Crystal structure of type II peptide deformylase from Staphylococcus aureus.

The first crystal structure of Class II peptide deformylase has been determined. The enzyme from Staphylococcus aureus has been overexpressed and purified in Escherichia coli and the structure determined by x-ray crystallography to 1.9 A resolution. The purified iron-enriched form of S. aureus peptide deformylase enzyme retained high activity over many months. In contrast, the iron-enriched form of the E. coli enzyme is very labile. Comparison of the two structures details many differences; however, there is no structural explanation for the dramatic activity differences we observed. The protein structure of the S. aureus enzyme reveals a fold similar, but not identical to, the well characterized E. coli enzyme. The most striking deviation of the S. aureus from the E. coli structure is the unique conformation of the C-terminal amino acids. The distinctive C-terminal helix of the latter is replaced by a strand in S. aureus which wraps around the enzyme, terminating near the active site. Although there are no differences at the amino acid level near the active site metal ion, significant changes are noted in the peptide binding cleft which may play a role in the design of general peptide deformylase inhibitors.

Expression, purification, and characterization of peptidyl-tRNA hydrolase from Staphylococcus aureus.

Bacterial peptidyl-tRNA hydrolase (Pth) activity ensures the rapid recycling of peptidyl-tRNAs that result from premature termination of translation. Pth has been shown to be essential for growth in Escherichia coli suggesting that its homologue in Staphylococcus aureus is a potential molecular therapeutic target for the development of antibacterial agents. In this report we describe the cloning of a DNA fragment (573 bp) containing the pth gene from a S. aureus (strain ISP3) genomic DNA library. Analysis of the predicted polypeptide sequence from the pth gene showed that the protein shared complete conservation of the three residues thought to be involved in the active site of E. coli Pth. The gene was cloned into a pQE-60 expression vector and expressed in E. coli, and the resulting His-tagged Pth protein was purified to greater than 95% purity from the soluble portion of the E. coli lysate in a single chromatographic step. His-tagged Pth was shown to be biologically active by its ability to hydrolyze diacetyl-[(3)H]Lys-tRNA(Lys) in a time- and concentration-dependent manner. Optimum hydrolyzing activity of Pth occurred at a pH value of 7.0 and a MgCl(2) concentration of 5 mM. The K(m) of the diacetyl-[(3)H]-Lys-tRNA(Lys) substrate for S. aureus Pth was determined to be 2.8 microM. A far UV circular dichroism spectrum revealed that His-tagged S. aureus Pth appears to have a structured core predominated by beta-sheet.

Microbiological Oxygenation of Bridgehead Azabicycloalkanes.

A series of N-substituted bridgehead azabicycloalkanes has been prepared and examined as substrates for microbiological oxygenation using the fungi Beauveria bassiana, Rhizopus nigricans, Aspergillus ochraceus, and Rhizopus arrhizus. Oxygenation using B. bassiana of N-tosyl-7-azabicyclo[2.2.1]heptane gave N-[p-(hydroxymethyl)benzenesulfonyl]-7-azabicyclo[2.2.1]heptane (56% yield), of N-(phenyloxycarbonyl)-7-azabicyclo[2.2.1]heptane gave the 2-endo-ol (56% yield, 51% ee), of N-BOC-7-azabicyclo[2.2.1]heptane gave the 2-endo-ol (10% yield), of N-Cbz-7-azabicyclo[2.2.1]heptane gave the 2-endo-ol (28%), of N-(phenyloxycarbonyl)-8-azabicyclo[3.2.1]octane gave the 3-endo-ol, and of N-(phenyloxycarbonyl)-9-azabicyclo[3.3.1]nonane gave the 3-exo-ol (30%) and 3-one (16%). Oxygenation using R. nigricans of N-BOC-7-azabicyclo[2.2.1]heptane gave the 2-endo-ol (62% yield, 28% ee) and the 2-exo-ol (27% yield, 42% ee). Oxidation of the N-BOC-7-azabicyclo[2.2.1]heptan-2-ols gives the 2-ketone, a synthetic intermediate useful for conversion to the natural product, epibatidine. Oxygenation of N-(phenyloxycarbonyl)-7-azabicyclo[2.2.1]heptane using R. arrhizus gives the 2-endo-ol (5% yield, 31% ee) and the 2-exo-ol (18% yield, 22% ee). Oxygenation of N-(phenyloxycarbonyl)-8-azabicyclo[3.2.1]octane using A. ochraceous gives the 3-endo-ol (36%) and the 3-one (4%).