Studies on the 5-enolpyruvylshikimate 3-phosphate synthase of Escherichia coli

1. A method for the purification of EPSP synthase of E. coli K12 has been developed. The purification procedure consisted of ammonium sulphate fractionation, ion-exchange chromatography and hydrophobic chromatography. The final step involved substrate elution from a phosphocellulose column. EPSP synthase was purified 843-fold and in 22% yield over the (NH4)2SO4 fraction. 2. E. coli EPSP synthase has been shown to be a monomeric enzyme. The subunit Mr was estimated to be 49,000 by SDS PAGE, and native Mr values of 42,000 and 55,000 were determined by gel filtration. Kinetic parameters for E. coli EPSP synthase are reported. The enzyme was inhibited by the herbicide glyphosate, inhibition was competitive with respect to phosphoenolpyruvate. 3. EPSP synthase has also been purified from an overproducing strain, E. coli AB2829/pKD501. The overproduced enzyme was purified 50-fold and in 30% yield over the crude extract fraction. EPSP synthase can be purified in milligram quantities from the overproducing strain. 4. The overproduced enzyme has been shown to be identical in its physical and kinetic properties to EPSP synthase purified from E. coli K12. The amino acid composition and N-terminal amino acid sequence of E. coli EPSP synthase are reported. 5. Chemical modification of EPSP synthase by 3-bromopyruvate has been examined. Although substrate protection against inactivation was observed, bromopyruvate did not appear to be an/ an active-site-directed reagent for E. coli EPSP synthase. 6. Phosphoserine aminotransferase has been purified from the overproducing strain, E. coli AB2829/pKD501. The purification procedure was similar to that developed for EPSP synthase; (NH4)2SO4 fractionation, ion-exchange chromatography and hydrophobic chromatography. The final step was ion-exchange chromatography on a mono-Q column. PSAT was purified approximately 7-fold over the crude extract fraction. 7. The subunit Mr of E. coli PSAT has been shown to be 39,000 and this enzyme appeared to be dimeric. The amino acid composition and N-terminal amino acid sequence of PSAT are reported. Some kinetic properties of this enzyme are also described

Missense translation errors in Saccharomyces cerevisiae

We describe the development of a novel plasmid-based assay for measuring the in vivo frequency of misincorporation of amino acids into polypeptide chains in the yeast Saccharomyces cerevisiae. The assay is based upon the measurement of the catalytic activity of an active site mutant of type III chloramphenicol acetyl transferase (CAT(III)) expressed in S. cerevisiae. A His195(CAC) --> Tyr195(UAC) mutant of CAT(III) is completely inactive, but catalytic activity can be restored by misincorporation of histidine at the mutant UAC codon. The average error frequency of misincorporation of histidine at this tyrosine UAC codon in wild-type yeast strains was measured as 0.5 x 10(-5) and this frequency was increased some 50-fold by growth in the presence of paromomycin, a known translational-error-inducing antibiotic. A detectable frequency of misincorporation of histidine at a mutant Ala195 GCU codon was also measured as 2 x 10(-5), but in contrast to the Tyr195 --> His195 misincorporation event, the frequency of histidine misincorporation at Ala195 GCU was not increased by paromomycin, inferring that this error did not result from miscognate codon-anticodon interaction. The His195 to Tyr195 missense error assay was used to demonstrate increased frequencies of missense error at codon 195 in SUP44 and SUP46 mutants. These two mutants have previously been shown to exhibit a translation termination error phenotype and the sup44(+) and sup46(+) genes encode the yeast ribosomal proteins S4 and S9, respectively. These data represent the first accurate in vivo measurement of a specific mistranslation event in a eukaryotic cell and directly confirm that the eukaryotic ribosome plays an important role in controlling missense errors arising from non-cognate codon-anticodon interactions

The crystal structure of E. coli pantothenate synthase confirms it as a member of the cytidyltransferase superfamily

BACKGROUND: Pantothenate synthetase (EC 6.3.2.1) is the last enzyme of the pathway of pantothenate (vitamin B(5)) synthesis. It catalyzes the condensation of pantoate with beta-alanine in an ATP-dependent reaction. RESULTS: We describe the overexpression, purification, and crystal structure of recombinant pantothenate synthetase from E. coli. The structure was solved by a selenomethionine multiwavelength anomalous dispersion experiment and refined against native data to a final R(cryst) of 22.6% (R(free) = 24.9%) at 1.7 A resolution. The enzyme is dimeric, with two well-defined domains per protomer: the N-terminal domain, a Rossmann fold, contains the active site cavity, with the C-terminal domain forming a hinged lid. CONCLUSIONS: The N-terminal domain is structurally very similar to class I aminoacyl-tRNA synthetases and is thus a member of the cytidylyltransferase superfamily. This relationship has been used to suggest the location of the ATP and pantoate binding sites and the nature of hinge bending that leads to the ternary enzyme-pantoate-ATP complex