Indoleglycerol Phosphate Research Articles

Up-promoter mutations in the trpBA operon of Pseudomonas aeruginosa

In Pseudomonas aeruginosa, the operon encoding tryptophan synthase (trpBA) is positively regulated by the TrpI protein and an intermediate in tryptophan biosynthesis, indoleglycerol phosphate (InGP). A gene fusion in which the trpBA promoter directs expression of the Pseudomonas putida xylE gene was constructed. By using a P. putida F1 todE mutant carrying this fusion on a plasmid, three cis-acting mutations that increased xylE expression enough to allow the todE strain to grow on toluene were isolated. The level of xylE transcript from the trpBA promoter was increased in all three mutants. All three mutations are base substitutions located in the -10 region of the trpBA promoter; two of these mutations make the promoter sequence more like the Escherichia coli RNA polymerase sigma 70 promoter consensus sequence. The activities of the wild-type and mutant trpBA promoters, as monitored by xylE expression, were assayed in P. putida PpG1 and in E. coli. The up-regulatory phenotypes of the mutants were maintained in the heterologous backgrounds, as was trpI and InGP dependence. These results indicate that the P. aeruginosa trpBA promoter has the key characteristics of a typical E. coli positively regulated promoter. The results also show that the P. aeruginosa and P. putida trpI activator gene products are functionally interchangeable.

Serine modulates substrate channeling in tryptophan synthase. A novel intersubunit triggering mechanism

Tryptophan synthase, an alpha 2 beta 2 complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the alpha subunit to the active site of the beta subunit. We now examine the kinetics of substrate channeling by tryptophan synthase directly by chemical quench-flow and stopped-flow methods. The conversion of indole-3-glycerol phosphate (IGP) to tryptophan at the active site proceeds at a rate of 24 s-1, which is limited by the rate of cleavage of IGP to produce indole (alpha reaction). In a single turnover experiment monitoring the conversion of radiolabeled IGP to tryptophan, only a trace of indole is detectable (less than or equal to 1% of the IGP), implying that the reaction of indole to form tryptophan must be quite fast (greater than or equal to 1000 s-1). The rate of reaction of indole from solution is much too slow (40 s-1 under identical conditions) to account for the negligible accumulation of indole in a single turnover. Therefore, the indole produced at the alpha site must be rapidly channeled to the beta site, where it reacts with serine to form tryptophan: channeling and the reaction of indole to form tryptophan must each occur at rates greater than or equal to 1000 s-1. Steady-state turnover is limited by the slow rate of tryptophan release (8 s-1). In the absence of serine, the cleavage of IGP to indole is limited by a change in protein conformation at a rate of 0.16 s-1. When the alpha beta reaction is initiated by mixing enzyme with IGP and serine simultaneously, there is a lag in the cleavage IGP and formation of tryptophan. The kinetics of the lag correspond to the rate of formation of the aminoacrylate in the reaction of serine with pyridoxal phosphate at the beta site, measured by stopped-flow methods (45 s-1). There is also a change in protein fluorescence, suggestive of a change in protein conformation, occurring at the same rate. Substitution of cysteine for serine leads to a longer lag in the kinetics of IGP cleavage and a correspondingly slower rate of formation of the aminoacrylate (6 s-1). Thus, the reaction of serine at the beta site modulates the alpha reaction such that the formation of the aminoacrylate leads to a change in protein conformation that is transmitted to the alpha site to enhance the rate of IGP cleavage 150-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro determination of the effect of indoleglycerol phosphate on the interaction of purified TrpI protein with its DNA-binding sites

Expression of the trpBA gene pair of Pseudomonas aeruginosa is regulated by the endogenous level of indoleglycerol phosphate (InGP) and the trpI gene product. The TrpI protein binds to the -77 to -32 region of the trpBA promoter. This region is divisible into two sites: site I, which is protected by TrpI in the presence and absence of InGP; and site II, which is protected by TrpI only in the presence of InGP. Recently, the trpI gene was subcloned into an expression vector and the protein was overproduced in Escherichia coli. The TrpI protein was purified to 80 to 95% purity. The molecular weight of native TrpI protein is estimated to be 129,000 by gel exclusion chromatography, and therefore it is likely a tetramer composed of 31,000-dalton monomers. Gel retardation assays with the purified TrpI protein demonstrated that InGP increases the affinity of TrpI for sites I and II approximately 17- and 14-fold, respectively. Binding of TrpI to site I is site II independent. However, the protein has low intrinsic affinity for site II and its binding to site II is site I dependent. Therefore, binding of TrpI to site II probably requires its interaction with a second TrpI molecule at site I.

Mechanism of the physiological reaction catalyzed by tryptophan synthase from Escherichia coli

The physiological synthesis of L-tryptophan from indoleglycerol phosphate and L-serine catalyzed by the alpha 2 beta 2 bienzyme complex of tryptophan synthase requires spatial and dynamic cooperation between the two distant alpha and beta active sites. The carbanion of the adduct of L-tryptophan to pyridoxal phosphate accumulated during the steady state of the catalyzed reaction. Moreover, it was formed transiently and without a lag in single turnovers, and glyceraldehyde 3-phosphate was released only after formation of the carbanion. These and further data prove first that the affinity for indoleglycerol phosphate and its cleavage to indole in the alpha subunit are enhanced substantially by aminoacrylate bound to the beta subunit. This indirect activation explains why the turnover number of the physiological reaction is larger than that of the indoleglycerol phosphate cleavage reaction. Second, reprotonation of nascent tryptophan carbanion is rate limiting for overall tryptophan synthesis. Third, most of the indole generated in the active site of the alpha subunit is transferred directly to the active site of the beta subunit and only insignificant amounts pass through the solvent. Comparison of the single turnover rate constants with the known elementary rate constants of the partial reactions catalyzed by the alpha and beta active sites suggests that the cleavage reaction rather than the transfer of indole or its condensation with aminoacrylate is rate limiting for the formation of nascent tryptophan.

Improved method for measuring the catalytic activity of tryptophan synthase α subunit in cell extracts

An improved method has been developed for measuring the catalytic activity of tryptophan synthase α-subunit in cell extracts using the indole-3-glycerol phosphate (InGP) → tryptophan reaction. The method involves the chemical and enzymatic synthesis of the substrate InGP immediately before use and avoids the preparation of salt-free hydroxylamine. The method is more convenient, safer and more reliable than the traditional method employing the InGP → indole reaction.

Deletion analysis of domain independence in the TRP1 gene product of Neurospora crassa

The trifunctional TRP1 gene from Neurospora crassa (N-TRP1) was subcloned into the yeast-Escherichia coli shuttle vector YEp13 and expressed in Saccharomyces cerevisiae. The three activities of the N-TRP1 gene product were detected in yeast mutants that lacked either N-(5'-phosphoribosyl) anthranilate (PRA) isomerase or both the glutamine amidotransferase function of anthranilate synthase and indole-3-glycerol phosphate (InGP) synthase. The protein was detected on immunoblots only as the full length 83 kda product indicating that the trifunctional gene product was expressed in yeast primarily in a fully active, undegraded form. By placing the subcloned N-TRP1 gene under the control of the inducible PHO5 promoter from yeast, the expression of all three activities was increased to more than ten fold that of wild-type yeast and the overproduced protein could be visualized by SDS-polyacrylamide gel electrophoresis of crude extract and Coomassie Blue staining. Using the expression system described the effect of selective deletion of regions of the coding sequence of the N-TRP1 gene on expression of the three activities was tested. Expression of either the F- or C-domains, catalyzing respectively the PRA isomerase or InGP synthase activities, did not depend on the presence of the other domain in the active polypeptide. Furthermore, normal dimer formation occurred with a protein active for InGP synthase in a deletion derivative lacking most of the PRA isomerase domain, ruling out the hypothesis that interaction between the active site regions for PRA isomerase and InGP synthase accounted for dimer formation in the trifunctional product.

The roles of indoleglycerol phosphate and the Trpl protein in the expression oftrpBA fromPseudomonas aeruginosa

The TrpI protein belongs to the LysR-family of procaryotic regulatory proteins. Members of this family share a characteristic similarity of their N-terminal amino acid sequences, and many of them are activators of divergently transcribed genes or operons. In Pseudomonas aeruginosa, the genes for tryptophan synthase, trpBA, are regulated by indoleglycerol phosphate (InGP) and TrpI. We demonstrate here that in the absence of InGP, the binding site of TrpI is located in the -52 to -77 region of the trpBA promoter; in the presence of InGP, the binding region is extended to the -32 region. In addition, two major, slow moving protein-DNA complexes are seen in gel retardation assays: the faster moving complex is formed in the absence of InGP and the amount of the slower moving complex is greatly enhanced in the presence of InGP. These results suggest that the binding of a second TrpI protein molecule, promoted by InGP, plays a crucial role in activating the expression of the trpBA gene pair.

Three-dimensional structure of the tryptophan synthase alpha 2 beta 2 multienzyme complex from Salmonella typhimurium.

The three-dimensional structure of the alpha 2 beta 2 complex of tryptophan synthase from Salmonella typhimurium has been determined by x-ray crystallography at 2.5 A resolution. The four polypeptide chains are arranged nearly linearly in an alpha beta beta alpha order forming a complex 150 A long. The overall polypeptide fold of the smaller alpha subunit, which cleaves indole glycerol phosphate, is that of an 8-fold alpha/beta barrel. The alpha subunit active site has been located by difference Fourier analysis of the binding of indole propanol phosphate, a competitive inhibitor of the alpha subunit and a close structural analog of the natural substrate. The larger pyridoxal phosphate-dependent beta subunit contains two domains of nearly equal size, folded into similar helix/sheet/helix structures. The binding site for the coenzyme pyridoxal phosphate lies deep within the interface between the two beta subunit domains. The active sites of neighboring alpha and beta subunits are separated by a distance of about 25 A. A tunnel with a diameter matching that of the intermediate substrate indole connects these active sites. The tunnel is believed to facilitate the diffusion of indole from its point of production in the alpha subunit active site to the site of tryptophan synthesis in the beta active site and thereby prevent its escape to the solvent during catalysis.

Isolation of auxotrophs and analysis of regulation of tryptophan biosynthesis in Zymomonas mobilis

Tryptophan auxotrophs were isolated and used to analyze the regulation of tryptophan biosynthesis in Zymomonas mobilis. Twelve tryptophan auxotrophs were cassified as trp E, B or A based on accumulation of, or growth on, indole and anthranilic acid. Trp B mutants were found to accumulate indole when grown on limiting, but not on excess tryptophan, suggesting that tryptophan plays a role in regulating its biosynthesis. Tryptophan synthase and indoleglycerol phosphate synthase specific activities were measured in the wild-type strain and two trp mutants grown in limiting or excess tryptophan. Neither activity was repressed by exogenous tryptophan.

Site-specific mutagenesis of the alpha subunit of tryptophan synthase from Salmonella typhimurium. Changing arginine 179 to leucine alters the reciprocal transmission of substrate-induced conformational changes between the alpha and beta 2 subunits.

Arginine 179 of the alpha subunit of tryptophan synthase of Salmonella typhimurium was changed to leucine by site-directed mutagenesis. The mutant alpha subunit was expressed in S. typhimurium, purified and crystallized as the alpha 2 beta 2 complex, and characterized by kinetic studies under steady-state reaction conditions. The rate of cleavage of indole 3-glycerol phosphate (alpha reaction) is reduced by 60% in the mutant alpha 2 beta 2 complex, whereas the rate of L-tryptophan synthesis from indole and L-serine (beta reaction) is unchanged. Thus, arginine 179 is not obligatory for catalysis, for binding of indole 3-glycerol phosphate, or for interaction of the alpha and beta 2 subunits. However, changing arginine 179 to leucine does have striking effects on ligand-dependent properties of this multienzyme complex. Ligands of the alpha subunit (DL-alpha-glycerophosphate and indole 3-propanol phosphate) which strongly inhibit the beta reaction of the native alpha 2 beta 2 complex have a slight stimulatory effect on the beta reaction of the mutant alpha 2 beta 2 complex. Likewise, L-serine, a ligand of the beta subunit which produces a 5-fold reduction in the Km for the alpha ligand indole 3-glycerol phosphate in the native alpha 2 beta 2 complex, has no effect on the mutant alpha 2 beta 2 complex. These results suggest that arginine 179 of the alpha subunit plays a role in the reciprocal transmission of substrate-induced conformational changes which occur between native alpha and beta 2 subunits in the alpha 2 beta 2 complex.

Organization of the functional domains of anthranilate synthase from Neurospora crassa. Limited proteolysis studies.

Treatment of the multifunctional alpha 2 beta 2 anthranilate synthase complex of Neurospora crassa with elastase produced two fragments of the complex, one possessing anthranilate synthase activity and the other having both indole-3-glycerol phosphate (InGP) synthase and N-(5'-phosphoribosyl)anthranilate (PRA) isomerase activities. Sequencing the NH2 terminus of the InGP synthase-PRA isomerase fragment revealed that cleavage was between positions 237 and 238 of the beta-subunit within a segment of the polypeptide chain which links the glutamine-binding (G) domain with the InGP synthase-PRA isomerase domains. The fragment containing anthranilate synthase activity has a molecular weight of 98,000, as estimated by gel filtration, and is composed of an apparently intact alpha-subunit (70 kDa) associated with the G-domain fragment (29 kDa) derived from the beta-subunit. The alpha X G-domain complex was resistant to further degradation by elastase. When either the alpha 2 beta 2 complex or the alpha X G-domain complex was incubated with trypsin, the alpha-subunit was degraded to a 66-kDa alpha-fragment with reduced enzymatic activity, which was resistant to further cleavage. In contrast, incubation of alpha-subunit alone with either elastase or trypsin resulted in its complete degradation, indicating that association of the alpha-subunit with either G-domain or beta-subunit protected the alpha-subunit from this extensive degradation. A model for the anthranilate synthase complex is proposed in which the trifunctional beta-subunit forms a dimer by the self-association of the InGP synthase-PRA isomerase domains; the G-domain is connected to the InGP synthase-PRA isomerase domain by a relatively disordered region of the peptide chain which, in the alpha 2 beta 2 complex, remains susceptible to proteases; and neither alpha-subunit nor G-domain significantly self-associates.

Isolation of the DNA sequence coding indole-3-glycerol phosphate synthetase and phosphoribosylanthranilate isomerase of Schizophyllum commune.

A Schizophyllum gene library was made in plasmid pRK9. Plasmids from this library were tested for their ability to complement several auxotrophic mutations of Escherichia coli. The goal was to isolate a Schizophyllum auxotrophic gene that could be used to transform a corresponding Schizophyllum auxotrophic mutant to prototrophy. Complementation was observed only for E. coli trpC indole 3-glycerol phosphate synthetase (IGPS) and phosphoribosyl-anthranilate isomerase (PRAI) mutations. Plasmids with a Schizophyllum sequence coding for both IGPS and PRAI activities were recovered from E. coli transformants. Expression of the Schizophyllum gene (TRP1) in E. coli is probably dependent on the Serratia marcescens promoter of plasmid pRK9. The DNA sequence containing the Schizophyllum TRP1 gene was not obviously rearranged in cloning.

Thermodynamics of the reactions catalyzed by the multifunctional enzyme complex tryptophan synthase

The intrinsic enthalpy changes (corrected for hydration of d-glyceraldehyde 3-phosphate) for the reactions catalyzed by the α and β 2 subunits of tryptophan synthase from Escherichia coli have been determined calorimetrically. Cleavage of indoleglycerol phosphate (α reaction) was found to be associated with a Δ H value of 54.0 ± 2.5 kJ mol −1 while condensation of indole with l-serine (β reaction) involved −80.3 ± 4.6 kJ mol −1′. By direct determination of the enthalpy concomitant with the overall synthesis of tryptophan from indoleglycerol phosphate and l-serine an enthalpy value of −13.4 ± 5.6 kJ mol −1 was observed. In view of the uncertainties of the literature data used for calculation of the hydration contribution, the agreement between the directly measured Δ H value of the overall reaction and the sum of the enthalpies of the α and β reactions is fair. Deamination of l-serine, a side reaction catalyzed preferentially by the isolated β 2 pyridoxal 5′-phosphate 2 subunit, was shown to be associated with an enthalpy change of −7.3 ± 0.4 kJ mol −1.

5-Azidoindole binding to the Escherichia coli tryptophan synthase α2β2 complex

The tryptophan synthase α 2 β 2 complex catalyzes tryptophan (Trp) biosynthesis from serine plus either indole (IN) or indole-3-glycerol phosphate (InGP). The photoreactive 5-azido analog in IN (AzIN), itself a substrate in the dark, was utilized to examine the substrate binding sites on this enzyme. When irradiated with AzIN at concentrations approaching IN saturation for the IN → Trp activity (0.1 m m), in the absence of serine, the enzyme was increasingly inactivated (up to 70–80%) concomitant with the progressive binding of a net of 2 mol AzIN per αβ equivalent. Little or no cooperativity in the binding of the 2 mol AzIN was observed. In contrast, there was minimal effect on the IN → InGP activity. Under these conditions AzIN appeared to be incorporated equally into each subunit. No significant inactivation nor binding occurred in the presence of serine. A quantitatively similar inactivation of InGP → Trp activity was observed over the same AzIN concentration range, suggesting common IN sites for Trp biosynthesis from either indole substrate. At higher concentrations (0.1–0.7 m m), no further inactivation occurred, although there was extensive additional binding (up to 10 mol/αβ equivalent). These data are consistent, although more clear-cut quantitatively, with the high- and low-affinity sites proposed from equilibrium dialysis studies. AzIN binding studies utilizing the isolated β 2 subunit confirmed earlier reports suggesting the existence of many nonspecific IN binding sites on this subunit.

The Quarternary Structure of Tryptophan Synthase from Escherichia coli

Single cysteine residues on the separated α and β2 subunits of tryptophan synthase were labelled with various fluorescent ligands suitable for fluorescence energy transfer measurements. Labelled α subunits, reconstituted into an αL2β2 complex, retained more than 75% activity. Labelled β2 subunits, either free or reconstituted into an α2βL2′ complex, retained 10–30% of their activity, depending on the label. The coenzyme pyridoxal 5′‐phosphate served as a natural fluorescent label of the active site of the β2 subunit. Different combinations of labelled subunits resulted in doubly labelled αL2βL2′ complexes that allowed five independent distances to be estimated by fluorescence energy transfer: within one β protomer, CysL–pyridoxal 5′‐phosphate = 1.4–3.2 nm; between two β protomers, CysL–CysL= 0.5–2.3 nm; between one α protomer and a β protomer, CysL–pyridoxal 5′‐phosphate = 1.9–3.2 nm, CysL–CysL= 3.1–4.4 nm; between two α protomers, CysL–CysL= 3.1–3.9 nm. Translational frictional ratios obtained from measurements of sedimentation and diffusion constants and partial specific volumes provided independent information on the shapes of the different particles in solution. Both the α and the β protomer are known to consist of two globular domains of known molecular weight. These were assumed to be approximately spherical and used to construct models first of the α and β2 subunits and then of the α2β2 complex. The data on specific distances and shape factors, together with considerations of symmetry and compactness allow many possible spatial arrangements of the domains to be eliminated. The radii of gyration, maximum dimensions and hydrated volumes calculated for the best models of the α and β2 subunits and the α2β2 complex agree reasonably well with the results of independent small‐angle X‐ray scattering studies [Wilhelm et al. (1982) Eur. J. Biochem. 129, 51–56. The active sites responsible for cleavage of indoleglycerol phosphate to indole and for condensation of indole with L‐serine are approximately 2 nm apart, ruling out the composite active sites hypothesis.

Cloning of the trp-1 gene from neurospora crassa by complementation of a trpC mutation in Escherichia coli.

Studies with a hybrid plasmid containing 4.0 kilobase pairs of Neurospora crassa DNA cloned into plasmid pBR322 indicated that the plasmid restored to prototrophy a trpC mutant of Escherichia coli which lacked phosphoribosyl anthranilate isomerase but not a trpC mutant which lacked indole glycerol phosphate synthetase, that the relevant transcription was initiated at a promoter within the N. crassa DNA, and that the phosphoribosyl anthranilate isomerase could be specified by a subcloned segment of the original DNA.

Organisation of the complex locus trp1 in the fission yeast Schizosaccharomyces pombe

65 trp1(-) alleles of Schizosaccharomyces pombe have been analysed for their interallelic complementation pattern, suppressibility by nonsense suppressors and position of the corresponding mutation site on an intragenic map of the trp1 locus. In addition to the three complementation classes previously described (Schweingruher and Dietrich 1973) as defective in phosphoribosylanthranilate isomerase (trp1A), indole glycerolphosphate synthetase (trp1B) and anthranilate synthetase (trp1C), two new complementation classes, trp1BC and trp1ABC, were found. The former is represented by a single allele which can only complement trp1A mutants. The latter is represented by six alleles which fail to complement tester mutants of the trp1A, trp1B or trp1C class. Classes trp1A, trp1B and trp1C correspond to mutations in three nonoverlapping regions mapping in the order trp1A, trp1B and trp1C All the alleles of the trp1BC and trp1ABC classes correspond to mutations in the trp1C region. Nonsense alleles of the opal (UGA) or ochre (UAA) type were found in the trp1A (2 alleles out of 17) and trp1ABC (5 alleles out of 6) classes only. These data indicate that the trp1 locus is transcribed as a single messenger RNA with transcription starting from the trp1C region. This messenger is probably translated in a single, multifunctional polypeptide, or at most in two polypeptides coded for by the trp1B-trp1C and by the trp1A regions. In addition, the polar effect of nonsense mutations in the trp1C region is cancelled by rare spontaneous mutations occuring at or very near the trp1 locus, which may act by creating an internal single for the initiation of transcription and/or translation.

Genetic evidence for a positive-acting regulatory factor mediating induction in the tryptophan pathway of Pseudomonas aeruginosa

A series of deletions of most or all of trpA, the distal member of the Pseudomonas aeruginosa tryptophan synthase structural gene pair, was constructed by BAL31 nuclease digestion and religation of a suitable plasmid. The residual trpB gene showed normal regulation, virtually ruling out the hypothesis of autogenous regulation proposed earlier on the basis of indirect evidence. On the other hand, SacII-mediated deletion of either or both of two small segments upstream from the trpBA promoter resulted in a fixed, low level of expression of the tryptophan synthase genes. Complementation studies implicated this region as the source of a diffusable, positive-acting regulatory factor responsible for the induction of the tryptophan synthase genes by their substrate, indoleglycerol phosphate.

Tryptophan synthase and production of L-tryptophan in regulatory mutants.

A 5-fluorotryptophan-resistant mutant of Brevibacterium flavum, No. 187, accumulated 2.6 g of indole 3-glycerol (InG) in addition to 8.0 g of l-tryptophan per liter in the culture medium. The addition of l-serine to the medium decreased the accumulation of InG and increased that of l-tryptophan up to a concentration of 10.3 g/liter, while the addition of l-tryptophan increased the InG accumulation, suggesting that InG was formed by hydrolysis of indole 3-glycerol phosphate (InGP), the substrate of tryptophan synthase (TS) which catalyzed the final step reaction of tryptophan biosynthesis. Then, in order to examine the mechanism of the InG accumulation, TS was purified from tryptophan auxotroph, TA-60. The reaction mechanism of TS was Ordered Bi Bi with Km’s of 0.63 and 0.038 mm for serine and InGP, respectively. Tryptophan, a product of the TS reaction, inhibited TS competitively with respect to serine and the Ki for tryptophan was estimated to be 2.0 mm. On the other hand, anthranilate synthase (AS), the...

Ordering tryptophan synthase genes of Pseudomonas aeruginosa by cloning in Escherichia coli.

The Pseudomonas aeruginosa tryptophan synthase genes, trpA and trpB, which are induced by their substrate indoleglycerol phosphate, were cloned along with their controlling region into the BamHI site of pBR322 to produce the 10.7-megadalton plasmid pZAZ5. SalI partial digestion and ligation yielded a smaller plasmid, pZAZ167, with the chromosomal insert reduced in size from 8.1 to 3.4 megadaltons. Both pZAZ5 and pZAZ167 display Pseudomonas-like regulation of the trpA and trpB genes. Deletion of an EcoRI fragment or a BglII fragment from pZAZ167 yielded plasmids pZAZ168 and pZAZ169; the former expresses trpB but not trpA, and the latter has lost both activities. A deleted form of pZAZ5 designated pZAZ101 was obtained by excising a BglII-BamHI segment and religating the trip gene segment in the opposite orientation. This plasmid expresses trpA and trpB constitutively. The physical maps of these plasmids establish the gene order: promoter-trpB-trpA.