Developmentally regulated expression of a sunflower 11S seed protein gene in transgenic tobacco

Summary 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.Abbreviations PRA N-(5'-phosphoribosyl)anthranilate - InGP indole-3-glycerol phosphate - SDS sodium dodecyl sulfate     

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.     

Three-dimensional structure of the bifunctional enzyme phosphoribosylanthranilate isomerase: indoleglycerolphosphate synthase from Escherichia coli refined at 2.0 A resolution.

The three-dimensional structure of the monomeric bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli has been refined at 2.0 A resolution, using oscillation film data obtained from synchrotron radiation. The model includes the complete protein (452 residues), two phosphate ions and 628 water molecules. The final R-factor is 17.3% for all observed data between 15 and 2 A resolution. The root-mean-square deviations from ideal bond lengths and bond angles are 0.010 A and 3.2 degrees, respectively. The structure of N-(5'-phosphoribosyl)anthranilate isomerase: indole-3-glycerol-phosphate synthase from E. coli comprises two beta/alpha-barrel domains that superimpose with a root-mean-square deviation of 2.03 A for 138 C alpha-pairs. The C-terminal domain (residues 256 to 452) catalyses the PRAI reaction and the N-terminal domain (residues 1 to 255) catalyses the IGPS reaction, two sequential steps in tryptophan biosynthesis. The enzyme has the overall shape of a dumb-bell, resulting in a surface area that is considerably larger than normally observed for monomeric proteins of this size. The active sites of the PRAI and the IGPS domains, both located at the C-terminal side of the central beta-barrel, contain equivalent binding sites for the phosphate moieties of the substrates N-(5'-phosphoribosyl) anthranilate and 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate. These two phosphate binding sites are identical with respect to their positions within the tertiary structure of the beta/alpha-barrel, the conformation of the residues involved in phosphate binding and the hydrogen-bonding network between the phosphate ions and the protein. The active site cavities of both domains contain similar hydrophobic pockets that presumably bind the anthranilic acid moieties of the substrates. These similarities of the tertiary structures and the active sites of the two domains provide evidence that N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from E. coli results from a gene duplication event of a monomeric beta/alpha-barrel ancestor.     

Nucleotide sequence of Saccharomyces cerevisiae genes TRP2 and TRP3 encoding bifunctional anthranilate synthase: indole-3-glycerol phosphate synthase

Saccharomyces cerevisiae anthranilate synthase:indole-3-glycerol phosphate synthase is a multifunctional hetero-oligomeric enzyme encoded by genes TRP2 and TRP3. TRP2, encoding anthranilate synthase Component I, was cloned by complementation of a yeast trp2 mutant. The nucleotide sequence of TRP2 as well as that of TRP3 were determined. The deduced anthranilate synthase Component I primary structure from yeast exhibits only limited similarity to that of the corresponding Escherichia coli subunit encoded by trpE. On the other hand, yeast anthranilate synthase Component II and indole-3-glycerol phosphate synthase amino acid sequences from TRP3 are clearly homologous with the corresponding sequences of the E. coli trpG and trpC polypeptide segments and thereby establish the bifunctional structure of TRP3 protein. Based on comparisons of TRP3 amino acid sequence with homologous sequences from E. coli and Neurospora crassa, an 11-amino acid residue connecting segment was identified which fuses the trpG and trpC functions of the bifunctional TRP3 protein chain. These comparisons support the conclusion that the amino acid sequence of connectors in homologous multifunctional enzymes need not be conserved. Connector function is thus not dependent on a specific sequence. Nuclease S1 mapping was used to identify mRNA 5' termini. Heterogeneous 5' termini were found for both TRP2 and TRP3 mRNA. TRP2 and TRP3 5'-flanking regions were analyzed for sequences that might function in regulation of these genes by the S. cerevisiae general amino acid control system. The 9 base pair direct repeat (Hinnebusch, A.G., and Fink, G.R. (1983) J. Biol. Chem. 258, 5238-5247) and inverted repeats were identified in the 5'-flanking sequences of TRP2 and TRP3.     

Post-translational modification of a tryptophan biosynthetic enzyme. Precursor-product relationships in vivo and carbon-energy source dependence

Purified preparations of the bifunctional, tryptophan biosynthetic enzyme N-(5'-phosphoribosyl)anthranilate isomerase/indole-3-glycerol phosphate synthase of Escherichia coli K12 contain five forms of the protein (molecular mass = 49 kDa) which can be separated by isoelectric focusing or two-dimensional gel electrophoresis. The stability of the enzyme and its different forms was studied in exponentially growing, ammonium-starved and energy-depleted cultures using a dual-labeling, pulse-chase method. Labeled enzyme was isolated by standard purification techniques and by immunoprecipitation from crude extracts. The results demonstrated the following: 1) post-translational modification occurred in vivo, 2) modification occurred to the same extent in growing and ammonium-starved cells, 3) modification was dependent on a carbon-energy source for the cells, and 4) the enzyme was not degraded in growing or nongrowing cells. The pulse-chase data also indicated that one form of the protein (band 1) was the precursor of three other forms (bands 3-5) and that the relative amount of one form (band 2) remained constant.     

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.     

Analysis of the sexual development-promoting region of Schizophyllum commune TRP1 gene

This study aims to elucidate the mechanism of sexual development of basidiomycetous mushrooms from mating to fruit body formation. Sequencing analysis showed the TRP1 gene of basidiomycete Schizophyllum commune encoded an enzyme with three catalytic regions of GAT (glutamine amidotransferase), IGPS (indole-3-glycerol phosphate synthase), and PRAI (5-phosphoribosyl anthranilate isomerase); among these three regions, the trp1 mutant (Trp^?) had a missense mutation (L→F) of a 338th amino acid residue of the TRP1 protein within the IGPS region. To investigate the function of IGPS region related to sexual development, dikaryons with high, usual, and no expression of the IGPS region of TRP1 gene were made. The dikaryotic mycelia with high expression of the IGPS formed mature fruit bodies earlier than those with usual and no expression of the IGPS. These results showed that the IGPS region in TRP1 gene promoted sexual development of S. commune.     

Protein engineering of a disulfide bond in a beta/alpha-barrel protein.

A disulfide bond has been introduced in the beta/alpha-barrel enzyme N-(5'-phosphoribosyl)anthranilate isomerase from Saccharomyces cerevisiae. The design of this disulfide bond was based on a model structure of this enzyme, built from the high-resolution crystal structure of the N-(5'-phosphoribosyl)anthranilate isomerase domain from Escherichia coli. The disulfide cross-link is spontaneously formed in vitro between residues 27 and 212, located in the structurally adjacent alpha-helices 1 and 8 of the outer helical ring of the beta/alpha-barrel. It creates a loop of 184 residues that account for 83% of the sequence of this enzyme, thus forming a quasi circular protein. The cross-linked mutant enzyme displays wild-type steady-state kinetic parameters. Measurements of the equilibrium constant for the reduction of this disulfide bond by 1,4-dithiothreitol show that its bond strength is comparable to that of other engineered protein disulfide bonds. The oxidized, cross-linked N-(5'-phosphoribosyl)anthranilate isomerase mutant is about 1.0 kcal/mol more stable than the wild-type enzyme, as estimated from its equilibrium unfolding transitions by guanidine hydrochloride.     

Crystallization and structure solution at 4 A resolution of the recombinant synthase domain of N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli complexed to a substrate analogue

The recombinant synthase domain of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli has been crystallized, and the structure has been solved at 4 A resolution. Two closely related crystal forms grown from ammonium sulphate diffract to 2 A resolution. One form (space group R32, a = 163 A, alpha = 29.5 degrees) contains the unliganded synthase domain; the second crystal form (space group P6(3)22, a = 144 A, c = 158 A) is co-crystallized with the substrate analogue N-(5'-phosphoribit-1-yl)anthranilate. The structure of the synthase-inhibitor complex has been solved by the molecular replacement method. This achievement represents the first successful use of a (beta alpha)8-barrel monomer as a trial model. The recombinant synthase domain associates as a trimer in the crystal, the molecules being related by a pseudo-crystallographic triad. The interface contacts between the three domains are mediated by those residues that are also involved in the domain interface of the bifunctional enzyme. This system provides a model for an interface which is used in both intermolecular and intramolecular domain contacts.     

Purification and characterization of the trifunctional beta-subunit of anthranilate synthase from Neurospora crassa

The trifunctional beta-subunit of anthranilate synthase complex of Neurospora crassa has been purified from a mutant which produces no detectable alpha-subunit. The isolated beta-subunit appeared to be a highly asymmetric dimer with a s20,w of 7.35 and an apparent molecular weight of 200,000 as determined by gel filtration on Sephacryl S-300 compared with a monomer molecular weight of approximately 84,000 Da as determined by sodium dodecyl sulfate-gel electrophoresis. The purified subunit was cleaved by elastase, trypsin, or chymotrypsin into fragments which retained the three enzyme activities. After elastase digestion, two active fragments were separated by gel filtration and ion exchange chromatography. A 30,000-Da fragment, which behaved as a monomer on gel filtration, interacted with free alpha-subunit to produce glutamine-dependent anthranilate synthase activity. A second 56,000-Da fragment, which behaved as an asymmetric dimer (apparent molecular weight 140,000) on gel filtration, retained both N-(5'-phosphoribosyl)anthranilate isomerase and indole-3-glycerol phosphate synthase activity. The failure to detect an NH2-terminal amino acid residue on either the intact beta-subunit or the 30,000-Da complementing fragment, while the 56,000-Da fragment possessed an NH2-terminal histidine residue, indicated that the complementing fragment was derived from the NH2-terminal sequence of the beta-subunit.     

Analysis of feedback-resistant anthranilate synthases from Saccharomyces cerevisiae.

The initial step of tryptophan biosynthesis is catalyzed by the enzyme anthranilate synthase, which in most microorganisms is subject to feedback inhibition by the end product of the pathway. We have characterized the TRP2 gene from a mutant Saccharomyces cerevisiae strain coding for an anthranilate synthase that is unresponsive to tryptophan. Sequence analysis of this TRP2(Fbr) (feedback-resistant) allele revealed numerous differences from a previously published TRP2 sequence. However, TRP2(Fbr) was found to differ in only one single-point mutation from its own parent wild type, a C-to-T transition resulting in a serine 76-to-leucine 76 amino acid substitution. Therefore, serine 76 is a crucial amino acid for proper regulation of the yeast enzyme. We constructed additional feedback-resistant enzyme forms of the yeast anthranilate synthase by site-directed mutagenesis of the conserved LLES sequence in the TRP2 gene. From analysis of these variants, we propose an extended sequence, LLESX10S, as the regulatory element in tryptophan-responsive anthranilate synthases from prokaryotic and eukaryotic organisms.     

Purification and characterization of the indole-3-glycerolphosphate synthase/anthranilate synthase complex of Saccharomyces cerevisiae

The indole-3-glycerolphosphate synthase/anthranilate synthase complex from Saccharomyces cerevisiae was purified to apparent homogeneity. The native complex with Mr approximately equal to 130 000 consists of two different subunits, the TRP2 gene product with Mr = 64 000 and the TRP3 gene product with Mr = 58 000. The larger polypeptide was identified as anthranilate synthase and is active in vitro with ammonia as cosubstrate without need of complex formation. The smaller polypeptide carries both glutamine amidotransferase activity and indole-3-glycerolphosphate synthase activity. Various steady-state kinetic parameters as well as the amino acid composition of the two polypeptides were determined.     

The TRP1 gene of Phytophthora parasitica encoding indole-3-glycerolphosphate synthase-N-(5'-phosphoribosyl)anthranilate isomerase: structure and evolutionary distance from homologous fungal genes.

The TRP1 gene was isolated from the genome of Phytophthora parasitica. It encodes bifunctional enzyme of the tryptophan biosynthetic pathway indole-3-glycerolphosphate synthase-N-(5'-phosphoribosyl)anthranilate isomerase (IGPS-PRAI). The gene was localized and sequenced using random in vitro insertions of omega interposon. The domain structure of the protein product was found to be similar to that of enteric bacteria but different from the structure of homologous enzymes in fungi. Two introns in the IGPS domain were found. This is unique in eukaryotic IGPS-encoding genes so far sequenced. Comparative analysis of the primary structure of IGPS and PRAI domains [neighbor-joining method of Saitou and Nei, Mol. Biol. Evol. 44 (1987) 406-425] confirmed a large phylogenetic distance of TRP1 from corresponding fungal genes. In the resulting distance tree Phytophthora sequences are located outside of the cluster which encompasses all known homologous proteins from fungi indicating that the lineage of oomycetes took a separate course of development before speciation within the fungal line of descent began. Two of the oligopeptide insertions engineered into the F domain of the protein product did not abolish the enzymatic activity of the protein.     

Measurement of the Five Enzymes which Convert Chorismate to Tryptophan in Wheat Plants (Triticum aestivum cv. Kalyansona)

Conditions are described for measuring anthranilate synthetase, anthranilate-PRPP-phosphoribosyl transferase, N-5′-phosphoribosyl anthranilate isomerase, indole-3-glycerol phosphate synthetase and tryptophan synthetase in crude extracts from Triticum aestivum (wheat) plants. Only the last enzyme has been measured before in extracts from green plants. The extractable quantities of each enzyme in all plant parts at all stages of growth were sufficient to synthesize the amount of tryptophan present within the same tissue in 48 h. Anthranilate synthetase activity was the lowest of the five enzyme activities and was the only one inhibited by tryptophan in vitro, indicating that this enzyme may be the control point in tryptophan biosynthesis in wheat plants.     

Mechanism of 3-Methylanthranilic Acid Derepression of the Tryptophan Operon in Escherichia coli

3-Methylanthranilic acid (3MA) inhibits growth and causes derepression of the tryptophan biosynthetic enzymes in wild-type strains of Escherichia coli. Previous reports attributed this effect to an inhibition of the conversion of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate to indole-3-glycerol phosphate and a consequent reduction in the concentration of endogenous tryptophan. Our studies have shown that 3MA-resistant mutants linked to the tryptophan operon have a feedback-resistant anthranilate synthetase; mutants with an altered indole-3-glycerol phosphate synthetase were not found. 3MA or 7-methylindole can be metabolized to 7-methyltryptophan, and 3MA, 7-methylindole, and 7-methyltryptophan lead to derepression of the tryptophan operon. Furthermore, 3MA-resistant mutants are also resistant to 7-methylindole derepression. These results strongly suggest that the primary cause of derepression by 3MA is through its conversion to 7-methyltryptophan, which can inhibit anthranilate synthetase, thereby decreasing the concentration of endogenous tryptophan. Unlike 5- or 6-methyltryptophan, 7-methyltryptophan does not appear to function as an active corepressor.     

An evolutionary comparison of Acinetobacter calcoaceticus trpF with trpF genes of several organisms

The deduced amino acid sequence of Acinetobacter calcoaceticus N-(5'-phosphoribosyl) anthranilate isomerase (PRAI), which is coded by trpF, was compared with TrpF of Caulobacter crescentus, Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Neurospora crassa, and Aspergillus nidulans. Sixty percent of identical or similar amino acids were located in alpha/beta TIM (triose-phosphate isomerase) barrels and in residues important in substrate binding and catalysis. In addition, the analysis of trpF genes presented here supports a model by which fusion between separate trpC and trpF genes arose in some cases by in-frame deletions.     

The catalytic mechanism of indole-3-glycerol phosphate synthase: crystal structures of complexes of the enzyme from Sulfolobus solfataricus with substrate analogue,substrate, and product

Indoleglycerol phosphate synthase catalyzes the ring closure of an N-alkylated anthranilate to a 3-alkyl indole derivative, a reaction requiring Lewis acid catalysis in vitro. Here, we investigated the enzymatic reaction mechanism through X-ray crystallography of complexes of the hyperthermostable enzyme from Sulfolobus solfataricus with the substrate 1-(o-carboxyphenylamino) 1-deoxyribulose 5-phosphate, a substrate analogue and the product indole-3-glycerol phosphate. The substrate and the substrate analogue are bound to the active site in a similar, extended conformation between the previously identified phosphate binding site and a hydrophobic pocket for the anthranilate moiety. This binding mode is unproductive, because the carbon atoms that are to be joined are too far apart. The indole ring of the bound product resides in a second hydrophobic pocket adjacent to that of the anthranilate moiety of the substrate. Although the hydrophobic moiety of the substrate moves during catalysis from one hydrophobic pocket to the other, the triosephosphate moiety remains rigidly bound to the same set of hydrogen-bonding residues. Simultaneously, the catalytically important residues Lys53, Lys110 and Glu159 maintain favourable distances to the atoms of the ligand undergoing covalent changes. On the basis of these data, the structures of two putative catalytic intermediates were modelled into the active site. This new structural information and the modelling studies provide further insight into the mechanism of enzyme-catalyzed indole synthesis. The charged epsilon-amino group of Lys110 is the general acid, and the carboxylate group of Glu159 is the general base. Lys53 guides the substrate undergoing conformational transitions during catalysis, by forming a salt-bridge to the carboxylate group of its anthranilate moiety.