No description available.

<font face="Arial">This study represented 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 the function and regulation of this important enzyme. </font>

<font face="Arial">This study represented 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 the function and regulation of this important enzyme. </font>

Proefschrift Universiteit van Amsterdam. / Met lit.-opg. en samenvatting in het Nederlands.

Thesis (Ph.D.) - James Cook University, 2004. / Typescript (photocopy). Bibliography: leaves 213-226.

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.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, June 1997. / Includes bibliographical references. Also available on the Internet.

Nitrile hydratase activity in Rhodococcus rhodochrous DAP 96253 can be induced with multiple inducers that include urea, cobalt (Co), iron (Fe) and nickel (Ni). When induced with Co/urea, cells of R. rhodochrous DAP 96253 expressed the highest level of nitrile hydratase activity (~200 units/min·mg-cdw) when compared with the other inducers tested. Cells induced with Co had the second highest nitrile hydratase activity (~7 units/min·mg-cdw), whereas in the uninduced cells, nitrile hydratase activity was lower than 1 unit/min·mg-cdw. Similarly in R. rhodochrous DAP 96622, when induced with Co/urea, the nitrile hydratase activity of R. rhodochrous DAP 96622 cells was around 50 units/min·mg-cdw which was the highest of all inducers tested. When induced with Co only, the nitrile hydratase activity of R. rhodochrous DAP 96622 was around 20 units/min·mg-cdw, and the nitrile hydratase activity of R. rhodochrous DAP 96622 uninduced was the same as the nitrile hydratase activity of uninduced R. rhodochrous DAP 96253. When Co/urea induced R. rhodochrous DAP 96253 cell lysate was examined on gradient SDS-PAGE and analyzed by Image Quant TL, the nitrile hydratase bands (both α and β subunits) accounted for more than 55% of the total cytosolic proteins. Whereas in Co/urea induced R. rhodochrous DAP 96622, the nitrile hydratase bands accounted for around 25% of the total cytosolic proteins. According to matrix-assisted laser desorption ionization time-of-flight mass spectrometry results, amidase in R. rhodochrous DAP 96253 was approximately 38 kDa from the nitrilase/cyanide hydratase family and amidase in R. rhodochrous DAP 96622 was 55 kDa from the amidase signature family. In addition, the nitrile hydratase regulation system in both R. rhodochrous DAP 96253 and DAP 96622 strains are different. Moreover, the nitrile hydratase regulation system in R. rhodochrous DAP 96253 is different from R. rhodochrous J1. Purified nitrile hydratase from R. rhodochrous DAP 96253 may form a protein complex with glutamine synthetase, resulting in a nitrile hydratase activity of approximately 1500 units/mg-proteins, and nitrile hydratase from R. rhodochrous DAP 96622 is not a protein complex and results in a nitrile hydratase activity of 950 units/mg-proteins.

Glutamine synthetase (GS; EC 6.3.1.2; L-glutamate ammonialigase) catalyzes the ATP-dependent conversion of glutamate and ammonia into glutamine. Due to its key role in nitrogen metabolism, including nucleotide, amino acid and urea biosynthesis, the enzyme has been ascribed an extraordinarily long evolutionary history. Thus, GS has been used as a molecular clock to establish phylogenetic relationship between different species. Through the National Center of Biotechnology Information (NCBI) using Basic Local Alignment Search Tool (BLAST) programs BLASTx (translated nucleotide-protein alignment) and BLASTn (nucleotide-nucleotide alignment) system, we obtained the complete cDNA of GS from tilapia cDNA liberary. Furthermore, the results of the alignment of tilapia GS sequence with that of other species indicated a close relationship between tilapia GS and other fishes. We also found that there is 79% homology between mammal and tilapia within the open read frame (ORF) of GS. However, sequence analysis by computer software revealed the fact that the size (0.5 kb) of GS 3¡¦untranslated region (3¡¦-UTR) of tilapia GS is different from that of mammals. Moreover, there is the complete distinct sequence of the 3¡¦-UTR of tilapia GS from that of mammals. The 3'-UTR of many eukaryotic mRNAs has been implicated in the control of mRNA stability, processing, polyadenylation, and translational regulation. Accordingly, to comprehend the role of 3¡¦-UTR in GS phylogenesis, we examine whether the 3'-UTR of tilapia GS is involved in the regulation of GS expression in mammals. We first generated the construct using pEGFP-N2 carrying the ORF (1.1kb) of tilapia GS gene (ORF-GFP) or the full length (1.6kb) of tilapia GS gene (Full-GFP). Transient or stable transfection of C6 gliomal cells with ORF-GFP indicated that GS mRNA and protein was expressed. When C6 cells were stably transfected with Full-GFP, the expression of GS mRNA, but not its protein, was found. Adenine/uridine-rich sequence elements (AREs) of the 3¡¦-UTR have been known to regulate mRNA stability of certain chemokines. Four AREs are also found in the 3¡¦-UTR of tilapia GS. We further generated the constructs with tilapia ORF-GFP and its 3¡¦-UTR containing 1-4 AREs (A1-GFP, A2-GFP, A3-GFP and A4-GFP). Stable transfection of C6 cells with the different constructs indicated that tilapia GS mRNA is normally transcripted, while there was no expression of GS proteins in stable transfectants. The findings suggest tilapia GS protein expression in mammals by its 3¡¦-UTR and unidentified evolutionary role of the 3¡¦-UTR region of GS.