Indole-3-Glycerol Phosphate Synthase From Mycobacterium tuberculosis: A Potential New Drug Target.

Tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis, affects millions of people worldwide. Several TB drugs have lost efficacy due to emerging drug resistance and new anti-TB targets are needed. Recent research suggests that indole-3-glycerol phosphate synthase (IGPS) in M. tuberculosis (MtIGPS) could be such a target. IGPS is a (ß/α)8 -barrel enzyme that catalyzes the conversion of 1-(o-carboxyphenylamino)-1-deoxyribulose 5'-phosphate (CdRP) into indole-glycerol-phosphate (IGP) in the bacterial tryptophan biosynthetic pathway. M. tuberculosis over expresses the tryptophan pathway genes during an immune response and inhibition of MtIGPS allows CD4 T-cells to more effectively fight against M. tuberculosis. Here we review the published data on MtIGPS expression, kinetics, mechanism, and inhibition. We also discuss MtIGPS crystal structures and compare them to other IGPS structures to reveal potential structure-function relationships of interest for the purposes of drug design and biocatalyst engineering.

Frustration and folding of a TIM barrel protein.

Triosephosphate isomerase (TIM) barrel proteins have not only a conserved architecture that supports a myriad of enzymatic functions, but also a conserved folding mechanism that involves on- and off-pathway intermediates. Although experiments have proven to be invaluable in defining the folding free-energy surface, they provide only a limited understanding of the structures of the partially folded states that appear during folding. Coarse-grained simulations employing native centric models are capable of sampling the entire energy landscape of TIM barrels and offer the possibility of a molecular-level understanding of the readout from sequence to structure. We have combined sequence-sensitive native centric simulations with small-angle X-ray scattering and time-resolved Förster resonance energy transfer to monitor the formation of structure in an intermediate in the Sulfolobus solfataricus indole-3-glycerol phosphate synthase TIM barrel that appears within 50 µs and must at least partially unfold to achieve productive folding. Simulations reveal the presence of a major and 2 minor folding channels not detected in experiments. Frustration in folding, i.e., backtracking in native contacts, is observed in the major channel at the initial stage of folding, as well as late in folding in a minor channel before the appearance of the native conformation. Similarities in global and pairwise dimensions of the early intermediate, the formation of structure in the central region that spreads progressively toward each terminus, and a similar rate-limiting step in the closing of the ß-barrel underscore the value of combining simulation and experiment to unravel complex folding mechanisms at the molecular level.

Structure and kinetics of indole-3-glycerol phosphate synthase from Pseudomonas aeruginosa: Decarboxylation is not essential for indole formation.

In tryptophan biosynthesis, the reaction catalyzed by the enzyme indole-3-glycerol phosphate synthase (IGPS) starts with a condensation step in which the substrate's carboxylated phenyl group makes a nucleophilic attack to form the pyrrole ring of the indole, followed by a decarboxylation that restores the aromaticity of the phenyl. IGPS from Pseudomonas aeruginosa has the highest turnover number of all characterized IGPS enzymes, providing an excellent model system to test the necessity of the decarboxylation step. Since the 1960s, this step has been considered to be mechanistically essential based on studies of the IGPS-phosphoribosylanthranilate isomerase fusion protein from Escherichia coli Here, we present the crystal structure of P. aeruginosa IGPS in complex with reduced CdRP, a nonreactive substrate analog, and using a sensitive discontinuous assay, we demonstrate weak promiscuous activity on the decarboxylated substrate 1-(phenylamino)-1-deoxyribulose-5-phosphate, with an ∼1000× lower rate of IGP formation than from the native substrate. We also show that E. coli IGPS, at an even lower rate, can produce IGP from decarboxylated substrate. Our structure of P. aeruginosa IGPS has eight molecules in the asymmetric unit, of which seven contain ligand and one displays a previously unobserved conformation closer to the reactive state. One of the few nonconserved active-site residues, Phe201 in P. aeruginosa IGPS, is by mutagenesis demonstrated to be important for the higher turnover of this enzyme on both substrates. Our results demonstrate that despite IGPS's classification as a carboxy-lyase (i.e. decarboxylase), decarboxylation is not a completely essential step in its catalysis.

Extremely stable indole-3-glycerol-phosphate synthase from hyperthermophilic archaeon Pyrococcus furiosus.

The gene-encoding Indole-3-glycerol phosphate synthase, a key enzyme involved in the cyclization of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate, from Pyrococcus furiosus was cloned and expressed in Escherichia coli. The gene product was produced in the soluble and active form. The recombinant protein, purified to apparent homogeneity, displayed highest activity at 100 °C and pH of 5.5. The recombinant enzyme followed Michaelis-Menten kinetics exhibiting apparent Vmax and Km values of 20 ± 0.5 µmol min-1 mg-1 and 140 ± 10 µM, respectively. The activation energy, determined from the linear Arrhenius plot, was 17 ± 0.5 kJ mol-1. A unique property of PfInGPS is its stability against denaturants and temperature. There was no significant change in activity even in the presence of 8 M urea or 5 M guanidine hydrochloride. Furthermore, recombinant PfInGPS was highly thermostable with a half-life of 200 min at 100 °C. To the best of our knowledge, this is the most stable indole-3-glycerol phosphate synthase characterized to date.

Relationship of Catalysis and Active Site Loop Dynamics in the (ßα)8-Barrel Enzyme Indole-3-glycerol Phosphate Synthase.

It is important to understand how the catalytic activity of enzymes is related to their conformational flexibility. We have studied this activity-flexibility correlation using the example of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (ssIGPS), which catalyzes the fifth step in the biosynthesis of tryptophan. ssIGPS is a thermostable representative of enzymes with the frequently encountered and catalytically versatile (ßα)8-barrel fold. Four variants of ssIGPS with increased catalytic turnover numbers were analyzed by transient kinetics at 25 °C, and wild-type ssIGPS was likewise analyzed both at 25 °C and at 60 °C. Global fitting with a minimal three-step model provided the individual rate constants for substrate binding, chemical transformation, and product release. The results showed that in both cases, namely, the application of activating mutations and temperature increase, the net increase in the catalytic turnover number is afforded by acceleration of the product release rate relative to the chemical transformation steps. Measurements of the solvent viscosity effect at 25 °C versus 60 °C confirmed this change in the rate-determining step with temperature, which is in accordance with a kink in the Arrhenius diagram of ssIGPS at ∼40 °C. When rotational diffusion rates of electron paramagnetic spin-labels attached to active site loop ß1α1 are plotted in the form of an Arrhenius diagram, kinks are observed at the same temperature. These findings, together with molecular dynamics simulations, demonstrate that a different degree of loop mobility correlates with different rate-limiting steps in the catalytic mechanism of ssIGPS.

Functional identification of the general acid and base in the dehydration step of indole-3-glycerol phosphate synthase catalysis.

The tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase is a proposed target for new antimicrobials and is a favored starting framework in enzyme engineering studies. Forty years ago, Parry proposed that the enzyme mechanism proceeds through two intermediates in a series of condensation, decarboxylation, and dehydration steps. X-ray crystal structures have suggested that Lys-110 (numbering according to the Sulfolobus solfataricus enzyme) behaves as a general acid both in the condensation and dehydration steps, but did not reveal an efficient pathway for the reprotonation of this critical residue. Our mutagenesis and kinetic experiments suggest an alternative mechanism whereby Lys-110 acts as a general acid in the condensation step, but another invariant residue, Lys-53, acts as the general acid in the dehydration step. These studies also indicate that the conserved residue Glu-51 acts as the general base in the dehydration step. The revised mechanism effectively divides the active site into discrete regions where the catalytic surfaces containing Lys-110 and Lys-53/Glu-51 catalyze the ring closure (i.e. condensation and decarboxylation) and dehydration steps, respectively. These results can be leveraged toward the development of novel inhibitors against this validated antimicrobial target and toward the rational engineering of the enzyme to produce indole derivatives that are highly prized by the pharmaceutical and agricultural industries.

Kinetic mechanism of indole-3-glycerol phosphate synthase.

The (ßα)(8)-barrel enzyme indole-3-glycerol phosphate synthase (IGPS) catalyzes the multistep transformation of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP) into indole-3-glycerol phosphate (IGP) in tryptophan biosynthesis. Mutagenesis data and crystal structure analysis of IGPS from Sulfolobus solfataricus (sIGPS) allowed for the formulation of a plausible chemical mechanism of the reaction, and molecular dynamics simulations suggested that flexibility of active site loops might be important for catalysis. Here we developed a method that uses extrinsic fluorophores attached to active site loops to connect the kinetic mechanism of sIGPS to structure and conformational motions. Specifically, we elucidated the kinetic mechanism of sIGPS and correlated individual steps in the mechanism to conformational motions of flexible loops. Pre-steady-state kinetic measurements of CdRP to IGP conversion monitoring changes in intrinsic tryptophan and IGP fluorescence provided a minimal three-step kinetic model in which fast substrate binding and chemical transformation are followed by slow product release. The role of sIGPS loop conformational motion during substrate binding and catalysis was examined via variants that were covalently labeled with fluorescent dyes at the N-terminal extension of the enzyme and mobile active site loop ß1α1. Analysis of kinetic data monitoring dye fluorescence revealed a conformational change that follows substrate binding, suggesting an induced-fit-type binding mechanism for the substrate CdRP. Global fitting of all kinetic results obtained with wild-type sIGPS and the labeled variants was best accommodated by a four-step kinetic model. In this model, both the binding of CdRP and its on-enzyme conversion to IGP are accompanied by conformational transitions. The liberation of the product from the active site is the rate-limiting step of the overall reaction. Our results confirm the importance of flexible active loops for substrate binding and catalysis by sIGPS.

CLIPS-1D: analysis of multiple sequence alignments to deduce for residue-positions a role in catalysis, ligand-binding, or protein structure.

BACKGROUND: One aim of the in silico characterization of proteins is to identify all residue-positions, which are crucial for function or structure. Several sequence-based algorithms exist, which predict functionally important sites. However, with respect to sequence information, many functionally and structurally important sites are hard to distinguish and consequently a large number of incorrectly predicted functional sites have to be expected. This is why we were interested to design a new classifier that differentiates between functionally and structurally important sites and to assess its performance on representative datasets. RESULTS: We have implemented CLIPS-1D, which predicts a role in catalysis, ligand-binding, or protein structure for residue-positions in a mutually exclusive manner. By analyzing a multiple sequence alignment, the algorithm scores conservation as well as abundance of residues at individual sites and their local neighborhood and categorizes by means of a multiclass support vector machine. A cross-validation confirmed that residue-positions involved in catalysis were identified with state-of-the-art quality; the mean MCC-value was 0.34. For structurally important sites, prediction quality was considerably higher (mean MCC = 0.67). For ligand-binding sites, prediction quality was lower (mean MCC = 0.12), because binding sites and structurally important residue-positions share conservation and abundance values, which makes their separation difficult. We show that classification success varies for residues in a class-specific manner. This is why our algorithm computes residue-specific p-values, which allow for the statistical assessment of each individual prediction. CLIPS-1D is available as a Web service at http://www-bioinf.uni-regensburg.de/. CONCLUSIONS: CLIPS-1D is a classifier, whose prediction quality has been determined separately for catalytic sites, ligand-binding sites, and structurally important sites. It generates hypotheses about residue-positions important for a set of homologous proteins and focuses on conservation and abundance signals. Thus, the algorithm can be applied in cases where function cannot be transferred from well-characterized proteins by means of sequence comparison.

Experimental assessment of the importance of amino acid positions identified by an entropy-based correlation analysis of multiple-sequence alignments.

The analysis of a multiple-sequence alignment (MSA) with correlation methods identifies pairs of residue positions whose occupation with amino acids changes in a concerted manner. It is plausible to assume that positions that are part of many such correlation pairs are important for protein function or stability. We have used the algorithm H2r to identify positions k in the MSAs of the enzymes anthranilate phosphoribosyl transferase (AnPRT) and indole-3-glycerol phosphate synthase (IGPS) that show a high conn(k) value, i.e., a large number of significant correlations in which k is involved. The importance of the identified residues was experimentally validated by performing mutagenesis studies with sAnPRT and sIGPS from the archaeon Sulfolobus solfataricus. For sAnPRT, five H2r mutant proteins were generated by replacing nonconserved residues with alanine or the prevalent residue of the MSA. As a control, five residues with conn(k) values of zero were chosen randomly and replaced with alanine. The catalytic activities and conformational stabilities of the H2r and control mutant proteins were analyzed by steady-state enzyme kinetics and thermal unfolding studies. Compared to wild-type sAnPRT, the catalytic efficiencies (k(cat)/K(M)) were largely unaltered. In contrast, the apparent thermal unfolding temperature (T(M)(app)) was lowered in most proteins. Remarkably, the strongest observed destabilization (ΔT(M)(app) = 14 °C) was caused by the V284A exchange, which pertains to the position with the highest correlation signal [conn(k) = 11]. For sIGPS, six H2r mutant and four control proteins with alanine exchanges were generated and characterized. The k(cat)/K(M) values of four H2r mutant proteins were reduced between 13- and 120-fold, and their T(M)(app) values were decreased by up to 5 °C. For the sIGPS control proteins, the observed activity and stability decreases were much less severe. Our findings demonstrate that positions with high conn(k) values have an increased probability of being important for enzyme function or stability.

Differences in the catalytic mechanisms of mesophilic and thermophilic indole-3-glycerol phosphate synthase enzymes at their adaptive temperatures.

Thermophilic enzymes tend to be less catalytically-active at lower temperatures relative to their mesophilic counterparts, despite having very similar crystal structures. An often cited hypothesis for this general observation is that thermostable enzymes have evolved a more rigid tertiary structure in order to cope with their more extreme, natural environment, but they are also less flexible at lower temperatures, leading to their lower catalytic activity under mesophilic conditions. An alternative hypothesis, however, is that complementary thermophilic-mesophilic enzyme pairs simply operate through different evolutionary-optimized catalytic mechanisms. In this communication, we present evidence that while the steps of the catalytic mechanisms for mesophilic and thermophilic indole-3-glycerol phosphate synthase (IGPS) enzymes are fundamentally similar, the identity of the rate-determining step changes as a function of temperature. Our findings indicate that while product release is rate-determining at 25°C for thermophilic IGPS, near its adaptive temperature (75°C), a proton transfer event, involving a general acid, becomes rate-determining. The rate-determining steps for thermophilic and mesophilic IGPS enzymes are also different at their respective, adaptive temperatures with the mesophilic IGPS-catalyzed reaction being rate-limited before irreversible CO2 release, and the thermophilic IGPS-catalyzed reaction being rate limited afterwards.

Structure of indole-3-glycerol phosphate synthase from Thermus thermophilus HB8: implications for thermal stability.

The three-dimensional structure of indole-3-glycerol phosphate synthase (IGPS) from the thermophilic bacterium Thermus thermophilus HB8 (TtIGPS) has been determined at 1.8 Å resolution. The structure adopts a typical (ß/α)(8)-barrel fold with an additional N-terminal extension of 46 residues. A detailed comparison of the crystal structure of TtIGPS with available structures of IGPS from the archaeon Sulfolobus solfataricus (SsIGPS) and the bacteria Thermotoga maritima (TmIGPS) and Escherichia coli (EcIGPS) has been performed. Although the overall folds of the proteins are the same, there are differences in amino-acid composition, structural rigidity, ionic features and stability clusters which may account for the high thermostability of the hyperthermophilic (SsIGPS and TmIGPS) and thermophilic (TtIGPS) proteins when compared with the mesophilic EcIGPS. The thermostability of IGPS seems to be established mainly by favourable interactions of charged residues, salt bridges and the spatial distribution of relatively rigid clusters of extensively interacting residues.

Coevolving residues of (beta/alpha)(8)-barrel proteins play roles in stabilizing active site architecture and coordinating protein dynamics.

Indole-3-glycerol phosphate synthase (IGPS) is a representative of (beta/alpha)(8)-barrel proteins-the most common enzyme fold in nature. To better understand how the constituent amino-acids work together to define the structure and to facilitate the function, we investigated the evolutionary and dynamical coupling of IGPS residues by combining statistical coupling analysis (SCA) and molecular dynamics (MD) simulations. The coevolving residues identified by the SCA were found to form a network which encloses the active site completely. The MD simulations showed that these coevolving residues are involved in the correlated and anti-correlated motions. The correlated residues are within van der Waals contact and appear to maintain the active site architecture; the anti-correlated residues are mainly distributed on opposite sides of the catalytic cavity and coordinate the motions likely required for the substrate entry and product release. Our findings might have broad implications for proteins with the highly conserved (betaalpha)(8)-barrel in assessing the roles of amino-acids that are moderately conserved and not directly involved in the active site of the (beta/alpha)(8)-barrel. The results of this study could also provide useful information for further exploring the specific residue motions for the catalysis and protein design based on the (beta/alpha)(8)-barrel scaffold.

Mapping the structure of folding cores in TIM barrel proteins by hydrogen exchange mass spectrometry: the roles of motif and sequence for the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus.

To test the roles of motif and amino acid sequence in the folding mechanisms of TIM barrel proteins, hydrogen-deuterium exchange was used to explore the structure of the stable folding intermediates for the of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS). Previous studies of the urea denaturation of sIGPS revealed the presence of an intermediate that is highly populated at approximately 4.5 M urea and contains approximately 50% of the secondary structure of the native (N) state. Kinetic studies showed that this apparent equilibrium intermediate is actually comprised of two thermodynamically distinct species, I(a) and I(b). To probe the location of the secondary structure in this pair of stable on-pathway intermediates, the equilibrium unfolding process of sIGPS was monitored by hydrogen-deuterium exchange mass spectrometry. The intact protein and pepsin-digested fragments were studied at various concentrations of urea by electrospray and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. Intact sIGPS strongly protects at least 54 amide protons from hydrogen-deuterium exchange in the intermediate states, demonstrating the presence of stable folded cores. When the protection patterns and the exchange mechanisms for the peptides are considered with the proposed folding mechanism, the results can be interpreted to define the structural boundaries of I(a) and I(b). Comparison of these results with previous hydrogen-deuterium exchange studies on another TIM barrel protein of low sequence identify, alpha-tryptophan synthase (alphaTS), indicates that the thermodynamic states corresponding to the folding intermediates are better conserved than their structures. Although the TIM barrel motif appears to define the basic features of the folding free energy surface, the structures of the partially folded states that appear during the folding reaction depend on the amino acid sequence. Markedly, the good correlation between the hydrogen-deuterium exchange patterns of sIGPS and alphaTS with the locations of hydrophobic clusters defined by isoleucine, leucine, and valine residues suggests that branch aliphatic side-chains play a critical role in defining the structures of the equilibrium intermediates.

Correlation of fitness landscapes from three orthologous TIM barrels originates from sequence and structure constraints.

Sequence divergence of orthologous proteins enables adaptation to environmental stresses and promotes evolution of novel functions. Limits on evolution imposed by constraints on sequence and structure were explored using a model TIM barrel protein, indole-3-glycerol phosphate synthase (IGPS). Fitness effects of point mutations in three phylogenetically divergent IGPS proteins during adaptation to temperature stress were probed by auxotrophic complementation of yeast with prokaryotic, thermophilic IGPS. Analysis of beneficial mutations pointed to an unexpected, long-range allosteric pathway towards the active site of the protein. Significant correlations between the fitness landscapes of distant orthologues implicate both sequence and structure as primary forces in defining the TIM barrel fitness landscape and suggest that fitness landscapes can be translocated in sequence space. Exploration of fitness landscapes in the context of a protein fold provides a strategy for elucidating the sequence-structure-fitness relationships in other common motifs.

How to make my blood boil.

Two recent papers comparing the structure of a hyperthermophilic protein with its mesophilic counterpart both conclude that large networks of ion-pairs are important for hyperthermostability. How and why is not yet clear.

2.0 A structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability.

BACKGROUND: Recent efforts to understand the basis of protein stability have focused attention on comparative studies of proteins from hyperthermophilic and mesophilic organisms. Most work to date has been on either oligomeric enzymes or monomers comprising more than one domain. Such studies are hampered by the need to distinguish between stabilizing interactions acting between subunits or domains from those acting within domains. In order to simplify the search for determinants of protein stability we have chosen to study the monomeric enzyme indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus (sIGPS), which grows optimally at 90 degrees C. RESULTS: The 2.0 A crystal structure of sIGPS was determined and compared with the known 2.0 A structure of the IGPS domain of the bifunctional enzyme from the mesophilic bacterium Escherichia coli (eIGPS). sIGPS and eIGPS have only 30% sequence identity, but share high structural similarity. Both are single-domain (beta/alpha)8 barrel proteins, with one (eIGPS) or two (sIGPS) additional helices inserted before the first beta strand. The thermostable sIGPS has many more salt bridges than eIGPS. Several salt bridges crosslink adjacent alpha helices or participate in triple or quadruple salt-bridge clusters. The number of helix capping, dipole stabilizing and hydrophobic interactions is also increased in sIGPS. CONCLUSIONS: The higher stability of sIGPS compared with eIGPS seems to be the result of several improved interactions. These include a larger number of salt bridges, stabilization of alpha helices and strengthening of both polypeptide chain termini and solvent-exposed loops.

Indole-3-glycerol-phosphate synthase from Sulfolobus solfataricus as a model for studying thermostable TIM-barrel enzymes.

Indole-3-glycerol-phosphate synthase, a thermophilic and thermostable enzyme from the archaeon Sulfolobus solfataricus, was purified and characterized. The sequence of the thermophilic enzyme was compared to the sequence of a homologous mesophilic enzyme from Escherichia coli. The secondary structure of the thermophilic enzyme was predicted taking into account the patterns of hydropathy, chain flexibility and amphipathicity and the CD spectrum. From this analysis it turned out that indole-3-glycerol-phosphate synthase from S. solfataricus can be considered a model for studying thermostable TIM-barrel enzymes. Some peculiarities of the amino-acid sequence of indole-3-glycerol-phosphate synthase from S. solfataricus are discussed in relation to the thermostability of the enzyme.

Phosphate group binding "cup" of PLP-dependent and non-PLP-dependent enzymes: leitmotif and variations.

Pyridoxal-5'-phosphate (PLP) is widely used by many enzymes in reactions where amino acids are interconverted. Whereas the role of the pyridoxal ring in catalysis is well understood, the functional role of the single phosphate group in PLP has been less studied. Here we construct unambiguous connection diagrams that describe the interactions among the three non-ester phosphate oxygen atoms of PLP and surrounding atoms from the protein binding site and from water molecules, the so-called phosphate group binding "cup". These diagrams provide a simple means to identify common recognition motifs for the phosphate group in both similar and different protein folds. Diagrams were constructed and compared in the cases of five newly determined structures of PLP-dependent transferases (fold type I enzymes) and, additionally, two non-PLP protein complexes (indole-3-glycerol phosphate synthase (IGPS) with bound indole-3-glycerol phosphate (IGP) and old yellow enzyme (OYE) with bound flavin mononucleotide (FMN)). A detailed comparison of the diagrams shows that three positions out of ten in the structure of the phosphate group binding "cup" contain invariant atoms, while seven others are occupied by conserved atom types. This level of similarity was also observed in the fold type III (TIM beta/alpha-barrel) enzymes that bind three different ligands: PLP, IGP and FMN.

Folding mechanism of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus: a test of the conservation of folding mechanisms hypothesis in (beta(alpha))(8) barrels.

As a test of the hypothesis that folding mechanisms are better conserved than sequences in TIM barrels, the equilibrium and kinetic folding mechanisms of indole-3-glycerol phosphate synthase (sIGPS) from the thermoacidophilic archaebacterium Sulfolobus solfataricus were compared to the well-characterized models of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli. A multifaceted approach combining urea denaturation and far-UV circular dichroism, tyrosine fluorescence total intensity, and tyrosine fluorescence anisotropy was employed. Despite a sequence identity of only 13%, a stable intermediate (I) in sIGPS was found to be similar to a stable intermediate in alphaTS in terms of its thermodynamic properties and secondary structure. Kinetic experiments revealed that the fastest detectable folding event for sIGPS involves a burst-phase (<5ms) reaction that leads directly to the stable intermediate. The slower of two subsequent phases reflects the formation/disruption of an off-pathway dimeric form of I. The faster phase reflects the conversion of I to the native state and is limited by folding under marginally stable conditions and by isomerization or rearrangement under strongly folding conditions. By contrast, alphaTS is thought to fold via an off-pathway burst-phase intermediate whose unfolding controls access to a set of four on-pathway intermediates that comprise the stable equilibrium intermediate. At least three proline isomerization reactions are known to limit their interconversions and lead to a parallel channel mechanism. The simple sequential mechanism deduced for sIGPS reflects the dominance of the on-pathway burst-phase intermediate and the absence of prolyl residues that partition the stable intermediate into kinetically distinguishable species. Comparison of the results for sIGPS and alphaTS demonstrates that the thermodynamic properties and the final steps of the folding reaction are better conserved than the early events. The initial events in folding appear to be more sensitive to the sequence differences between the two TIM barrel proteins.

The crystal structure of indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus in three different crystal forms: effects of ionic strength.

Indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus is a monomeric enzyme with the common (beta/alpha)8-fold. Recently, its three-dimensional structure was solved in an orthorhombic crystal form, grown by using 1.3 M ammonium sulfate as precipitating agent. Here we describe the X-ray structure analysis of two new crystal forms of this enzyme that were obtained at medium and low ionic strength, respectively. Hexagonal crystals with space group P3(1)21 and cell dimensions a = 62.4 A, b = 62.4 A, c = 122.9 A, gamma = 120 degrees grew in 0.1 M Mes buffer at pH 6.0 with 30% polyethylene glycol monomethylether as precipitant and 0.2 M ammonium sulfate as co-precipitant. A second crystal form with space group P2(1)2(1)2(1) and cell constants a = 62.6 A, b = 74.0 A, c = 74.2 A was obtained using polyethylene glycol and ethylene glycol as precipitants in 0.1 M Mes buffer at pH 6.5. Both structures were solved by molecular replacement and refined at 2.5 A and 2.0 A resolution, respectively. Although the global folds are almost identical, alternative conformations are observed in flexible loop regions, mostly stabilized by crystal contacts. In none of the three crystal forms is the so-called phosphate binding site empty, suggesting that this position has high affinity for anions with tetrahedrally arranged oxygen atoms. Differences in ionic strength of the crystallization buffer have only minor effects on number and specificity of intramolecular salt bridges. The crystal packing, on the other hand, seems to be influenced by the ionic strength of the solvent, since the number of intermolecular salt bridges in the low ionic strength crystal forms is significantly higher.