Substrate recognition by the leucyl/phenylalanyl-tRNA-protein transferase. Conservation within the enzyme family and localization to the trypsin-resistant domain.

The leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase) from Escherichia coli catalyzes a peptidyltransferase reaction that results in the N-terminal aminoacylation of acceptor proteins using Leu-, Phe-, and Met-tRNAs as amino acid donors. We demonstrated that L/F-transferase homologs are widely distributed throughout the eubacteria, supporting our proposal that the enzyme family is ancient and catalyzes early peptide bond synthesis. However, here we present data suggesting that the L/F-transferase is not a homolog of the peptidyltransferase enzymes involved in cell wall peptidoglycan biosynthesis in Gram-positive species, such as Staphylococcus aureus. A sequence comparison of the known L/F-transferase homologs began to identify the essential residues required to catalyze a peptidyltransferase reaction and revealed that <20% of the residues were invariant within the L/F-transferase family. Despite this sequence variation, substrate specificity was broadly conserved, and L/F-transferase homologs from Providencia stuartii, Vibrio cholerae, Neisseria gonorrhoeae, and the cyanobacterium Synechocystis sp. all complemented an E. coli aat mutant (lacking L/F-transferase activity) for the degradation of N-end rule substrates. In vitro comparison of the most divergent L/F-transferase homologs, from E. coli and the cyanobacterium Synechocystis sp., revealed near-complete conservation of both substrate specificity and secondary structure. Finally, we demonstrated that variants of the E. coli L/F-transferase, lacking either 33 or 78 N-terminal residues, retained measurable peptidyltransferase activity and wild type substrate specificity. Overall, our results identified an essential core of an L/F-transferase and revealed that a peptidyltransferase catalyst may be constructed from approximately 120 amino acids.

Aminoacyl-tRNA recognition by the leucyl/phenylalanyl-tRNA-protein transferase.

We employ mutant and mischarged aminoacyl-tRNAs to characterize aminoacyl-tRNA recognition by the leucyl/phenylalanyl-tRNA-protein transferase (L/Ftransferase). Wild type Met-tRNAMetm (CAU anticodon) and mischarged Met-tRNAVal-1 (CAU anticodon) are substrates for the L/F-transferase during the NH2-terminal aminoacylation of alpha-casein, whereas Val-tRNAVal-1 (UAC), Val-tRNAMetm (UAC), and Arg-tRNAMetm (CCG, A20) are not. Mutations in the anticodon and extra arm of tRNALeu-1 do not measurably effect its ability to serve as a substrate for the L/F-transferase, and the dissociation constants of the complexes between L/F-transferase and either wild type Leu-tRNALeu-4 (UAA) or mutant Leu-tRNALeu-4 (CUA) are each 0.4 +/- 0.2 microM. The dissociation constants for the complexes between the L/F-transferase and uncharged tRNA, leucine methyl ester, and puromycin are all 10-1,000-fold greater than that of the Leu-tRNA.L/F-transferase complex. Dissociation of the Leu-tRNA.L/F-transferase complex is slow, relative to the rate calculated assuming that association is diffusion controlled. Finally, deoxyoligonucleotide.aminoacyl-tRNA hybrids (dO.AA-tRNAs) are employed to characterize the determinants of the Leu-tRNALeu-4 acceptor stem recognized by the L/F-transferase. A dO.AA-tRNA completely lacking acceptor stem base pairs remains a substrate for the L/F-transferase, whereas a dO.AA-tRNA containing a 2-base pair single-stranded region, at its 3' terminus, does not.

The yeast plasma membrane proton pumping ATPase is a viable antifungal target. I. Effects of the cysteine-modifying reagent omeprazole.

The yeast plasma membrane proton pumping ATPase (H(+)-ATPase) was investigated as a potential molecular target for antifungal drug therapy by examining the inhibitory effects of the sulfhydryl-reactive reagent omeprazole on cell growth, glucose-induced medium acidification and H(+)-ATPase activity. Omeprazole inhibits the growth of Saccharomyces cerevisiae and the human pathogenic yeast Candida albicans in a pH dependent manner. Omeprazole action is closely correlated with inhibition of the H(+)-ATPase and is fungicidal. Glucose-dependent medium acidification is correspondingly blocked by omeprazole and appears to require the H(+)-ATPase to proceed through its reaction cycle. A strong correlation is observed between inhibition of medium acidification and H(+)-ATPase activity in plasma membranes isolated from treated cells. The inhibitory properties of omeprazole are blocked by pre-treatment of activated drug with beta-mercaptoethanol, which is consistent with the expected formation of a sulfhydryl-reactive sulfenamide derivative. Mutagenesis of the three putative membrane sector cysteine residues (C148S, C312S, C867A) in the S. cerevisiae H(+)-ATPase suggests that covalent modification of the conserved C148 residue may be important for inhibition of ATPase activity and cell growth. Other mutations (M128C and G158D/G156C) mapping near C148 support the importance of this region by modulating omeprazole inhibition of the H(+)-ATPase. These findings suggest that the plasma membrane H(+)-ATPase may serve as an important molecular target for antifungal intervention.

The leucyl/phenylalanyl-tRNA-protein transferase. Overexpression and characterization of substrate recognition, domain structure, and secondary structure.

Previous work has shown that, in the bacterium Escherichia coli, the aat gene is essential for the degradation of proteins bearing amino-terminal Arg and Lys residues via the N-end rule pathway of protein degradation. We now show that the aat gene encodes directly the leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase). This enzyme catalyzes the transfer of Leu, Phe, and, less efficiently, Met and Trp, from aminoacyl-tRNAs, to the amino terminus of acceptor proteins. We have used the cloned aat gene to overexpress and purify an affinity tagged L/F-transferase. The recombinant L/F-transferase is as active as the previously purified wild type enzyme and contains no detectable RNA component. We have used the recombinant enzyme to demonstrate that both the solubility and substrate specificity, for aminoacyl-tRNA substrates, of the L/F-transferase are dependent on ionic strength conditions and that the modified nucleotides found in natural tRNAs are not essential for recognition by the enzyme. Limited digestion of the L/F-transferase with trypsin removes the proline rich NH2 terminus of the enzyme identifying a globular core, and circular dichroism demonstrates that the L/F-transferase is predominantly alpha-helical. Finally, a region of sequence conservation between the L/F-transferase and the NH2-terminal protein acetylases has been identified.

[The effect of bacteriolytic preparation lysoamidase on Staphylococcus aureus 209P cells]. / Deistvie bakterioliticheskogo preparata lizoamidaza na kletki Staphylococcus aureus 209P.

The effect of the bacteriolytic preparation "Lysoamidase" on Staphylococcus aureus 299 P was studied. The maximum activity of the preparation was observed at pH 8.0 ionic strength 0.01-0.02 M and 50-60 degrees of the incubation medium. The electron microscopic examination revealed that "Lysoamidase" hydrolyzed the cell wall in one or several points with the following osmotic shock and extrusion of the cytoplasm. In an isotonic solution (1 M sucrose) "Lysoamidase" caused protoplast formation.

[The role of the carotenoid pigments of staphylococcus in the response of cells to temperature changes]. / Rol' karotinoidnykh pigmentov stafilokokka v reaktsii kletok na izmenenie temperatury

A study was made of the reaction of the membranous apparatus of the pigmented strains of staphylococcus 209-P and its four apigmented variants to the action of various temperatures measured by the exit from the cells of the low molecular components. Permeability of the cell membranes in case of the action of the extreme temperatures of the 209-P strain altered much more than that of the apigment variants. It is supposed that the carotinoid pigments of the apathogenic staphylococci took part in the formation of functional lability of the bacterial membranes.