Co-crystallization of Staphylococcus aureus peptide deformylase (PDF) with potent inhibitors.

In bacteria the biosynthesis of all nascent polypeptides begins with N-formylmethionine. The post-translational removal of the N-formyl group is carried out by peptide deformylase (PDF). Processing of the N-formyl group from critical bacterial proteins is required for cell survival. This formylation/deformylation cycle is unique to eubacteria and is not utilized in eucaryotic cytosolic protein biosynthesis. Thus, inhibition of PDF would halt bacterial growth, spare host cell-function, and would be a novel mechanism for a new class of antibiotic. Diffraction-quality Se-met crystals of S. aureus PDF were prepared that belong to space group C222(1) with unit cell parameters of a = 94.1 b = 121.9 c = 47.6 A. Multiple anomalous dispersion data were collected at the Advanced Photon Source 17-ID beamline and used to solve the PDF structure to 1.9 A resolution. Crystals were also prepared with three PDF inhibitors: thiorphan, actinonin and PNU-172550. The thiorphan and actinonin co-crystals belong to space group C222(1) with similar unit-cell dimensions. Repeated attempts to generate a complex structure of PDF with PNU-172550 from the orthorhombic space group were unsuccessful. Crystallization screening identified an alternate C2 crystal form with unit-cell dimensions of a = 93.4 b = 42.5 c = 104.1 A, beta = 93 degrees.

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.

Marcfortine and paraherquamide class of anthelmintics: discovery of PNU-141962.

Three distinct chemical classes for the control of gastrointestinal nematodes are available: benzimidazoles, imidazothiazoles, and macrocyclic lactones. The relentless development of drug resistance has severely limited the usefulness of such drugs and the search for a new class of compounds preferably with a different mode of action is an important endeavor. Marcfortine A (1), a metabolite of Penicillium roqueforti, is structurally related to paraherquamide A (2), originally isolated from Penicillium paraherquei. Chemically the two compounds differ only in one ring; in marcfortine A, ring G is six-membered and carries no substituents, while in paraherquamide A, ring G is five-membered with methyl and hydroxyl substituents at C14. Paraherquamide A (2) is superior to marcfortine A as a nematocide. 2-Desoxoparaherquamide A (PNU-141962, 53) has excellent nematocidal activity, a superior safely profile, and is the first semi-synthetic member of this totally new class of nematocides that is a legitimate candidate for development. This review describes the chemistry, efficacy and mode of action of PNU-141962.

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%).