Enzymatic properties of mutant Escherichia coli tryptophan synthase alpha-subunits

Thirty-nine mutant tryptophan synthase alpha subunits have been purified and analyzed (in the presence of the beta 2-subunit) for their enzymatic (kcat, Km) behavior in the reactions catalyzed by the alpha 2.beta 2 complex, the fully constituted form of this enzyme. The mutant alpha subunits, obtained by in vitro random, saturation mutagenesis of the encoding trpA gene, contain single amino acid substitutions at sites within the first 121 residues of the alpha polypeptide. Four categories of altered residues have been tentatively assigned roles in the catalytic functions of this enzyme: 1) catalytic residues (Glu49 and Asp60); 2) residues involved in substrate binding or orientation (Phe22, Thr63, Gln65, Tyr102, and Leu105); 3) residues involved in alpha.beta subunit interactions (Gly51, Pro53, Asp56, Asp60, Pro62, Ala67, Phe72, Thr77, Pro78, Tyr102, Asn104, Leu105, and Asn108); and 4) residues with no apparent catalytic roles. Catalytic residue alterations result in no detectable activity in the alpha-subunit specific reactions. Substrate binding/orientation roles are detected enzymatically primarily as rate defects; alterations only at Tyr102 result in apparent Km effects. alpha.beta interaction roles are detected as rate defects in all tryptophan synthase reactions plus Km increases for the alpha-subunit substrate, indole-3-glycerol phosphate, only when L-serine is present at the beta 2-subunit active site. A substitution at only one site, Asn104, appears to be unique in its potential effect on intersubunit channeling of indole, the product of the alpha-subunit specific reaction, to the beta 2-subunit active site.     

Homodimers of mutant tryptophan synthase alpha-subunits in Escherichia coli.

Tryptophan synthase alpha-subunit from Escherichia coli functionally exists as a heterotetramer of alpha(2)beta(2) with beta-subunit. While wild-type and mutant (F139W, T24M/F139W, and T24L/F139W) alpha-subunits were expressed as a monomer from recombinant plasmids in Escherichia coli, T24A/F139W, T24S/F139W, and T24K/F139W mutant alpha-subunits were abnormally expressed as soluble homodimers in addition to monomers. Monomers of dimer-forming mutant alpha-subunits retain high affinity to beta-subunit, high activity in stimulating catalytic activities of beta-subunit, and nearly intact content of secondary structure, indicating that the global structures of these monomers are identical to that of F139W alpha-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 alpha-subunit folding.     

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

The fluorescence of tyrosine has been used to monitor a folding process of tryptophan synthase alpha-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 approximately 40% each by Tyr(4) and Tyr(115), and to the remaining approximately 20% by Tyr(173) and Tyr(175). Y173F and Y175F mutant proteins showed an increase in their fluorescence intensity by approximately 40% and approximately 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 alpha-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 alpha-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.     

Crystallization and X-ray crystallographic studies of wild-type and mutant tryptophan synthase alpha-subunits from Escherichia coli

The alpha-subunit of Escherichia coli tryptophan synthase (aTS), a component of the tryptophan synthase alpha2beta2 complex, is a monomeric 268-residues protein (Mr = 28,600). alphaTS by itself catalyzes the cleavage of indole-3-glycerol phosphate to glyceraldehyde-3-phosphate and indole, which is converted to tryptophan in tryptophan biosynthesis. Wild-type and P28L/Y173F double mutant alpha-subunits were overexpressed in E. coli and crystallized at 298 K by the hanging-drop vapor-diffusion method. X-ray diffraction data were collected to 2.5 angstroms resolution from the wild-type crystals and to 1.8 angstroms from the crystals of the double mutant, since the latter produced better quality diffraction data. The wild-type crystals belonged to the monoclinic space group C2 (a = 155.64 angstroms, b = 44.54 angstroms, c = 71.53 angstroms and beta = 96.39 degrees) and the P28L/Y173F crystals to the monoclinic space group P21 (a = 71.09 angstroms, b = 52.70, c = 71.52 angstroms, and beta = 91.49 degrees). The asymmetric unit of both structures contained two molecules of aTS. Crystal volume per protein mass (V(m)) and solvent content were 2.15 angstroms3 Da(-1) and 42.95% for the wild-type and 2.34 angstroms3 Da(-1) and 47.52% for the double mutant.     

A molecular characterization of spontaneous frameshift mutagenesis within the trpA gene of Escherichia coli

Spontaneous frameshift mutations are an important source of genetic variation in all species and cause a large number of genetic disorders in humans. To enhance our understanding of the molecular mechanisms of frameshift mutagenesis, 583 spontaneous Trp+ revertants of two trpA frameshift alleles in Escherichia coli were isolated and DNA sequenced. In order to measure the contribution of methyl-directed mismatch repair to frameshift production, mutational spectra were constructed for both mismatch repair-proficient and repair-defective strains. The molecular origins of practically all of the frameshifts analyzed could be explained by one of six simple models based upon misalignment of the template or nascent DNA strands with or without misincorporation of primer nucleotides during DNA replication. Most frameshifts occurred within mononucleotide runs as has been shown often in previous studies but the location of the 76 frameshift sites was usually outside of runs. Mismatch repair generally was most effective in preventing the occurrence of frameshifts within runs but there was much variation from site to site. Most frameshift sites outside of runs appear to be refractory to mismatch repair although the small number of occurrences at most of these sites make firm conclusions impossible. There was a dense pattern of reversion sites within the trpA DNA region where reversion events could occur, suggesting that, in general, most DNA sequences are capable of undergoing spontaneous mutational events during replication that can lead to small deletions and insertions. Many of these errors are likely to occur at low frequencies and be tolerated as events too costly to prevent or repair. These studies also revealed an unpredicted flexibility in the primary amino acid sequence of the trpA product, the alpha subunit of tryptophan synthase.     

Substrate interactions with the alpha-subunit of the Escherichia coli tryptophan synthase. A study of the activity of mutant alpha-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.     

Solubilization and renaturation of overexpressed aggregates of mutant tryptophan synthase alpha-subunits

Certain Escherichia coli tryptophan synthase mutant alpha-subunits encoded from mutagenized trpA-containing plasmids were overexpressed as insoluble aggregates which were seen as large, intracellular inclusion bodies. The insoluble aggregates were solubilized to various degrees by several neutral, chaotropic salts. The order of effectiveness of these salts (KSCN, NaI greater than NaNO3, LiBr greater than CaCl2) followed that for the Hofmeister series. Optimum conditions for the use of KSCN resulted in a maximum 70 to 75% solubilization of the aggregate forms for all mutant alpha-subunits examined. Removal of KSCN by dialysis resulted in the recovery of biological activity and of certain characteristic structural properties. Such salts may be a useful alternative for other recombinant protein aggregates which resist complete renaturation by commonly used treatments with guanidine or urea.     

Solubilization and renaturation of overexpressed aggregates of mutant tryptophan synthase alpha-subunits.

Certain Escherichia coli tryptophan synthase mutant alpha-subunits encoded from mutagenized trpA-containing plasmids were overexpressed as insoluble aggregates which were seen as large, intracellular inclusion bodies. The insoluble aggregates were solubilized to various degrees by several neutral, chaotropic salts. The order of effectiveness of these salts (KSCN, NaI greater than NaNO3, LiBr greater than CaCl2) followed that for the Hofmeister series. Optimum conditions for the use of KSCN resulted in a maximum 70 to 75% solubilization of the aggregate forms for all mutant alpha-subunits examined. Removal of KSCN by dialysis resulted in the recovery of biological activity and of certain characteristic structural properties. Such salts may be a useful alternative for other recombinant protein aggregates which resist complete renaturation by commonly used treatments with guanidine or urea.     

Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits.

Random chemical mutagenesis, in vitro, of the 5' portion of the Escherichia coli trpA gene has yielded 66 mutant alpha subunits containing single amino acid substitutions at 49 different residue sites within the first 121 residues of the protein; this portion of the alpha subunit contains four of the eight alpha helices and three of the eight beta strands in the protein. Sixty-two of the subunits were examined for their heat stabilities by sensitivity to enzymatic inactivation (52 degrees C for 20 min) in crude extracts and by differential scanning calorimetry (DSC) with 29 purified proteins. The enzymatic activities of mutant alpha subunits that contained amino acid substitutions within the alpha and beta secondary structures were more heat labile than the wild-type alpha subunit. Alterations only in three regions, at or immediately C-terminal to the first three beta strands, were stability neutral or stability enhancing with respect to enzymatic inactivation. Enzymatic thermal inactivation appears to be correlated with the relative accessibility of the substituted residues; stability-neutral mutations are found at accessible residual sites, stability-enhancing mutations at buried sites. DSC analyses showed a similar pattern of stabilization/destabilization as indicated by inactivation studies. Tm differences from the wild-type alpha subunit varied +/- 7.6 degrees C. Eighteen mutant proteins containing alterations in helical and sheet structures had Tm's significantly lower (-1.6 to -7.5 degrees C) than the wild-type Tm (59.5 degrees C). In contrast, 6 mutant alpha subunits with alterations in the regions following beta strands 1 and 3 had increased Tm's (+1.4 to +7.6 degrees C). Because of incomplete thermal reversibilities for many of the mutant alpha subunits, most likely due to identifiable aggregated forms in the unfolded state, reliable differences in thermodynamic stability parameters are not possible. The availability of this group of mutant alpha subunits which clearly contain structural alterations should prove useful in defining the roles of certain residues or sequences in the unfolding/folding pathway for this protein when examined by urea/guaninidine denaturation kinetic analysis.     

Relative activities and stabilities of mutant Escherichia coli tryptophan synthase alpha subunits

In vitro mutagenesis of the Escherichia coli trpA gene has yielded 66 mutant tryptophan synthase alpha subunits containing single amino acid substitutions at 49 different residue sites and 29 double and triple amino acid substitutions at 16 additional sites, all within the first 121 residues of the protein. The 66 singly altered mutant alpha subunits encoded from overexpression vectors have been examined for their ability to support growth in trpA mutant host strains and for their enzymatic and stability properties in crude extracts. With the exception of mutant alpha subunits altered at catalytic residue sites Glu-49 and Asp-60, all support growth; this includes those (48 of 66) that have no enzymatic defects and those (18 of 66) that do. The majority of the enzymatically defective mutant alpha subunits have decreased capacities for substrate (indole-3-glycerol phosphate) utilization, typical of the early trpA missense mutants isolated by in vivo selection methods. These defects vary in severity from complete loss of activity for mutant alpha subunits altered at residue positions 49 and 60 to those, altered elsewhere, that are partially (up to 40 to 50%) defective. The complete inactivation of the proteins altered at the two catalytic residue sites suggest that, as found via in vitro site-specific mutagenesis of the Salmonella typhimurium tryptophan synthetase alpha subunit, both residues probably also participate in a push-pull general acid-base catalysis of indole-3-glycerol phosphate breakdown for the E. coli enzyme as well. Other classes of mutant alpha subunits include some novel types that are defective in their functional interaction with the other tryptophan synthetase component, the beta 2 subunit. Also among the mutant alpha subunits, 19 were found altered at one or another of the 34 conserved residue sites in this portion of the alpha polypeptide sequence; surprisingly, 10 of these have wild-type enzymatic activity, and 16 of these can satisfy growth requirements of a trpA mutant host. Heat stability and potential folding-rate alterations are found in both enzymatically active and defective mutant alpha subunits. Tyr-4. Pro-28, Ser-33, Gly-44, Asp-46, Arg-89, Pro-96, and Cys-118 may be important for these properties, especially for folding. Two regions, one near Thr-24 and another near Met-101, have been also tentatively identified as important for increasing stability.     

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

The alpha-subunit of tryptophan synthase (alphaTS) 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 alpha-subunit of tryptophan synthase from Escherichia coli at 2.8 and 1.8A resolution, respectively. The structure of wild-type alphaTS from E. coli was similar to that of the alpha(2)beta(2) complex structure from Salmonella typhimurium. As compared with both structures, the conformational changes are mostly in the interface of alpha- and beta-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 alpha-subunit core to the alpha-subunit surface involved in interactions with the beta-subunit and increases structural stability.     

In vitro mutagenesis of Escherichia coli citrate synthase to clarify the locations of ligand binding sites

In vitro mutagenesis techniques have been used to investigate two structure-function questions relating to the allosteric citrate synthase of Escherichia coli. The first question concerns the binding site of alpha-keto-glutarate, which is a structural analogue of the substrate oxaloacetate and yet has been suggested to be an allosteric inhibitor of the enzyme. Using oligonucleotide-directed mutagenesis of the cloned E. coli citrate synthase gene, we prepared missense mutants, designated CS226H----Q and CS229H----Q, in which histidine residues at positions 226 and 229, respectively, were replaced by glutamine. In the homologous pig heart citrate synthase it is known (Wiegand, G., and Remington, S. J. (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 97-117) that the equivalent of His-229 helps to bind oxaloacetate, while the equivalent of His-226 is nearby. Kinetic and ligand binding measurements showed that CS226H----Q had a reduced affinity for oxaloacetate and alpha-ketoglutarate, while CS229H----Q bound oxaloacetate even less effectively, and was not inhibited by alpha-ketoglutarate at all under our conditions. This parallel loss of binding affinities for oxaloacetate and alpha-ketoglutarate, in two mutants altered in residues at the active site of E. coli citrate synthase, strongly suggests that inhibition of this enzyme by alpha-ketoglutarate is not allosteric but occurs by competitive inhibition at the active site. The second question investigated was whether the known inhibition by acetyl-CoA of binding of NADH, an allosteric inhibitor of E. coli citrate synthase, occurs heterotropically, as an indirect result of acetyl-CoA binding at the active site, or directly, by competition at the allosteric NADH binding site. Using existing restriction sites in the cloned E. coli citrate synthase gene, we prepared a deletion mutant which lacked 24 amino acids near what is predicted to the acetyl-CoA-binding portion of the active site. The mutant protein was inactive, and acetyl-CoA did not bind to the active site but still inhibited NADH binding. Thus acetyl-CoA can interact with both the allosteric and the active sites of this enzyme.     

Escherichia coli mutant trpA34 has an Asp----Asn change at active site residue 60 of the tryptophan synthetase alpha chain

Asp-60 is believed to be a catalytically essential residue of the tryptophan synthetase alpha chain of Escherichia coli (Nagata, S., Hyde, C.C., and Miles, E.W. (1989) J. Biol. Chem. 264, 6288-6296). Surprisingly, mutations altering Asp-60 were not observed in the many trpA missense mutants characterized in the 1960s. However, there was one genetic class of trpA missense mutants, represented by trpA34, for which protein structure analyses failed to detect an amino acid substitution. DNA sequence analyses have now shown that the trpA34 mutation was in codon 60 and that it resulted in replacement of Asp-60 by Asn. This finding provides additional support for the conclusion that the tryptophan synthetase alpha chain contains only a small number of absolutely essential residues.     

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

Guanidine hydrochloride-induced denaturation and thermal denaturation of three kinds of tryptophan synthase alpha subunit have been compared by circular dichroism measurements. The three alpha subunits are from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid in which the C-terminal domain comes from E. coli (alpha-2 domain) and the N-terminal domain comes from S. typhimurium (alpha-1 domain). Analysis of denaturation by guanidine hydrochloride at 25 degrees C showed that the alpha-2 domain of S. typhimurium was more stable than the alpha-2 domain of E. coli, but the alpha-1 domain of S. typhimurium was less stable than the alpha-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 degrees C and coincided with that obtained from guanidine hydrochloride-induced denaturation.     

In vitro construction and characterization of phoA-lacZ gene fusions in Escherichia coli.

Using recombinant DNA techniques, we have constructed phoA-lacZ gene fusions. Two of the fusions encode hybrid proteins containing approximately half of alkaline phosphatase at the amino terminus joined to beta-galactosidase. For the one fusion strain analyzed in detail, it was shown that the hybrid protein is found in the membrane fraction of cells. In its membrane location, the beta-galactosidase activity of the hybrid is not sufficient to support cell growth on lactose. Unexpectedly, fusions containing phoA and lacZ joined in the wrong translational reading frame were also obtained. These fusions direct the phosphate-regulated synthesis of beta-galactosidase, apparently via a translation restart mechanism. Thus, when gene fusions are constructed, the presence of properly regulated beta-galactosidase activity does not necessarily indicate that a hybrid protein is being produced.     

Fusion of trpB and trpA of Escherichia coli yields a partially active tryptophan synthetase polypeptide

The separate alpha and beta polypeptides of the tryptophan synthetase of bacteria are represented in fungi by a fusion polypeptide in which the first third is homologous to bacterial alpha chains and the remainder is homologous to bacterial beta chains. In the yeast polypeptide, a short nonhomologous "connector" joins the two homologous segments. The chromosomal order of all bacterial genes that specify tryptophan synthetase beta and alpha chains, respectively, is trpB-trpA. Fusion of these genes in their present arrangement would result in the synthesis of a polypeptide with a segmental order, N-beta-alpha-C, opposite that observed in fungi. To investigate possible explanations for the apparent transposition that occurred in the evolution of the fungal gene we have made two fusions of trpB and trpA of Escherichia coli in their natural orientation. We find that the fusion proteins are synthesized but both are less active catalytically than the wild type bacterial protein. In addition, the fusion proteins associate abnormally, they are activated only slightly by wild type alpha or beta 2, and they are less sensitive than the wild type protein to inhibition by antibodies to alpha or beta 2. The fusion proteins have normal substrate affinities. Our findings suggest that the altered structures of the fusion proteins affect catalytic ability and the locations of the alpha and/or beta chain combining sites. This structural distortion may have prevented the natural selection of direct gene fusions during the course of the fungal gene's evolution.     

Isolation and characterization of an Escherichia coli mutant having temperature-sensitive farnesyl diphosphate synthase.

The screening of a collection of highly mutagenized strains of Escherichia coli for defects in isoprenoid synthesis led to the isolation of a mutant that had temperature-sensitive farnesyl diphosphate synthase. The defective gene, named ispA, was mapped at about min 10 on the E. coli chromosome, and the gene order was shown to be tsx-ispA-lon. The mutant ispA gene was transferred to the E. coli strain with a defined genetic background by P1 transduction for investigation of its function. The in vitro activity of farnesyl diphosphate synthase of the mutant was 21% of that of the wild-type strain at 30 degrees C and 5% of that at 40 degrees C. At 42 degrees C the ubiquinone level was lower (66% of normal) in the mutant than in the wild-type strain, whereas at 30 degrees C the level in the mutant was almost equal to that in the wild-type strain. The polyprenyl phosphate level was slightly higher in the mutant than in the wild-type strain at 30 degrees C and almost the same in both strains at 42 degrees C. The mutant had no obvious phenotype regarding its growth properties.