Indoleglycerol Phosphate Synthetase Research Articles

Tryptophan analog resistance mutations in Chlamydomonas reinhardtii.

Forty single gene mutations in Chlamydomonas reinhardtii were isolated based on resistance to the compound 5'-methyl anthranilic acid (5-MAA). In other organisms, 5-MAA is converted to 5'-methyltryptophan (5-MT) and 5-MT is a potent inhibitor of anthranilate synthase, which catalyzes the first committed step in tryptophan biosynthesis. The mutant strains fall into two phenotypic classes based on the rate of cell division in the absence of 5-MAA. Strains with class I mutations divide more slowly than wild-type cells. These 17 mutations map to seven loci, which are designated MAA1 to MAA7. Strains with class II mutations have generation times indistinguishable from wild-type cells, and 7 of these 23 mutations map to loci defined by class I mutations. The remainder of the class II mutations map to 9 other loci, which are designated MAA8-MAA16. The maa5-1 mutant strain excretes high levels of anthranilate and phenylalanine into the medium. In this strain, four enzymatic activities in the tryptophan biosynthetic pathway are increased at least twofold. These include the combined activities of anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, indoleglycerol phosphate synthetase and anthranilate synthase. The slow growth phenotypes of strains with class I mutations are not rescued by the addition of tryptophan, but the slow growth phenotype of the maa6-1 mutant strain is partially rescued by the addition of indole. The maa6-1 mutant strain excretes a fluorescent compound into the medium, and cell extracts have no combined anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase and indoleglycerol phosphate synthetase activity. The MAA6 locus is likely to encode a tryptophan biosynthetic enzyme. None of the other class I mutations affected these enzyme activities. Based on the phenotypes of double mutant strains, epistatic relationships among the class I mutations have been determined.

Structural organization of the TRP1 gene of Phycomyces blakesleeanus: implications for evolutionary gene fusion in fungi

The complete nucleotide (nt) sequence of the cloned TRP1 gene from Phycomyces blakesleeanus is reported. The gene encodes a trifunctional polypeptide that represents a sequential fusion of glutamine amidotransferase (TrpG), indoleglycerolphosphate synthetase (TrpC), and phosphoribosylanthranilate isomerase (TrpF) activities from the amino- to the carboxy terminus. This genetic organization is characteristic of filamentous fungi in general. The transcription start sites and the polyadenylation sites of the gene have been mapped. The two predominant TRP1 transcripts have a short leader of 13 and 18 nt, while minor species with significantly longer leaders are also detectable. Approximately 50 bp and 70 bp upstream from the major transcription start points, sequences that match the canonical eukaryotic TATA and CAAT boxes, respectively, are found. Two major polyadenylation signals are located approximately 50 and 70 nt downstream from the translational stop. Three closely clustered minor polyadenylation sites map to roughly 120–150 bp 3′ to the termination codon. The TRP1 gene is the first and only Phycomyces gene that has been cloned and sequenced. The information regarding the promoter and terminator of a Phycomyces gene derived from these studies should benefit strategies aimed at gene manipulations in this organism.

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.

Inactivation and partial degradation of phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthetase in nongrowing cultures of Escherichia coli

The stability of tryptophan biosynthetic enzyme activities was examined in cultures of repressor-negative (trpR) strains of Escherichia coli K-12 incubated under conditions of nutrient starvation of chloramphenicol inhibition. The results show that four of the five activities examined are stable under most nongrowing conditions, whereas one activity, indoleglycerol phosphate (InGP) synthetase, carried by the trpC protein, is unstable under most conditions tested. Phosphoribosylanthranilate (PRA) isomerase activity, which is also carried by the trpC protein, is unstable during starvation for ammonium, cysteine, or sulfate but is stable under other nongrowing conditions where InGP synthetase is not. InGP synthetase activity but not PRA isomerase activity is also diminished about twofold in cultures using glycerol as a carbon-energy source. These results indicate that one or both activities of the trpC protein is specifically inactivated under several culture conditions. Experiments with antibodies to the trpC protein show that sulfate-starved and ammonium-starved cultures contain 20 to 40% less immunologically reactive trpC protein than unstarved cultures. This indicates that the trpC protein is probably partially degraded under these conditions. During recovery from sulfate starvation or ammonium starvation, cultures slowly regain normal levels of InGP synthetase and PRA isomerase activities, suggesting that inactivation may be reversible.

Some Physical Characteristics of the Enzymes of l-Tryptophan Biosynthesis in Higher Plants

Anthranilate synthetase, phosphoribosyltransferase, phosphoribosyl anthranilate isomerase, and indoleglycerol phosphate synthetase were examined in partially purified extracts of the monocotyledon, Zea mays and the dicotyledon, Pisum sativum. The plant extracts were chromatographed on DEAE-cellulose and Sephadex G150. The molecular weights of the enzymes were determined and found to be similar to those observed for many bacteria. None of the plant tryptophan enzyme activities was aggregated in vitro as is also the case with most bacteria. This is in contrast with the complex aggregation patterns observed in other eucaryotic organisms that have been examined (fungi and Euglena gracilis). The tryptophan enzymes from peas and corn were generally similar but some differences in stability were observed.

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.

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.

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.

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.

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.

Structure of the trpC cistron specifying indoleglycerol phosphate synthetase, and its localization in the tryptophan operon of Escherichia coli.

Structure of the trpC cistron specifying indoleglycerol phosphate synthetase, and its localization in the tryptophan operon of Escherichia coli.

Aromatic biosynthesis in yeast: I. The synthesis of tryptophan and the regulation of this pathway

1. 1. The enzymatic functions of the tryptophan path of Saccharomyces cerevisiae have been found to be the same as reported for Aerobacter aerogenes and Escherichia coli. 2. 2. It was concluded that the locus tr-3 codes for a polypeptide necessary for two enzymic functions, anthranilate synthetase and indoleglycerol phosphate synthetase. Evidence is presented showing that the gene tr-4 codes for anthranilate phosphoribosylpyrophosphate phosphoribosyltransferase and tr-1 for phosphoribosylanthranilic acid isomerase. It was deduced that tr-2 codes for a plypeptide necessary for anthranilate synthetase. 3. 3. The stability of the anthranilate synthetase of Saccharomyces cerevisiae was markedly dependent on pH with an optimum stability in Tris buffer at pH 7.25. The enzyme from Aerobacter aerogenes was relatively pH stable. This finding is used to support the view that in Saccharomyces cerevisiae anthranilate synthetase and indoleglycerol phosphate synthetase form an aggregate enzyme molecule. 4. 4. In yeast, anthranilate synthetase is controlled by end-product inhibition and repression. The control of anthranilic acid synthesis by exogenous tryptophan is less efficient for Saccharomyces cerevisiae than for Escherichia coli.

Increased Activity of Tryptophan Biosynthetic Enzymes in Histidine Mutants of Neurospora crassa

Carsiotis, M. (University of Cincinnati College of Medicine, Cincinnati, Ohio), and Ann M. Lacy. Increased activity of tryptophan biosynthetic enzymes in histidine mutants of Neurospora crassa. J. Bacteriol. 89:1472-1477. 1965.-The activities of tryptophan synthetase and indoleglycerol phosphate synthetase, the last two enzymes in the tryptophan biosynthetic pathway, are elevated twofold or more in all histidine mutants of Neurospora crassa. The elevation occurs only when the mutants are grown in a histidine-limited medium. Several possible mechanisms for this elevation were disproved experimentally. The cause of the elevated enzyme activities remains unknown.