Indole-3-glycerol Phosphate Synthase Research Articles

Regulation of Tryptophan Biosynthesis in the n-Alkane Utilising Yeast Candida maltosa

Summary The enzymes of tryptophan pathway were partially purified from Candida maltosa and the regulation patterns were established. Ammonia-dependent anthranilate synthase (ASN) (M r 40,000), anthranilate phosphoribosyltransferase (PRT) (M r 100,000), phosphoribosylanthranilate isomerase (PRAI) (M r 30,000), and tryptophan synthase (TS) (M r 140,000) are separated from each other, whereas glutamine-dependent anthranilate synthase (ASG) is active only in complex (M r 75,000) with indole-3-glycerol-phosphate synthase (InGPS). The formation of three enzymes (ASN, PRT, ASG-InGPS) was regulated by the general control of amino acid biosynthesis, whereas synthesis of PRAI and TS was constitutive. Tryptophan only affected both anthranilate synthase activities. The K m -values of ASN were 0.083 mi for chorismate and 10 mM for ammonium. Inhibition of the enzyme reaction by tryptophan was competitive with respect to chorismate (K i = 0.007 mM). The K m -values of ASG were estimated to be 0.043 mM for chorismate and 1.0 mM for glutamine; a K i of 0.004 mM was obtained for tryptophan. A number of tryptophan analogues also inhibited the two enzyme reactions.

Charakterisierung von 5-Fluor- und 5-Methyltryptophan-resistenten Mutanten von Hansenula henricii

Strains of Hansenula henricii resistant to growth inhibition by various tryptophan analogs were selected. DL-5-methyltryptophan resistant cells appeared with a frequency of about 5 x 10- 6 (2 X 10- 7 for DL-5-fluoro-tryptophan) resistance. All strains resistant to fluoro-tryptophan have an defect in permeation for this tryptophan analogue. The Hansenula mutants resistant to methyl-tryptophan can be devided into four groups. To the first group belong mutants, which are permeation-resistant for 5-methyl-tryptophan. Strains of group II posess an anthranilate synthase, whose inhibition by tryptophan is decreased. Mutants of the third group show anthranilate synthase level increased to about 200 per cent; specific activity of the anthranilate phosphoribosylpyrophosphate transferase and of the indoleglycerolphosphate synthase were not altered. To the fourth group belong mutants, which contain anthranilate synthase derepressed by added tryptophan antimetabolites, L-tryptophan and several other derivatives of indole. An increase of the activity of the other tryptophan synthesizing enzymes was not observed in this case.

Limited proteolysis of N-(5′-phosphoribosyl)anthranilate isomerase: Indoleglycerol phosphate synthase from Escherichia coli yields two different enzymically active, functional domains

The native enzyme must be denatured either by sodium dodecyl sulfate or by urea before limited proteolysis can occur. Under these conditions only one or two peptide bonds are hydrolyzed by each of the following proteases: Staphylococcal V8 protease, trypsin and elastase. The amino-terminal amino acid sequences were determined to identify the cleavage sites. The new sequences comprise approximately 20% of the entire polypeptide chain, and show good agreement with the nucleotide sequence of the trpC gene. Both V8 protease † † Abbreviations used: V8 protease, extracellular protease from Staphylococcus aureus strain V8: PRA, N-(5′-phosphoribosyl)anthranilate; InGP, indoleglycerol phosphate; CdRP, 1-(2-carboxyphenyl-amino)-1-deoxyribulose 5-phosphate; SDS, sodium dodeoyl sulfate. and elastase yield large carboxy-terminal fragments, about two thirds of the size of the parent enzyme, and corresponding small amino-terminal fragments. Trypsin cleaves a single peptide bond in the last one third of the polypeptide chain. After separation of the fragments, removal of dodecyl sulfate and renaturation, only the large fragments fold to stable structures. The small fragments precipitate. The large amino-terminal fragment catalyzes only the synthesis of indoleglycerol phosphate and precipitates when solutions are frozen and thawed. The large carboxy-terminal fragment catalyzes only the isomerization of N-(5′-phosphoribosyl)anthranilate and is stable towards freezing and thawing. These studies prove that the intact bifunctional enzyme consists of two autonomously folding, functional domains. They also support the notion that the bifunctional enzyme may have arisen by the fusion of separate ancestral genes, and that stabilization of the intrinsically labile indoleglycerol phosphate synthase domain by interdomain interactions is functionally advantageous.

N-(5-Phosphoribosyl)anthranilate isomerase-indoleglycerol-phosphate synthase. 2. Fast-reaction studies show that a fluorescent substrate analog binds independently to two different sites

The mechanism of binding of reduced 1-(2-carboxyphenylamino)-1-deoxyribulose 5-phosphate (rCdRP) to two different binding sites on the bifunctional enzyme is determined by kinetic studies, using temperature-jump and stopped-flow equipment with fluorescence detection. Two rapid binding processes and a comparatively slow isomerization process are observed over a wide range of enzyme and rCdRP concentrations. Kinetic measurements with low concentrations of rCdRP show that the isomerization is coupled only to the more rapid of the two binding reactions that involves the active site of indoleglycerol-phosphate synthase. The slower of the two binding reactions represents rCdRP binding in one step to the active site of (phosphoribosyl)anthranilate isomerase. The simplest mechanism explaining quantitatively the dependence of the relaxation times on concentration consists of rCdRP binding to two sites on the enzyme that are intrinsically different and independent, even to the extent that a ligand-induced isomerization of one site is not transmitted to the other site. Simulation studies show that the concentration dependences of the amplitudes of the three relaxation processes are also consistent with the mechanism. The results are discussed in terms of two autonomous domains of folding of the polypeptide chain.

Isolation and characterization of two tryptophan biosynthetic enzymes, indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, from Bacillus subtilis.

Two of the enzymes responsible for tryptophan biosynthesis in Bacillus subtilis have been extensively purified. These proteins are indole-3-glycerol phosphate synthase and N-(5'-phosphoribosyl) anthranilate isomerase. By comparison to the non-differentiating enteric bacteria in which these two enzymes are fused into a single polypeptide, the isolation of the indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase from B. subtilis has demonstrated that the two proteins are separate species in this organism. The two enzymes were clearly separable by anion-exchange chromatography without any significant loss of activity. Molecular weights were determined for both enzymes by gel filtration and sodium dodecyl sulfate-slab gel electrophoresis, and indicated that the indoleglycerol phosphate synthase is the slightly larger of the two proteins. The minimum molecular weight for indoleglycerol phosphate synthase was 23,500, and that for phosphoribosyl anthranilate isomerase was 21,800. Both enzymes have been examined as to conditions necessary to achieve maximal activity of their individual functions and to maintain that activity.

Regulation of enzyme synthesis in the tryptophan pathway of Acinetobacter calcoaceticus.

In Acinetobacter calcoaceticus the seven genes coding for the enzymes responsible for tryptophan synthesis map at three chromosomal locations. Two three-gene clusters, one (trpGDC) specifying the small subunit of anthranilate synthase, phosphoribosyl transferase, and indoleglycerol phosphate synthase and the other (trpFBA) specifying phosphoribosyl anthranilate isomerase and both tryptophan synthase subunits, are not linked to each other or to the trpE gene specifying the large anthranilate synthase subunit. When regulation of trp gene expression is studied in the wild type, only the level of the trpF gene product decreases upon addition of tryptophan to the medium. Tryptophan starvation of tryptophan auxotrophs, however, results in increased levels of all the tryptophan enzymes; this and additional evidence suggests that the expression of all the trp genes is subject to repression. The trpGDC genes are coordinately controlled, and the trpE gene is regulated in parallel with them. The trpFBA genes are controlled neither coordinately nor in parallel with the other trp genes, but respond proportionally when compared with each other. So far, two types of constitutive mutants have been found. The first class of mutants apparently occurs in the structural gene for a repressor protein; this repressor locus is unlinked to any of the biosynthetic trp genes and affects only the expression of trpE and the trpGDC cluster. The second class contains mutants closely linked to the trpGDC region; they overproduce only the gene products of this cluster.

Free tryptophan pool and tryptophan biosynthetic enzymes in Saccharomyces cerevisiae.

The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase)have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties.

A rapid spectrophotofluorometric assay for indoleglycerol phosphate synthase

A rapid and sensitive spectrophotofluorometric assay for indoleglycerol phosphate synthase is presented. The method is based upon the fluorescence of the reaction product indoleglycerol phosphate and has two advantages over previously reported assays. It is more sensitive and is useful for measuring enzyme activities in extracts containing materials that prohibit the use of other methods.

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.

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.

Characterization of mutants with single and multiple defects in the tryptophan biosynthetic pathway in Bacillus subtilis.

Sixty-five tryptophan auxotrophs which map in a cluster on the genome of Bacillus subtilis were characterized on the basis of (i) growth response, (ii) accumulation of intermediate compounds, and (iii) determination of enzymatic defects. They could be placed into six phenotypic classes. Certain of the mutants exhibited pleiotropic effects on more than one enzymatic activity in a manner different from those effects reported for the tryptophan pathway in other organisms. Invariably, mutations in the second gene, that coding for phosphoribosyl transferase activity, were found to lack the indoleglycerol phosphate synthase activity specified by the third gene in the cluster; however, this polarity did not extend to genes more distal in the cluster. Furthermore, mutations in the gene which codes for phosphoribosyl-anthranilate isomerase not only led to a loss of this enzyme but also to a loss of phosphoribosyl transferase and indoleglycerol phosphate synthase. In contrast, mutations in either of the loci coding for these latter functions had no apparent effect on isomerase activity. No polarity of the conventional type was found, e.g., none of the mutations in any gene led to polarized effects on the levels of the enzymes specified by the other genes of the cluster. These observations indicated a possible in vivo aggregation involving the transferase, isomerase, and synthase enzymes, with the isomerase acting as the "key" enzyme in the aggregate.