No description available.

No description available.

This thesis reports the work done on two research projects which were carried out independently of each other but converge on the central theme of β-lactam biosynthesis. Chapter 1 provides an overview of biosynthesis in secondary metabolism, with special emphasis on current knowledge about the β-lactams. The first project, covered from Chapters 2 to 5, was part of our group's continuing effort to understand the structure and mechanism of Ring Expandase-Hydroxylase (REXH), an enzyme involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium. REXH is a bifunctional enzyme, converting penicillin N to DAOC and thence to DAC. [diagram omitted from transcription] Chapter 3 discusses the investigation of purification protocols for native REXH and soluble recombinant REXH, as well as an improved refolding method for recombinant REXH expressed as inclusion bodies. Chapter 4 describes two new alternative substrates for REXH, viz. carba-DAOC and DAC, whilst the y-lactam analogue of penicillin N was not found to be a substrate for REXH. Chapter 5 summarises some structural investigations of REXH employing methods such as electrospray mass spectrometry, selective proteolysis and inhibition kinetics. [diagram omitted from transcription] The second project, covered from Chapters 6 to 9, represents the first biosynthetic studies on valclavam, an antifungal produced by Streptomyces antibioticus. Valclavam belongs to the family of clavams which includes clavulanic acid as its most well studied member. [diagram omitted from transcription] Chapter 7 details the development of methods for the bioassay, fermentation and isolation of valclavam. It also describes the isolation of a stable degradation fragment of valclavam which led to the revision of the structures of valclavam and Tü 1718B (another metabolite from the same organism). Chapter 8 gives an account of the whole-cell feeding experiments which strongly suggest that the primary metabolic precursors for valclavam are L-valine, L-arginine, L-methionine and glycerol. Chapter 9 reports the discovery of two enzymic activities, belonging to those of clavaminic acid synthase and proclavaminic acid amidino hydrolase, which are likely to be involved in the biosynthesis of valclavam. Together, the results of Chapters 8 and 9 point to an extensive overlap between the clavulanic acid pathway in Streptomyces clavuligerus and the valclavam pathway in Streptomyces antibioticus.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1995. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1995. / Added t.p. with thesis statement inserted. Includes bibliographical references.

No description available.

No description available.

No description available.

Streptococcus pneumoniae is a major pathogen that causes Otitis Media infections and bacterial meningitis in children as well as community acquired pneumonia in adults. Clinical isolates of S. pneumoniae exhibiting resistance to Beta-lactam antibiotics are being isolated with increased frequency in many countries. Streptococcus pneumoniae strains resistant to Beta-lactam drugs have modified forms of penicillin-binding proteins that exhibit reduced affinity for binding to chemotherapeutic Beta-lactams. Penicillin binding proteins are membrane-bound enzymes that catalyze the terminal step in cell wall synthesis, and are targets for Beta-lactam drugs. Seventeen clinical isolates and six vaccine strains of Streptococcus pneumoniae were characterized using conventional phenotypic methods, susceptibility to antimicrobial agents, capsular serotyping, and by different biochemical and genotyping methods. One strain, Sp D2, was resistant to penicillin and other Beta-lactams used in the study, to erythromycin, and to Trimethoprim/Sulfamethoxazole. Sp D2 exhibited a unique protein profile in 1D SDS-PAGE gels of whole-cell proteins. Cells of Sp D2 were fractionated, and the cytoplasmic membrane fraction was obtained by ultracentrifugation and analyzed using a 1D SDS-PAGE gel. A protein band with a mass of ~50 kDa was excised and subjected to Trypsin In-Gel Digestion, followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. The resulting MALDI-TOF-MS data (peptide mass fingerprints) did not produce any significant matches with proteins in any of the published S. pneumoniae genome databases. The 50 kDa protein was further subjected to N-terminal and internal sequence analysis and database searching, and the protein could not be identified by significant matches. Sp D2 did not react with any anti-pneumococcal polysaccharide capsular antibodies, and is designated as a non-typeable strain. Sp D2 exhibited a positive reaction in the Bile Solubility Test, the Optochin Test, and also positive reactions in PCR assays for the presence of the pneumococcal surface protein gene (PsaA), the autolysin gene (LytA), and the pneumolysin gene (Ply); which confirms that Sp D2 is a strain of S. pneumoniae.

1. We have explored a formal [3+2] strategy that is synthetically useful for constructing 3,4-disubstituted succinimides with ethyl bromoacetate derivatives or methyl glyoxylate in one step. 2. We reported a new approach to (E)-£]-aryl-£\,£]-unsaturated amides. Instead of using aldehydes, phosphorus, silicon containing compounds and metal catalysts for the synthesis of double bonds, £\-sulfonyl acetamide and various benzyl bromides were used as starting materials