No description available.

No description available.

No description available.

No description available.

β-lactamase enzymes catalyze the irreversible hydrolysis of the four-membered cyclic amide ring characteristic of β-lactam antibiotics rendering them inactive and useless against pathogenic bacteria. Understanding structure activity relationships between β-lactam antibiotics and β-lactamases is important for designing novel β-lactams, β-lactamase inhibitors, and β-lactam-based fluorescent probes for rapid diagnosis of β-lactam antibiotic resistant infections. The first half of this study focuses on the class A β-lactamase BlaC from Mycobacterium tuberculosis (Mtb) and addresses intermolecular interactions between BlaC and substrates, inhibitors, and biosensors that influence their kinetic parameters with BlaC and activities against Mtb. The substrate structure activity relationship explained the molecular basis for differential innate resistance of Mtb to faropenem, biapenem, and tebipenem by showing the interactions between BlaC and the lactams that govern differential acyl-intermediate stability and affinity. The inhibitor structure activity relationship revealed features of the BlaC active site that can be exploited to enhance binding and inhibition of BlaC by benzoxaboroles, and demonstrates their utility as potentiators of β-lactam antibiotic activity against Mtb. BlaC-specific β-lactam based fluorescent probes were designed and optimized for Mtb detection. Their utility was demonstrated by detecting down to 10 colony forming units of bacillus Mycobacterium bovis Calmette–Guérin (BCG) in human sputum. The second half of this study focuses on the New Delhi Metallo-β-lactamase-1 (NDM-1), which is rapidly generating bacterial resistance to nearly all β-lactams. The NDM-1 gene encodes a class B1 metallo-β-lactamase enzyme. Purified recombinant NDM-1 was biochemically and biophysically characterized. The crystal structures of apo and monometalated NDM-1 provided structural insight into metal binding and the promiscuous enzymatic activity of NDM-1. Mechanistic details of the NMD-1 reaction were examined by comparing crystal structures of NDM-1 in complex with an unhydrolyzed β-lactam substrate and with hydrolyzed products. These structures were used for quantum mechanics / molecular mechanics simulations to estimate the free energy along the β-lactamase reaction coordinate. The results suggest that NDM-1 uses bulk water as the nucleophile that attacks the β-lactam ring, and a coordinated hydroxide ion or water molecule as the catalytic base depending on pH.

The actinomycetes are gram-positive bacteria that produce more than two-thirds of the known biologically active microbial natural products, including many commercially important antibiotics, anti-cancer agents, other pharmacologically useful agents, animal health products and agrochemicals. The prevailing utilization of antibiotics continues to be the mainstay against microbial infections and a majority ofthe over six thousand antibiotics discovered thus far are from Streptomyces spp. One of the most well-characterized antibiotic biosynthetic pathway is the one involving the biosynthesis of the penicillins, cephalosporins and cephamycins. This pathway involves two initial steps which are common in filamentous fungi, lower eukaryotes and prokaryotes. The penam nucleus of penicillins and the cephem nucleus of both cephamycins andcephalosporins are formed by the condensation of the three precursor amino acids L-a-aminoadipic acid, Lcysteine and L-valine by a mechanism designated as 'non-ribosomal peptide synthesis', which involves activation and condensation of the three component amino acids and epimerization of the L- to D-valine to form a linear acyclic tripeptide called o-(L-a-aminoadipyl)-L-cysteinyl-Dvaline (ACV) by the action of a peptide synthetase. ACV is then cyclized to form isopenicillin N, an intermediate that contains an L-a-aminoadipyl side-chain attached to the penem nucleus (Fig. 1.2) by isopenicilin N synthase (IPNS or Cyclase) and this encompasses the creation of the Beta-lactam and thiazolidine rings. A broad range of ~-lactam producing Streptomyces spp were grown, the DNA extraction procedure optimised and total chromosomal DNA isolated. A bioinformatics analysis of known IPNS gene sequences allowed the synthesis of PCR primers for the iso-penicillin N synthase gene. IPNS genes and lPNS-like genes were successfully amplified from the total DNA of ten strains including two novel thermophilic strains, A. and B. Sequencing was carried out on the genes from S. hygroscopicus, S. tanashiensis and the two thermophiles A and B. This allowed development of the conditions for gene shuffiing of the IPNS gene which was carried out pairwise and resulted in the reconstitution of shuffied genes of the correct size. The resulting mixed gene sequences were cloned into the pTrcHis2-TOPO expression vector and the plasmid DNA screened and assayed for IPNS activity using HPLC which showed ten fold increase in IPNS activity as a result of the shuffiing.

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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.

No description available.

No description available.