Proefschrift Universiteit van Amsterdam. / Met samenv. i.h. Nederlands.

Magister Scientiae - MSc / This study represents the first structural investigation of any type III glutamine synthetase (GS). The GS, GlnA, from the medically important opportunistic human pathogen Bacteroides fragilis was studied with a view to better understanding its structure/functioning in relation to the extensively characterised GSIs. GSIIIs are the most recently discovered family of GSs and are the most phylogenitically distant GSs from the GSIs. Images (160) of negatively stained rGlnA, expressed in E.coli YMC11 (glnA-), were recorded at 50K magnification using a Leo 912 operating at 120kV with energy filtering coupled to a 4 megapixel CCD camera. An angular refinement based reconstruction strategy was adopted using SPIDER. A reconstruction based on an ab initio starting model, derived by a common-lines based simultaneous minimization of rotationally invariant K-mean clustered class averages, converged to the same structure as a reconstruction based on a GSI starting model to a resolution of 2.1nm as determined by Fourier shell correlation). In contrast to preliminary EM observations, which identified GlnA as a hexamer, this work has revealed a dodecameric structure, with subunits (82.8KDa) arranged in two opposing hexagonal rings with distinct handedness. This is similar to the quaternary structure of GSIs and GlnTs except that the complex is 50% longer and the two rings are not symmetrically related. They differ not only in diameter (16.5 or 15.0nm) but also the degree of separation of subunits and as such the particle possesses only C6 and not D6 symmetry. The finding that particles lie in a preferred orientation, with the larger ring in contact with the carbon support, accounted for this asymmetry, through partial staining. Hexameric views, with similar overall arrangement but larger size in comparison to GSI, were also observed. However, it was uncertain whether these were true hexamers resulting from dissociation of the dodecamers or were a consequence of partial staining. Homology modelling was also undertaken in an attempt to predict the structure of GlnA based on GSI, with a view to interpreting the low resolution EM structure. Due to the failures of state of the art algorithms in detecting the distant homologies between GS families, manual profile-based alignment strategies, incorporating structural information, were employed. Through the first full length alignments of GS sequences from all four families, conservation of all active site residues, core active site αβ barrel fold motifs, and additional previously unreported regions was demonstrated. Docking of these homology models into the 3D structure confirmed the presence of the αβ barrel fold predicted by the bioinformatic analysis of the sequences alone, thus, identifying the indentations between subunits in the volume as putative active sites. In addition to providing unequivocal proof that GlnA is a GS and confirming the presence of putative αβ barrel active site folds, this work has made steps towards understanding the regulation of this enzyme. It is hypothesised that GlnA occurs as both active hexamer and an inactive dodecamer, the interconversion of which, is thought to represent a means of reversible post-translational regulation. / South Africa

No description available.

Strain MS14, exhibiting antifungal activity, was classified to belong to Burkholderia contaminans. Occidiofungin produced by strain MS14 is an octapeptide dedicated to a broad range of antifungal activities of the bacterium. The 58.2-kb genomic fragment containing 18 open reading frames (ORFs), named occidiofungin (occ) gene cluster, is required for occidiofungin production. Putative proteins encoded by five nonribosomal peptide synthetase genes (occA – occE) of the gene cluster were predicted to contain the catalytic modules responsible for the biosynthesis of occidiofungin. Transcription of all the ORFs identified in the region except ORF1 and ORF16 was regulated by both ambR1 and ambR2, the LuxR-type regulatory genes located at the left border of the cluster. The functional ambR1 gene was essential for transcription of ambR2, and constitutive expression of ambR2 did not restore the phenotype of the mutant MS14GG44(ambR1::nptII). Sequence analysis revealed that the occ gene cluster shared high similarity (99% nucleotide coverage and 91% identity) to an uncharacterized DNA region of B. ambifaria strain AMMD. The gene cluster was not found in other Burkholderia strains available in GenBank (nucleotide coverage < 24%). Analysis of G+C composition and prediction using “IslandPick” indicate that the occ gene cluster has possibly been horizontally transferred between bacteria. In addition, the absence of the gene cluster in clinical strains of Burkholderia indicates that occidiofungin is not required for potential human pathogenesis. The findings have provided insights into the development of antifungal medicines and agricultural fungicides based on occidiofungin.

The aminoacyl-tRNA synthetases are a family of enzymes involved in the process of translation, more specifically, ligating amino acids to their cognate tRNA molecules. Recent evidence suggests that aminoacyl-tRNA synthetases are capable of aminoacylating proteins, some of which are involved in the autophagy pathway. Here, we test the conditions under which E. coli and human threonyl-tRNA synthetases, as well as hisidyl-tRNA synthetase aminoacylate themselves. These reactions are ATP dependent, stimulated by Mg2+, and are inhibited by increasing cognate tRNA concentrations. These data represent the foundation for future aminoacylation experiments, specifically delving into the relationship between the autophagy pathway and the aminoacylation of proteins. Additionally, we provide evidence of the inhibitory abilities of the compound EHTS-0 on both E. coli and human threonyl-tRNA synthetases. Further, we also show that an EHTS-0 analog, EHTS-1, also significantly inhibits E. coli threonyl-tRNA synthetase but not the human enzyme. These data could be useful in determining the potential for EHTS-0 and EHTS-1 as possibly anti-angiogenic drugs.

This investigation concerned determining certain factors which affect the activity of cyclopropane synthetase in L. plantarum. In vitro experiments showed the enzme to be sensitive to ionic strength and subject to product inhibition by S-adenosylhomocysteine nucleusidase, which relieves this inhibition by degrading SAH to adenine and ribosylhomocysteine.

No description available.

No description available.

Thesis advisor: Steven D. Bruner / Thesis advisor: Larry W. McLaughlin / Nonribosomal peptides are therapeutically important natural products produced through pathways that utilize large multimodular enzymes, termed nonribosomal peptide synthetases (NRPSs). Central to the assembly line methodology, the monomer building blocks and the growing polymer chain are covalently linked to dedicated peptidyl carrier protein domains as phosphopantetheinyl thioesters. Although structures of multidomain NRPS fragments have been solved recently, the active conformation of the carrier domains with their attached phosphopantetheinyl arms has not been determined. Significant conformational changes in carrier domains are likely to occur as the domains shuttle peptidyl phosphopantetheinyl thioesters between the active sites of the partner domains. This thesis focuses on the application of the synthetic isosteric non-hydrolyzable CoA analogs to manipulate carrier domain geometry of NRPS assemblies through. The synthetic conjugates are designed to deliver an inhibitor moiety to a domain of interest. Using this strategy, various complexes have been designed to direct the phosphopantetheinyl arm to active sites of adenylation domains and thioesterase domains in catalytically relevant conformations. The structurally restrained multidomain NRPS assemblies are useful for elucidating the complex structure and mechanism of NRPSs. An X-ray crystal structure of a peptidyl carrier-thioesterase NRPS didomain fragment from enterobactin synthetase has been solved with a phosphopantetheinyl analog which forms a cross-link between the two domains. This structure provides, for the first time, detailed insights into the phosphopantetheinyl arm interaction with an NRPS partner domain, as well as an active confirmation of a mutidomain NRPS in the holo-form. In addition, the hydrolytically stable CoA analogs have been successfully used as probes in the structural and mechanistic study of a CoA-utilizing enzyme DpgC, a unique cofactor-independent dioxygenase involved in vancomycin biosynthesis. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Abhishek Chatterjee / Abstract Unnatural amino acid (UAA) incorporation is a powerful tool used by biochemists to discover the nature of protein structure and function. The evolution of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs enables site-specific incorporation of UAAs proteins inside of living cells. The goal of this study was to further expand the repertoire of genetically encoded unnatural amino acids in E. coli as well as eukaryotes. We first attempted to engineer an aaRS, previously evolved for p-borono-phenylalanine (pBoF), to specifically charge 3-acetyl-p-borono-phenylalanine (AcpBoF). A randomized library of the pBoF-specific synthetases was generated and it was subjected to established selection schemes in a bacterial host. This report also describes the development of a yeast-based selection system to alter the substrate specificity of bacterial leucyl-tRNA synthetase, for genetic code expansion in eukaryotes. / Thesis (MS) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.