Indoleglycerol Phosphate Research Articles

Subunit structure of anthranilate synthetase from Neurospora crassa.

Freshly purified preparations of anthranilate synthetase complex from Neurospora crassa appeared to be homogeneous on polyacrylamide disc gels and were composed of two distinct subunits, 94,000 and 70,000 daltons, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Carboxymethylation of the complex or treatment with guanidine hydrochloride and urea before sodium dodecyl sulfate treatment did not alter the subunit pattern. When the purified complex was iodinated with 125I- or methylated with [14C]dimethylsulfate, no labeled components other than the two subunits stained with Coomassie blue were detected after electrophoresis in the presence of sodium dodecyl sulfate. Although some purified preparations were stable, most were unstable upon storage. Analysis of the unstable preparations on nondenaturing and sodium dodecyl sulfate polyacrylamide disc gels revealed that the complex in these preparations was progressively fragmented to smaller components and subunits upon repeated freeze-thaw treatment or prolonged incubation at or above 4 degrees. Distinct fragments were generated ranging in size down to 25,000 daltons, and some fragments retained some of the activities associated with the anthranilate synthetase complex. On the basis of these and earlier studies, we conclude that anthranilate synthetase from Neurospora crassa is composed of two distinct subunits in an alpha2beta2 structure; one subunit is a trifunctional peptide which contains the catalytic sites for the phosphoribosylanthranilate isomerase and indoleglycerol phosphate synthetase reactions, and associates with the second subunit to form glutamine-dependent anthranilate synthetase. The smaller subunits and components previously reported for this complex are apparently due to protease activity present in purified preparations.

Tryptophan biosynthetic pathway in the Enterobacteriaceae: some physical properties of the enzymes.

Several physical properties of the first four enzymatic activities of the tryptophan pathway were examined using gel filtration and ion exchange chromatography. Five different patterns were noted. Differences in the anthranilate synthetase (AS) and phosphoribosylanthranilate transferase (PRT) defined these patterns. In all the organisms studied phosphoribosylanthranilate isomerase and indoleglycerol phosphate synthetase co-eluted from both diethylaminoethyl-cellulose and G-200 and thus probably are contained in a single polypeptide of 50,000 daltons. An AS-PRT complex was found in Citrobacter species, Enterobacter cloacae, and Erwinia dissolvens. In all the other bacteria examined AS and PTR were separate molecules. In Serratia marcescens, S. marinorubra, and Enterobacter liquefaciens, AS was 140,000 daltons and PRT was 45,000 daltons. In Erwinia carotavora and Enterobacter hafniae the AS was the same size as the Serratia species but the PRT was larger at 67,000 daltons. Two Proteus species had an AS and PRT of the same size as E. carotavora and E. halfniae but the Proteus AS was different in that it partially dissociated upon gel filtration. Aeromonas formicans was unique in its possession of an AS with a molecular weight of 220,000. The PRT of A. formicans was found to elute at 67,000 daltons. Possible paths of evolution of the tryptophan enzymes are discussed in terms of the results of this study. The results presented here are also considered with respect to existing taxonomic schemes of the enteric bacteria.

Tryptophan synthase from yeast. Purification by affinity chromatography, physical properties.

Tryptophan synthase was purified from Saccharomyces cerevisiae with the aid of affinity chromatography up to a specific activity of 525 units/g protein. Electrophoresis on polyacrylamide gel showed one main protein component. Gel filtration on Sephadex G‐150 indicates an average molecluar weight of 143000. Dissociation with dodecylsulfate followed by dodecyl‐sulfate‐acrylamide gel electrophoresis leads to the conclusion that the enzyme is probably composed of four subunits of equal size. The Km‐values with respect to its substrates indoleglycerol phosphate, indole and l‐serine were estimated. l‐Serine was shown to increase the Km‐value for indoleglycerol phosphate about 7‐fold. Several indole analogues inhibit the tryptophan synthase reaction (B) (i.e. indole + serine → tryptophan); indoleacrylic acid is the most effective. The pH‐optimum of tryptophan synthase is around 6.9 to 7.0. Sulfhydryl modification by 5,5′‐dithiobis(2‐nitrobenzoic acid) and p‐chloromercuriphenylsulfonic acid results in a large loss of tryptophan synthase activity, which cannot be prevented by the substrates l‐serine and indole. The tryptophan synthase inactivating system from yeast affects the catalysis of both reactions (A) (i.e. indoleglycerol phosphate → indole + glyceraldehyde 3‐phosphate) and (B).

Enzymes of the Tryptophan Pathway in Three Bacillus Species

The tryptophan synthetic pathway was characterized in three species of Bacillus, B. subtilis, B. pumilus, and B. alvei. They share the common features of a pathway which is subject to tryptophan repression, contains no unexpected complexes among the five enzymes, exhibits dissociable anthranilate synthase enzymes which do not require phosphoribosyl transferase for amidetransfer activity, contains separate indoleglycerol phosphate synthase and phosphoribosylanthranilate isomerase enzymes, and contains similar tryptophan synthetase multimers. In looking at these characteristics in detail however, differences among the three species became apparent, as, for example, in the complementation observed between the alpha and beta(2) components of tryptophan synthetase, and the dissociation patterns of the large and small components of anthranilate synthase. The results demonstrate some pitfalls in attempting to compare multimeric enzymes in crude extracts from different organisms.

Formation of hybrid anthranilate synthetase in vitro from components of Aspergillus and Neurospora.

Glutamine-dependent anthranilate synthetase was produced in vitro by mixing the extracts of a trypA and a trypC mutant of Aspergillus nidulans. Neither mutant alone possessed this activity. The enzyme formed in the mixture had the properties of the wild-type anthranilate synthetase which, together with N-(5′-phosphoribosyl) anthranilate (PRA) isomerase and indole 3-glycerol phosphate (InGP) synthetase, is found in a 10S multienzyme complex. Extracts of the trypA69 mutant contained a 6.5S protein as the active component—presumably the trypC + product—which in addition showed PRA isomerase and InGP synthetase activity. Extracts of the trypC801 mutant lacked all three enzyme activities but contained a 4.5S component—the trypA + gene product—which in vitro showed ammonia-dependent anthranilate synthetase activity. These mutants are analogous in their properties to certain tryp-2 and tryp-1 mutants of Neurospora. When complementary extracts of the two genera were mixed (Aspergillus trypA with Neurospora tryp-1 or Aspergillus trypC with Neurospora tryp-2), a “hybrid” glutamine-dependent anthranilate synthetase was obtained which showed less than half the activity produced in homologous combinations.

Mapping of the tryptophan genes of Acinetobacter calcoaceticus by transformation.

Auxotrophs of Acinetobacter calcoaceticus blocked in each reaction of the synthetic pathway from chorismic acid to tryptophan were obtained after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. One novel class was found to be blocked in both anthranilate and p-aminobenzoate synthesis; these mutants (trpG) require p-aminobenzoate or folate as well as tryptophan (or anthranilate) for growth. The loci of six other auxotrophic classes requiring only tryptophan were defined by growth, accumulation, and enzymatic analysis where appropriate. The trp mutations map in three chromosomal locations. One group contains trpC and trpD (indoleglycerol phosphate synthetase and phosphoribosyl transferase) in addition to trpG mutations; this group is closely linked to a locus conferring a glutamate requirement. Another cluster contains trpA and trpB, coding for the two tryptophan synthetase (EC 4.2.1.20) subunits, along with trpF (phosphoribosylanthranilate isomerase); this group is weakly linked to a his marker. The trpE gene, coding for the large subunit of anthranilate synthetase, is unlinked to any of the above. This chromosomal distribution of the trp genes has not been observed in other organisms.

Partial characterization of phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, and indole glycerol phosphate synthase from Serratia marcescens.

The molecular organization of the enzymes phosphoribosyl (PR) transferase, phosphoribosyl anthranilate (PRA) isomerase, and indole glycerol phosphate (InGP) synthase of the tryptophan biosynthetic pathway of Serratia marcescens was investigated and compared with that reported in other enteric bacteria. PRA isomerase and InGP synthase activities were found to reside in a single polypeptide chain, a situation analogous to that in Escherichia coli, Salmonella typhimurium, and Aerobacter aerogenes. This bifunctional enzyme was purified to near homogeneity. Its molecular weight was estimated to be 48,000. PR transferase was found unassociated with PRA isomerase and InGP synthase after gel filtration and ion-exchange chromatography. Whereas in other enteric organisms PR transferase has been reported to form an aggregate with anthranilate synthase, it is a distinct entity in S. marcescens.

Tryptophan synthetase in Euglena gracilis strain G.

The five enzyme activities in the synthesis of l-tryptophan have been obtained in extracts of Euglena gracilis. One of these, tryptophan synthetase, has been studied in detail. The general catalytic properties of tryptophan synthetase, including the range of reactions catalyzed and its substrate and cofactor affinities, are similar to those reported for other organisms. The Euglena enzyme has two properties never previously observed for tryptophan synthetase. First, the rate of catalysis of the conversion of indole-glycerol phosphate to l-tryptophan remained at its maximal value and was unaffected by the ionic environment up to 0.3 m KCl. In contrast, the conversion of indole to tryptophan showed a sharp maximum at 0.08 m KCl. Second, the enzyme is a component of a complex that includes every enzyme in the pathway committed to tryptophan biosynthesis with the exception of anthranilate synthetase, the regulatory enzyme.

Inhibition of tryptophan synthetase by indoleacrylic acid.

Indoleacrylic acid (5 x 10(-4)m in the growth medium) inhibits the growth of mycelia of Neurospora crassa and causes the cells to accumulate indoleglycerol phosphate. Measurement of kinetic parameters of partially purified tryptophan synthetase in the presence of indoleacrylic acid showed the following patterns of inhibition: in Reaction 1, the physiological reaction, indoleglycerol phosphate + serine [Formula: see text] tryptophan + glyceraldehyde-3-phosphate (unusual with respect to indoleglycerol phosphate, partially competitive with respect to serine); in Reaction 2, indole + serine [Formula: see text] tryptophan (unusual with respect to indole, partially competitive with respect to serine); in Reaction 3, indoleglycerol phosphate [Formula: see text] indole + glyceraldehyde-3-phosphate (no inhibition observed). The effects of indoleacrylic acid on whole cells were completely reversed by equimolar concentrations of tryptophan but not by indole. The addition of a 10-fold molar excess of serine to the medium completely reversed the effects. The accumulation of indoleglycerol phosphate appears to be the result of the partial blockage of tryptophan synthetase, the resulting deprivation of tryptophan, and the consequent release of its physiological feedback control on anthranilate synthetase.

Physiological and enzymatic aspects of histidine-mediated control of the tryptophan pathway.

It would thus appear that in Saccharomyces cerevisiae there are two forms of histidine-mediated control on the tryptophan pathway. In some strains histidine increases anthranilate synthetase and indole glycerol phosphate synthetase activities, while tryptophan synthetase decreases. In other strains histidine affects coordinately all enzymatic activities involved in tryptophan biosynthesis. The two groups of strains also differ in the formation, during the growth of the enzymatic activities involved in tryptophan biosynthesis. This difference in the relative rates at which the two enzymes are formed may explain the accumulation of intermediates in the cultural media of some strains. The derepression of anthranilate synthetase and indole glycerol phosphate synthetase activities by histidine is particularly manifest in the auxotrophic his3 strains that show these activities very depressed in histidine starvation; large amounts of this amino acid stimulate them to a considerably greater extent than in prototrophic strains.

Reaction of the α Subunit of the Escherichia coli Tryptophan Synthetase with 1,5-Difluoro-2,4-dinitrobenzene

1,5-Difluoro-2,4-dinitrobenzene introduces an intramolecular cross-linkage into the Escherichia coli tryptophan synthetase α subunit between residues cysteine 80 and lysine 108, concomitant with a loss of its aldolytic activity on indoleglycerol phosphate. The stereochemical relationship of these residues and other residue pairs cross-linked by other bifunctional reagents to the activity of the enzyme is discussed.

Tryptophan synthase: a two-component enzyme from pea plants (Pisum sativum cv. Alaska)

Evidence is presented showing that tryptophan synthase (L-serine hydro-lyase (adding indole), EC. 4.2.1.20) from pea is composed of two components, A and B, analogous to the protein subunits reported for bacteria, algae, and tobacco enzyme. Separation of the two components was achieved by gel filtration on Sephadex G-100. In contrast with enzyme from tobacco the two components could not be separated by ammonium sulfate precipitation. However, the A component was removed by heating at 70 °C for 5 min. The B component, alone, catalyzed the condensation of L-serine plus indole to tryptophan (reaction 2) at a significant level. A combination of the A and B components was necessary for the formation of tryptophan from indole-glycerol phosphate (reaction 1); and conversion of indole-glycerol phosphate to indole (reaction 3). Reaction 3 could only be demonstrated with partially purified enzyme extract, and the reaction proceeded well in the absence of added pyridoxal phosphate but was increased by its presence. The relative rates of the reactions were in the ratio of 30:100:1.5 for reactions 1, 2, and 3, which is in agreement with studies with microbial TSase. However, in contrast, some free indole was isolated from reaction 1. The reversal of reaction 3 could not be detected. It is suggested that reaction 1 is probably the one of physiological significance as is believed to be the case in microorganisms.

Regulation of Tryptophan Biosynthetic Enzymes in Neurospora crassa

The formation of enzymatic activities involved in the biosynthesis of tryptophan in Neurospora crassa was examined under various conditions in several strains. With growth-limiting tryptophan, the formation of four enzymatic activities, anthranilic acid synthetase (AAS), anthranilate-5-phosphoribosylpyrophosphate phosphoribosyl transferase (PRAT), indoleglycerol phosphate synthetase (InGPS), and tryptophan synthetase (TS) did not occur coordinately. AAS and TS activities began to increase immediately, whereas PRAT and InGPS activities began to increase only after 6 to 12 hr of incubation. In the presence of amitrole (3-amino-1,2,4-triazole), the formation of TS activity in a wild-type strain was more greatly enhanced than were AAS and InGPS activities. With a tr-3 mutant, which ordinarily exhibits an elevated TS activity, amitrole did not produce an increase in TS activity greater than that observed on limiting tryptophan. With tr-3 mutants, the increased levels of TS activity could be correlated with the accumulation of indoleglycerol in the medium; prior genetic blocks which prevented or reduced the synthesis of indoleglycerol also reduced the formation of TS activity. The addition of indoleglycerol to cultures of a double mutant (tr-1, tr-3) which could not synthesize indoleglycerol markedly stimulated the production of TS activity but not PRAT activity; the production of TS activity reached the same level with limiting or with excess tryptophan. A model explaining these and other related observations on enzyme formation in N. crassa is proposed.

Structural and Functional Roles of the Cysteine Residues in the α Subunit of the Escherichia coli Tryptophan Synthetase

Abstract The reaction pattern of the three sulfhydryl groups of the α subunit of the Escherichia coli tryptophan synthetase with N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) at pH 7.0 is profoundly altered in the presence of 10 mm indole. The effect of indole is upon both the rate of reaction, and upon its over-all extent. In the absence of indole, only cysteinyl sulfhydryl groups 80 and 117 of the α subunit are available for reaction with N-ethylmaleimide, but in 10 mm indole all three thiol groups become available for reaction. Similar results were obtained with DTNB (i.e. in the absence of indole a single sulfhydryl group reacts rapidly with DTNB, any further reaction occurring very slowly; in the presence of indole all three sulfhydryl groups react at a slower although uniform rate). The effects of indole in the 7.5 to 10.0 mm concentration range could not be mimicked with 20% sucrose, 20% ethanol, 20% ethylene glycol, or 20% dioxane, solvents known to be effective perturbants of protein tertiary structure. The presence of indole also had no effect on the optical rotatory dispersion of the enzyme, and caused no detectable change in the 16 ± 2% α helical structure found for this protein. Indole 3-glycerol phosphate (InGP) at concentrations twice the enzyme's Km for this substrate in the InGP aldolase reaction catalyzed by the free α subunit, protected all three sulfhydryl groups from reaction with DTNB in the presence of indole. Examination of the indole concentration dependence of its effect on sulfhydryl group reactivity, and determination of its Km in the reverse InGP aldolase reaction (synthesis of InGP from indole in the presence of excess d-glyceraldehyde 3-phosphate) showed that the two processes reached half-saturation at indole concentrations of 3.6 and 4.1 mm, respectively. It is concluded that the effect of indole on sulfhydryl group reactivity reflects enzyme conformational changes related to the mechanism of catalysis of the InGP aldolase reaction by the independent α subunit. Indole-induced conformational changes in α subunit structure may be involved in the regulation and catalysis of the last steps in tryptophan synthesis by the α2β2-tryptophan synthetase complex.

Structural and Functional Roles of the Cysteine Residues in the α Subunit of the Escherichia coli Tryptophan Synthetase

Abstract Cysteine 80 and cysteine 117 of the α subunit of the Escherichia coli tryptophan synthetase have been cross-linked by use of the bifunctional reagent N,N'-bis(maleimidomethyl) ether (BME). The sites of reaction were identified from the amino acid compositions of radioactive peptides purified from tryptic and tryptic-chymotryptic digests of α subunit which had reacted with 3H-BME to the extent of 1 mole of cross-linking reagent per mole of enzyme. Hydrodynamic studies showed that no significant dimer formation had occurred. The electrophoretic and immunochemical properties of the enzyme are identical with those of untreated control preparations. Although the intramolecularly cross-linked monomers are apparently homogeneous by these criteria, BME-treated α subunits are functionally heterogeneous in that 70% of the independent indoleglycerol phosphate activity of the α subunit has been lost after reaction of 1 mole of BME with 1 mole of α subunit. In addition, 70% of the protein in such preparations cannot form a functional α2β2-tryptophan synthetase complex with the β2 subunit, as measured indirectly by enzymatic activity studies and directly in studies on α2β2 complex formation in sucrose density gradient centrifugation experiments. Two possible explanations for the observed functional heterogeneity of BME cysteine 80 to cysteine 117 cross-linked α subunit preparations are advanced. These mechanisms may be interdependent. Two new asymmetric centers are introduced by the addition of the protein cysteinyl thiols across the two maleimide double bonds of the bifunctional reagent. A population of α subunits cross-linked between these 2 cysteinyl residues might contain as many as four different conformational forms of BME cross-link. The sulfhydryl group region of the enzyme is known from previous studies to be closely related to the active site of the subunit in the indoleglycerol phosphate aldolase reaction, and to be important in the association of the α subunit with β2 subunits to form the α2β2-tryptophan synthetase complex. Alternatively, the order of addition of the two cysteinyl thiols to the BME molecule is not known, and it is possible that differences in either protein active site structure, or in the stereochemical course of the cross-linking reaction may be the consequence of initial reaction of BME at one or the other of the 2 cysteinyl residues. Such differences in structure between the active and inactive subunits are nonetheless not great enough to allow the physical separation of the different forms involved.

Intra-cistronic complementation among trpC mutants of Escherichia coli.

Some merodiploids containing two different trpC alleles grow in the absence of added tryptophan. The trpC gene product is the bifunctional enzyme indoleglycerol phosphate synthetase but the ability to grow on minimal medium does not require that the alleles be dificient in different functions. This indication of complementation is supported by the evidence that the trpC alleles, of the merodiploids, can be transferred by transduction or by the trp-colV,B episome. Genetic recombination between episome and chromosome is seen to occur, but at a rate too low to explain the observed results. The nature of the interaction between the trpC alleles is not readily explained since indoleglycerol phosphate synthetase appears to consist of a single polypeptide chain in in vitro studies.

Genetic control of a multienzyme complex: subunit structures of mutationally altered forms.

IN Neurospora crassa, anthranilate synthetase, N- (5’-phosphoribosyl) anthranilate (PRA) isomerase, and indole-3-glycerol phosphate (InGP) synthetase are organized in a multienzyme complex whose structure and function are jointly controlled by the tryp-2 and tryp-2 loci (DEMOSS and WEGMAN 1965; DEMOSS, JACKSON and CHALMERS 1967). The wild-type complex has a molecular weight of 240,000 and is composed of six subunits of two distinct types (GAERTNER and DEMOSS 1969). It was proposed that these subunits are the gene products of the tryp-2 and tryp-2 loci and that complex formation is an obligate step in the expression of anthranilate synthetase. This suggestion is supported by the finding that an active complex with a sedimentation constant of 10s can be produced in vitro by mixing extracts of certain zryp-2 and tryp-2 mutants (ARROYO-BEGOVICH and DEMOSS 1969). Mutations at both loci independently affect the three catalytic activities and the overall integrity of the complex (DEMOS, JACKSON and CHALMERS 1967). TO understand the relationship between structure and activity we have isolated, by genetic methods, various mutationally altered gene products and have studied their interaction with wild-type subunits in uitro. The results demonstrate that the expression of anthranilate synthetase is dependent upon the specific association of the products of the tryp-1 and tryp-2 genes and have allowed us to infer the subunit structures of the altered complexes in tryp-1 and tryp-2 mutants. MATERIALS AND METHODS

Catalytic Facilitation in Vitro by Two Multienyzme Complexes from Neurospora crassa

Abstract The conversions of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) to 3-enolpyruvylshikimate 5-phosphate (ES-5-P) and of N-(5'-phosphoribosyl)anthranilate (PR-anthranilate) to indole-3-glycerol phosphate (InGP) are catalyzed in vitro by two multienzyme complexes from Neurospora crassa. The rates of the over-all sequential reactions catalyzed by the two complexes were found to be greater than the rates of reactions initiated later in the sequence with known intermediates. Under conditions of saturation with the substrate, the rate of formation of ES-5-P from DAHP was 10 times the rate of formation of ES-5-P from shikimate, and the rate of formation of InGP from PR-anthranilate was twice the rate of formation of InGP from 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate. With both complexes, addition of the intermediate did not increase or inhibit the more rapid over-all reaction rates. We conclude that for the over-all reaction sequences the catalytic efficiencies of the enzymic components are increased in these multienzyme complexes either by a mechanism of intermediate substrate localization or by an activation of enzymic activities. Although these cases of catalytic facilitation in vitro have not as yet been correlated directly with the phenomenon of metabolic compartmentalization or channeling as observed in vivo, the activation of the components of a specific multienzyme complex or the localization of intermediate substrates within a specific complex are both mechanisms by which the cell could effect a compartmentalization or channeling of biochemical intermediates.

In vitro formation of an active multienzyme complex in the tryptophan pathway of Neurospora crassa.

A multienzyme complex that catalyzes the anthranilate synthetase, phosphoribosylanthranilate (PRA) isomerase, and indoleglycerolphosphate (InGP) synthetase reactions was produced in vitro when extracts from a tryp-1 mutant and a tryp-2 mutant of Neurospora crassa were mixed. The sedimentation values and the molecular weights for the interacting components obtained from the mutants were estimated by sucrose gradients and by gel filtration on Sephadex columns. The component coded for by the tryp-2 gene (a-component) which is present in the tryp-1-17 mutant has a sedimentation coefficient of 4.5S and molecular weight of 70,000. The component coded for by the tryp-1 gene (i-component) which is present in the tryp-2-6 mutant has a sedimentation coefficient 7.5S and a molecular weight of 170,000. The complex formed in vitro had similar properties to those previously reported for the wild-type complex. A Q(10) value of 3 and an apparent activation energy of 18,000 cal/mole were calculated for the formation of the complex from the two components.

Untersuchungen zur Indol-3-essigsäure-Bildung aus Indol,Indol-3-glycerin und Indol-3-glycerinphosphat

Indole or indoleglycerol phosphate were incubated with crude or dialyzed enzyme extracts ( p H 5.4-8.0) of pea seedlings. Neither with nor without addition of serine-like cosubstrates, any formation of auxin or an auxin precursor could be detected by means of the Avena curvature test or thin-layer chromatography. In presence of indole, the auxin content of crude homogenates ( p H 6.8) was slightly enhanced; that was due to an indole-induced inhibition of the breakdown of IAA produced by bacteria during the incubation period. Indoleglycerol was not altered by wheat tissues and was physiologically inactive in the Triticum section test. Wir sind Herrn Dr. F. Lingens für seine Information über die IGP-Synthese und Frl. I. Dörr für ihre gute technische Hilfe zu großem Dank verpflichtet.