Although the absence of catalysis for the tau(1) (fast) phase leaves its assignment ambiguous, our previous mutational analysis demonstrated its connection to the unique cis peptide bond preceding proline 28. The acceleration of the tau(2) (medium) and tau(3) (slow) refolding phases by cyclophilin demonstrated that cis/trans prolyl isomerization is also the source of these phases. A collection of proline mutants, which covered all of the remaining 18 trans proline residues of alphaTS, was constructed to obtain specific assignments for these phases. Almost all of the mutant proteins retained the complex equilibrium and kinetic folding properties of wild-type alphaTS; only the P217A, P217G and P261A mutations caused significant changes in the equilibrium free energy surface. Both the P78A and P96A mutations selectively eliminated the tau(1) folding phase, while the P217M and P261A mutations eliminated the tau(2) and tau(3) folding phases, respectively. The redundant assignment of the tau(1) phase to Pro28, Pro78 and Pro96 may reflect their mutual interactions in non-random structure in the unfolded state. The non-native cis isomers for Pro217 and Pro261 may destabilize an autonomous C-terminal folding unit, thereby giving rise to kinetically distinct unfolded forms. The nature of the preceding amino acid, the solvent exposure, or the participation in specific elements of secondary structure in the native state, in general, are not determinative of the proline residues whose isomerization reactions can limit folding.

A cis-prolyl peptide bond isomerization dominates the folding of the alpha subunit of Trp synthase, a TIM barrel protein.

The cis/trans isomerization of prolyl peptide bonds has been suggested to dominate the folding of the alpha subunit of tryptophan synthase from Escherichia coli (alphaTS). To test the role of the unique cis isomer between Asp27 and Pro28, the folding properties of P28A, P28G and G(3)P28G, a three-glycine insertion mutant between Asp27 and Gly28, were investigated using urea as a denaturant. Circular dichroism analysis demonstrated that none of the mutations perturb the secondary structure significantly, although the aromatic side-chain packing is altered for P28A and P28G. All three mutant proteins inherited the three-state thermodynamic behavior observed in wild-type alphaTS, ensuring that the fundamental features of the energy surface are intact. Kinetic studies showed that neither alanine nor glycine substitutions at Pro28 results in the elimination of any slow-refolding phases. By contrast, the G(3)P28G mutant eliminates the fastest of the slow-refolding phases and one of the two unfolding phases. Double-jump experiments on G(3)P28G confirm the assignment of the missing refolding phase to the isomerization of the Asp27-Pro28 peptide bond. These results imply that the local stability conveyed by the tight, overlapping turns containing the cis peptide bond is sufficient to favor the cis isomer for several non-prolyl residues. The free energy required to drive the isomerization reaction is provided by the formation of the stable intermediate, demonstrating that the acquisition of structure and stability is required to induce subsequent rate-limiting steps in the folding of alphaTS.

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.