Tryptophan Synthase from Saccharomyces cerevisiae Is a Dimer of Two Polypeptide Chains of Mr 76000 Each

Enzymes can be reconstituted with high yield after dissociation and deactivation in strong denaturants. This finding can be utilized to separate proteases and other impurities (with molecular weights differing under denaturing conditions from the subunit molecular weight of the enzyme to be purified) from the desired enzyme. Applying this approach to tryptophan synthase from yeast, a homogeneous preparation with a molecular weight of 154000 Jr 5000 and a specific activity of 1200 & 100 Yanofsky units/mg has been obtained. The latter value exceeds the previously reported maximum activity by a factor of 10, and characterizes the enzyme in its native state. The K , values for D-glyceraldehyde 3-phosphate and indole of the indole-glycerolphosphate synthesis (reaction 2 ) and the pH optimum, as well as the K,,, values for L-serine, indole, and pyridoxal5'-phosphate of the L-tryptophan condensation (reaction 3) have been determined.At least two tryptophan-synthase-inactivating proteinases from yeast have been well characterized in the past. Therefore, the low activity of previous preparations may be assumed to be caused by proteolytic cleavage. To prove this hypothesis, tryptophan synthase was prepared following a conventional procedure. Using ultracentrifugation and gel chromatography under quasi-physiological conditions, this enzyme has been found to be a homogeneous species of M , = 75000 f 3000 and = 3.3 k 0.2 S, which is still capable of catalyzing both reactions 2 and 3 with a specific activity for the latter reaction of 120 Yanofsky units/mg. Comparing the K, values for all relevant ligands, only the K, value for pyridoxal 5'-phosphate shows a marked difference with respect to the value observed for the native enzyme. At -18 "C the nicked enzyme is stable over a period of several weeks.As measured by sedimentation equilibrium in 6M guanidine . HCl at pH 2.3, the molecule consists of two large polypeptide fragments, M,,I = 22000 k 3000 and Mr,2 = 50000 + 5000.These results suggest tryptophan synthase from yeast to be a dimer of two bifunctional subunits; obviously the specific proteinases preferentially attack a region forming a link between two domains of the enzyme corresponding to the ci and / 3 subunits of the Escherichia coli enzyme.Tryptophan synthase isolated from different microorganisms catalyzes the last step in tryptophan biosynthesis :

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“…Similar results have been obtained using hydrophobic interaction chromatography with pentyl-Sepharose [46].…”

Section: Discussionsupporting

confidence: 76%

Purification of Tryptophan Synthase from Saccharomyces cerevisiae and Partial Activity of Its Nicked Subunits

1979

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“…Tryptophan synthases of fungi are homodimers (e.g. Dettwiler and Kirschner 1979), and the two functional domains are linked by non-conserved connector sequences.…”

Section: Introductionmentioning

confidence: 99%

The tryptophan synthase-encoding trpB gene of Aspergillus nidulans is regulated by the cross-pathway control system

Eckert¹,

Kübler²,

Hoffmann³

et al. 2000

Mol Gen Genet

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The tryptophan synthase-encoding gene, trpB, of Aspergillus nidulans was cloned and characterized. It was mapped to chromosome I, between the gene medA, which is required for sexual and asexual development, and an ORF encoding a protein with significant similarity to subunit B of vacuolar ATP synthases. The 5' untranslated region was found to be at least 142 nucleotides (nt) long, the poly(A) addition site was localized at position + 216 relative to the stop codon by sequencing of several independent cDNA clones. The trpB gene contains two exons separated by an intron of 105 nt, which is located close to the 5' end of the ORF. Directly upstream of the transcriptional start site, one well conserved potential binding site for the cross-pathway control transcriptional activator CPCA was found. The level of trpB transcript was shown to be regulated by cross-pathway control. A knockout mutant for trpB displays tryptophan auxotrophy, no trpB transcript is detectable, and development is perturbed to an extent that is dependent on the amount of tryptophan added to the medium. The trpB gene encodes a protein of 723 amino acids, with a calculated molecular weight of 77.6 kDa. The deduced amino acid sequence shows 72.6% similarity to the tryptophan synthase of Neurospora crassa. Most amino acid residues essential for catalytic activity in the tryptophan synthase of Salmonella typhimurium are conserved. The linker region joining the two domains of the enzyme is 13 residues longer than the longest connector found so far in tryptophan synthases from fungi.

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“…In contrast to the synthesis of separate alpha and beta subunits in bacteria, fungi such as Saccharomyces cerevisiae and Neurospora crassa produce active tryptophan synthase as a dimer of a single polypeptide, which contains fused TSA and TSB domains (Matchett and DeMoss 1975;Dettwiler and Kirschner 1979;Zalkin and Yanofsky 1982;Burns and Yanofsky 1989). This provides a mechanism for the coordinate regulation of subunit synthesis, a problem solved in bacteria by organizing the tryptophan synthase genes into an operon (Yanofsky and Crawford 1987).…”

Section: Introductionmentioning

confidence: 99%

Characterization of tryptophan synthase alpha subunit mutants of Arabidopsis thaliana

Radwanski

Barczak

1996

Mol Gen Genet

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Three mutations in the Arabidopsis thaliana gene encoding the alpha subunit of tryptophan synthase were isolated by selection for resistance to 5-methylanthranilate or 5-fluoroindole, toxic analogs of tryptophan pathway intermediates. Plants homozygous for trp3-1 and trp3-2 are light-conditional tryptophan auxotrophs, while trp3-100 is a more leaky mutant. Genetic complementation crosses demonstrated that the three mutations are allelic to each other, and define a new complementation group. All three mutants have decreased steady-state levels of tryptophan synthase alpha protein, and the trp3-100 polypeptide exhibits altered electrophoretic mobility. All three mutations were shown to be in the TSA1 (tryptophan synthase alpha subunit) structural gene by several criteria. Firstly, the trp3-1 mutation is linked to TSA1 on the bottom of chromosome 3. Secondly, the trp3-1 mutation was complemented when transformed with the wild-type TSA1 gene. Finally, DNA sequence analysis of the TSA1 gene revealed a single transition mutation in each trp3 mutant.

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Tryptophan Synthase from Saccharomyces cerevisiae Is a Dimer of Two Polypeptide Chains of Mr 76000 Each

Cited by 38 publications

References 40 publications

Purification of Tryptophan Synthase from Saccharomyces cerevisiae and Partial Activity of Its Nicked Subunits

Purification of Tryptophan Synthase from Saccharomyces cerevisiae and Partial Activity of Its Nicked Subunits

The tryptophan synthase-encoding trpB gene of Aspergillus nidulans is regulated by the cross-pathway control system

Characterization of tryptophan synthase alpha subunit mutants of Arabidopsis thaliana

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