A rationally designed monomeric variant of anthranilate phosphoribosyltransferase from Sulfolobus solfataricus is as active as the dimeric wild-type enzyme but less thermostable.

The anthranilate phosphoribosyltransferase from Sulfolobus solfataricus (ssAnPRT) forms a homodimer with a hydrophobic subunit interface. To elucidate the role of oligomerisation for catalytic activity and thermal stability of the enzyme, we loosened the dimer by replacing two apolar interface residues with negatively charged residues (mutations I36E and M47D). The purified double mutant I36E+M47D formed a monomer with wild-type catalytic activity but reduced thermal stability. The single mutants I36E and M47D were present in a monomer-dimer equilibrium with dissociation constants of about 1 microM and 20 microM, respectively, which were calculated from the concentration-dependence of their heat inactivation kinetics. The monomeric form of M47D, which is populated at low subunit concentrations, was as thermolabile as monomeric I36E+M47D. Likewise, the dimeric form of I36E, which was populated at high subunit concentrations, was as thermostable as dimeric wild-type ssAnPRT. These findings show that the increased stability of wild-type ssAnPRT compared to the I36E+M47D double mutant is not caused by the amino acid exchanges per se but by the higher intrinsic stability of the dimer compared to the monomer. In accordance with the negligible effect of the mutations on catalytic activity and stability, the X-ray structure of M47D contains only minor local perturbations at the dimer interface. We conclude that the monomeric double mutant resembles the individual wild-type subunits, and that ssAnPRT is a dimer for stability but not for activity reasons.

Purification, characterization and crystallization of thermostable anthranilate phosphoribosyltransferase from Sulfolobus solfataricus.

Anthranilate phosphoribosyltransferase (TrpD; EC 2.4.2.18) from the hyperthermophilic archaeon Sulfolobus solfataricus (ssTrpD) was expressed in Escherichia coli, purified and crystallized. Analytical gel permeation chromatography revealed a homodimeric composition of the enzyme. The steady-state kinetic characteristics suggest tight binding of the substrate anthranilic acid and efficient catalysis at the physiological growth temperature of S. solfataricus. Crystals of ssTrpD diffract to better than 2.6 A resolution and preliminary X-ray characterization was carried out. The crystals are suitable for structure determination.

Subunit communication in the anthranilate synthase complex from Salmonella typhimurium.

The anthranilate synthase-phosphoribosyl transferase complex of the tryptophan biosynthetic pathway in Salmonella typhimurium is an allosteric, heterotetrameric (TrpE2-TrpD2) enzyme whose multiple activities are negatively feedback-regulated by L-tryptophan. A hybrid complex containing one catalytically active, feedback-insensitive and one catalytically inactive, feedback-sensitive mutant TrpE subunit was assembled in vitro and used to investigate communication between regulatory and catalytic sites located on different subunits. The properties of the hybrid complex demonstrate that the binding of a single inhibitor molecule to one TrpE subunit is sufficient for the propagation of a conformational change that affects the active site of the companion subunit.

Purification and characterization of yeast anthranilate phosphoribosyltransferase.

Anthranilate phosphoribosyltransferase from Saccharomyces cerevisiae has been purified to homogeneity from an overproducing strain. Analytical ultracentrifugation demonstrated that the enzyme is a dimer of Mr = 83,000 +/- 4,000 (S20.w = 4.7 S). Moreover, as shown by active enzyme sedimentation, the enzyme remains dimeric even at low concentrations. The presence of yeast phosphoribosylanthranilate isomerase in the gradient does not lead to complex formation between the two enzymes as might be expected if phosphoribosyl anthranilate, the very labile product of the anthranilate phosphoribosyltransferase, were channelled to phosphoribosylanthranilate isomerase in vivo. The steady-state-kinetic behaviour of the enzyme suggests that catalysis involves a ternary enzyme-substrate complex, with KANTm = 1.6 microM, and KPRib-PPm = 22.4 microM. The enzyme has been used to generate phosphoribosylanthranilate in situ for kinetic studies of phosphoribosylanthranilate isomerase from Escherichia coli: KPRAm = 5 microM, kcat = 40 s-1.

Characterization and regulation of anthranilate synthetase from a chloramphenicol-producing streptomycete.

In Streptomyces sp. 3022a, anthranilate synthetase is composed of two non-identical subunits. The major subunit (molecular weight, 72,000) converts chorismic acid to anthranilic acid, using ammonia as the source of the amino group. The smaller subunit (molecular weight 28,000 to 29,000) confers on the enzyme the ability to use glutamine instead of ammonia as a substrate. In this study, reactivity with glutamine reached its maximum at pH 7.2 to 7.6, whereas that with ammonia increased linearly through pH 9.0 without reaching a maximum. Activity was increased and stabilized by adding glutamine and magnesium chloride to the buffer system. Both activities of the enzyme were inhibited by anthranilic acid and by tryptophan. Synthesis was repressed by histidine, anthranilic acid, tryptophan, and p-aminobenzoic acid. When activity was repressed by anthranilic acid and by tryptophan, there was a concomitant increase in the activity of arylamine synthetase, an enzyme involved in chloramphenicol production. Stimulating arylamine synthetase, however, did not increase antibiotic synthesis.

Purification and properties of a third form of anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase from the Enterobacteriaceae.

Anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase was purified from the bacterium Erwinia carotovora, a member of the Enterobacteriaceae. The enzyme was homogeneous according to the criteria of gel electrophoresis and NH2-terminal amino acid sequence analysis. The molecular weight of the enzyme as determined on a calibrated Sephadex G-200 column was 67,000 +/- 2,000. Sodium dodecyl sulfate-polyacrylamide gels gave a subunit molecular weight of 40,000 +/- 1,000, suggesting that the enzyme was a dimer. A comparison of the NH2-terminal sequence of the enzyme with the (previously determined) homologue from Serratia marcescens, a monomer with a molecular weight of 45,000, showed that the larger Serratia subunit came into register with amino acid 14 of the Erwinia subunit. The register for the length of the known overlap, 26 amino acids, was highly conserved.

A dimer of a single polypeptide chain catalyzes the terminal four reactions of the L-tryptophan pathway in Euglena gracilis.

In Euglena gracilis the terminal four enzyme activities of the tryptophan biosynthetic pathway were found to be associated with a protein with an estimated molecular weight of 325,000 +/- 20,000. The protein was purified approximately 2,000-fold with relatively proportional recoveries of all four enzyme activities. The purified material was homogeneous by the criteria of analytical disc gel electrophoresis and gel isoelectric focusing. Disc gel electrophoresis after denaturation with sodium dodecyl sulfate gave a single protein band with a molecular weight of 155,000 +/- 5,000. Disc gel electrophoresis in 8 M urea also gave rise to a single protein band. We interpret these results as evidence for a single species of subunit. The pathway in Euglena is the only one known to the present in which the terminal enzyme, tryptophan synthase, is not a separate molecular species.

Anthranilate synthase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferases from Salmonella typhimurium. Purification of the enzyme complex and analysis of multiple forms.

1. The anthranilate synthase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase enzyme complex (chorismate pyruvatelyase (amino-accepting), EC 4.1.3.27) - (N-(5'-phosphoribosyl)-anthranilate: pyrophosphate phosphoribosyltransferase, EC 2.4.2.18), from Salmonella typhimurium has been purified with high yields to homogeneity. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme complex revealed one major band containing 96% of the protein. The final yield of enzyme complex activity ranged from 30 to 60%. The absorbance spectrum of enzyme complex showed a peak at 280 nm and fine structure with peaks at 253, 259, 266 and 269 nm. These latter wavelengths correspond closely with the known absorbance maxima of phenylalanine. 2. When purified enzyme complex was subjected to standard gel electrophoresis, a four band pattern of protein peaks was consistently observed. The major enzyme complex band was apparently the native tetramer, having a molecular weight 280 000 and containing ammonia- and glutamine-dependent anthranilate synthase activity. The other three bands were molecular weight isomers of the major enzyme complex band. Two forms of molecular weight isomers were present: dimers and an aggregate of the native enzyme complex. The molecular weight isomers of the enzyme complex may represent forms generated by aggregation and denaturation of the native enzyme complex. 3. A new and highly sensitive spectrophotometric assay for phosphoribosyl-transferase is described. The method is based upon the difference in extinction coefficients between anthranilate and N-(5'-phosphoribosyl)anthranilate.

Purification, subunit structure and partial amino-acid sequence of anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase from the enteric bacterium Serratia marcescens.

The enzyme anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase from Serratia marcescens was purified to apparent homogeneity. The purification procedure included ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex gel filtration and hydroxyapatite chromatography. The molecular weight of the native protein as determined on a calibrated Sephadex G-200 column was 45000. Dodecylsulfate-polyacrylamide gel electrophoresis in the presence of reducing agent revealed a subunit molecular weight of 43000 +/- 900, suggesting that the enzyme exists as a monomer. The sequence of the amino-terminal 38 residues revealed that three amino amino acids, glutamine (six residues), glutamic acid (five residues) and serine (five residues) comprised 42% of the sequence composition.