Deletion plasmids from transformants of Pseudomonas aeruginosa trp cells with the RSF1010-trp hybrid plasmid and high levels of enzyme activity from the gene on the plasmid.
A RSF1010-trp hybrid plasmid which contained the tryptophan operon of Escherichia coli was introduced into Pseudomonas aeruginosa trp cells by transformation. From the Trp+ transformants several deletion plasmids were obtained, and their physical maps with restriction endonucleases were constructed. P. aeruginosa trp cells with these plasmids showed at first more than 100 times higher levels of tryptophan synthetase beta activity over that of the control P. aeruginosa wild-type cells, but these levels were drastically decreased by 1 week of successive transfers of cultures. This decrease in enzyme activity was found to be due to the change on the plasmids but not to the host cells. The production of E. coli tryptophan synthetase beta enzyme in P. aeruginosa cells was proved by immunological test. (+info)
Tissue-specific expression of the beta-subunit of tryptophan synthase in Camptotheca acuminata, an indole alkaloid-producing plant.
Camptothecin is an anticancer drug produced by the monoterpene indole alkaloid pathway in Camptotheca acuminata. As part of an investigation of the camptothecin biosynthetic pathway, we have cloned and characterized a gene from C. acuminata encoding the beta-subunit of tryptophan (Trp) synthase (TSB). In C. acuminata TSB provides Trp for both protein synthesis and indole alkaloid production and therefore represents a junction between primary and secondary metabolism. TSB mRNA and protein were detected in all C. acuminata organs examined, and their abundance paralleled that of camptothecin. Within each shoot organ, TSB was most abundant in vascular tissues. Within the root, however, TSB expression was most abundant in the outer cortex. TSB has been localized to chloroplasts in Arabidopsis, but there was little expression of TSB in C. acuminata tissues where the predominant plastids were photosynthetically competent chloroplasts. Expression of the promoter from the C. acuminata TSB gene in transgenic tobacco plants paralleled expression of the native gene in C. acuminata in all organs except roots. TSB is also highly expressed in C. acuminata during early seedling development at a stage corresponding to peak accumulation of camptothecin, consistent with the idea that Trp biosynthesis and the secondary indole alkaloid pathway are coordinately regulated. (+info)
Identifying the structural boundaries of independent folding domains in the alpha subunit of tryptophan synthase, a beta/alpha barrel protein.
Two equilibrium intermediates have previously been observed in the urea denaturation of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, an eight-stranded beta/alpha barrel protein. In the current study, a series of amino-terminal fragments were characterized to probe the elementary folding units that may be in part responsible for this complex behavior. Stop-codon mutagenesis was used to produce eight fragments ranging in size from 105-214 residues and containing incremental elements of secondary structure. Equilibrium studies by circular dichroism indicate that all of these fragments are capable of adopting secondary structure. All except for the shortest fragment fold cooperatively. The addition of the fourth, sixth, and eighth beta-strands leads to distinct increases in structure, cooperativity, and/or stability, suggesting that folding involves the modular assembly of betaalphabeta supersecondary structural elements. One-dimensional NMR titrations at high concentrations of urea, probing the environment around His92, were also performed to test for the presence of residual structure in the fragments. All fragments that contained the first four betaalpha units of structure exhibited a cooperative unfolding transition at high concentrations of urea with significant but reduced stability relative to the full-length protein. These results suggest that the residual structure in alphaTS requires the participation of hydrophobic residues in multiple beta-strands that span the entire sequence. (+info)
The progressive development of structure and stability during the equilibrium folding of the alpha subunit of tryptophan synthase from Escherichia coli.
The urea-induced equilibrium unfolding of the alpha subunit of tryptophan synthase (alphaTS), a single domain alpha/beta barrel protein, displays a stable intermediate at approximately 3.2 M urea when monitored by absorbance and circular dichroism (CD) spectroscopy (Matthews CR, Crisanti MM, 1981, Biochemistry 20:784-792). The same experiment, monitored by one-dimensional proton NMR, shows another cooperative process between 5 and 9 M urea that involves His92 (Saab-Rincon G et al., 1993, Biochemistry 32:13,981-13,990). To further test and quantify the implied four-state model, N <--> I1 <--> I2 <--> U, the urea-induced equilibrium unfolding process was followed by tyrosine fluorescence total intensity, tyrosine fluorescence anisotropy and far-UV CD. All three techniques resolve the four stable states, and the transitions between them when the FL total intensity and CD spectroscopy data were analyzed by the singular value decomposition method. Relative to U, the stabilities of the N, I1, and I2 states are 15.4, 9.4, and 4.9 kcal mol(-1), respectively. I2 partially buries one or more of the seven tyrosines with a noticeable restriction of their motion; it also recovers approximately 6% of the native CD signal. This intermediate, which is known to be stabilized by the hydrophobic effect, appears to reflect the early coalescence of nonpolar side chains without significant organization of the backbone. I1 recovers an additional 43% of the CD signal, further sequesters tyrosine residues in nonpolar environments, and restricts their motion to an extent similar to N. The progressive development of a higher order structure as the denaturant concentration decreases implies a monotonic contraction in the ensemble of conformations that represent the U, I2, I1, and N states of alphaTS. (+info)
The Rhizobium etli trpB gene is essential for an effective symbiotic interaction with Phaseolus vulgaris.
A mutant strain (CTNUX4) of Rhizobium etli carrying Tn5 unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a trpB (tryptophan synthase)-homologous gene. When tested on the roots of Phaseolus vulgaris, strain CTNUX4 was able to induce only small, slightly pink, ineffective (Fix-) nodules. However, under free-living conditions, strain CTNUX4 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) unless tryptophan was added to the growth medium. These data and histological observations indicate that the lack of tryptophan biosynthesis affects the symbiotic behavior of R. etli. (+info)
Catalytic mechanism of the tryptophan synthase alpha(2)beta(2) complex. Effects of pH, isotopic substitution, and allosteric ligands.
The mechanism of the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium is explored by determining the effects of pH, of temperature, and of isotopic substitution on the pyridoxal phosphate-dependent reaction of L-serine with indole to form L-tryptophan. The pH dependence of the kinetic parameters indicates that three ionizing groups are involved in substrate binding and catalysis with pK(a)1 = 6.5, pK(a)2 = 7.3, and pK(a)3 = 8.2-9. A significant primary isotope effect (approximately 3.5) on V and V/K is observed at low pH (pH 7), but not at high pH (pH 9), indicating that the base that accepts the alpha-proton (betaLys-87) is protonated at low pH, slowing the abstraction of the alpha-proton and making this step at least partially rate-limiting. pK(a)2 is assigned to betaLys-87 on the basis of the kinetic isotope effect results and of the observation that the competitive inhibitors glycine and oxindolyl-L-alanine display single pK(i) values of 7.3. The residue with this pK(a) (betaLys-87) must be unprotonated for binding glycine or oxindolyl-L-alanine, and, by inference, L-serine. Investigations of the temperature dependence of the pK(a) values support the assignment of pK(a)2 to betaLys-87 and suggest that the ionizing residue with pK(a)1 could be a carboxylate, possibly betaAsp-305, and that the residue associated with a conformational change at pK(a)3 may be betaLys-167. The occurrence of a closed to open conformational conversion at high pH is supported by investigations of the effects of pH on reaction specificity and on the equilibrium distribution of enzyme-substrate intermediates. (+info)
Autogenous regulation of the inducible tryptophan synthase of Pseudomonas putida.
Mutants blocked before indole-3-glycerol phosphate formation in the tryptophan biosynthetic pathway of P. putida ("early-blocked" mutants) are unable to use indole as a source of tryptophan for growth on minimal medium. The uninduced level of tryptophan synthase [EC 188.8.131.52; L-serine hydro-lyase (adding indole)] in such mutants was thought to be responsible for this property. We have shown that levels of indole higher than those previously tested will support growth of these mutants. In addition, the growth rate of these mutants on a given indole concentration was shown to be proportional to the synthase level induced under the same conditions. This apparent induction of tryptophan synthase by indole in "early-blocked" mutants was shown to be caused by formation of the normal effector molecule, indole-3-glycerol-P, from indole. Secondary mutations occur in "early-blocked" trp strains, which enable them to grow on low concentrations of indole. One type of "indole-utilization" mutation occurs in the trpA gene, inactivating its product. Tryptophan synthase is readily induced by low concentrations of indole in these mutants, even though they are unable to convert indole to indole-3-glycerol-P. We propose that the alpha-chain of the synthase has an autogenous regulatory function, serving as the repressor or the indole-3-glycerol-P recognition component of the repressor of the trpAB operon (synthase alpha-and beta-chains). Our hypothesis holds that the trpA type of "indole-utilization" mutation alters the repressor (synthase alpha-chain) so that indole as well as indole-3-glycerol-P serves as an effector molecule for tryptophan synthase induction. (+info)
The aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate: the effects of the R386A and R292V mutations on this exchange reaction.
1H-NMR was used to follow the aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate. The effect of the concentrations of both the amino acids and the cognate keto acids on exchange rates was determined for wild-type and the R386A and R292V mutant forms of aspartate aminotransferase. The wild-type enzyme is found to be highly stereospecific for the exchange of the alpha-protons of L-aspartate and L-glutamate. The R386A mutation which removes the interaction of Arg-386 with the alpha-carboxylate group of aspartate causes an approximately 10,000-fold decrease in the first order exchange rate of the alpha-proton of L-aspartate. The R292V mutation which removes the interaction of Arg-292 with the beta-carboxylate group of L-aspartate and the gamma-carboxylate group of L-glutamate causes even larger decreases of 25,000- and 100,000-fold in the first order exchange rate of the alpha-proton of L-aspartate and L-glutamate respectively. Apparently both Arg-386 and Arg-292 must be present for optimal catalysis of the exchange of the alpha-protons of L-aspartate and L-glutamate, perhaps because the interaction of both these residues with the substrate is essential for inducing the closed conformation of the active site. (+info)