A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2017. / Glutathione transferase P1-1 (GSTP1-1) is an enzyme belonging to the glutathione transferases superfamily of enzymes responsible for xenobiotic detoxification metabolism in the cells. It has been shown recently that GSTP1-1 performs a distinct function from its family members in that it acts as a carrier of the glutathione in the reactivation and glutathionylation of oxidised peroxiredoxin 6 (Prdx6). Prdx6 is a peroxidase belonging to the peroxiredoxin superfamily. The family functions to reduce organic peroxides which are sources of oxidative stress. Prdx6, however, differs from its family members as it is a bi-functional enzyme and it only contains one cysteine in its catalytic centre. The interaction of GSTP1-1 with Prdx6 has proven to be vital for the functioning of the Prdx6. The recombinant Prdx6 and GSTP1-1 proteins have been over-expressed and purified to homogeneity. The secondary structure of the proteins was studied using circular dichroism which has shown that GSTP1-1 is predominantly alpha helical and Prdx6 is mainly alpha helical with aspects of a beta sheet. The tertiary structural analysis has been carried out using tryptophan fluorescence which revealed that in both proteins the tryptophans are partially exposed to solvent. Furthermore, the quaternary structure was analysed using size exclusion-HPLC which indicated that the proteins are homodimeric in solution (both ~50 kDa). This study will present the findings on the overall characterisation and the implications of the findings on the interaction of these proteins. / LG2018

Cyanobacteria produce a myriad of structurally unique secondary metabolites with useful bioactive properties. Heterologous expression of a variety of microbial natural compounds has been used to harness their diversity and facilitate their combinatorial biosynthesis. However, these genetic techniques have not been developed for secondary metabolite-producing cyanobacteria. Therefore the genetically manipulable Escherichia coli and Synechocystis sp. PCC6803 were engineered in order to develop effective heterologous hosts and promoters for the expression of cyanobacterial-derived compounds. The phosphopanthetheinyl transferase (PPT), Sppt, from Synechocystis sp. PCC6803 was characterised to determine its ability to activate carrier proteins from secondary metabolite pathways. Despite in silico evidence which suggested Sppt was able to activate a wide range of carrier proteins, biochemical analysis revealed that it is dedicated for fatty acid synthesis. Consequently, E. coli and Synechocystis sp. PCC6803 were engineered to encode a broad-range PPT, from the filamentous cyanobacteria Nodularia spumigena NSOR10, for the activation of carrier proteins from nonribosomal peptide synthesis. Cyanobacterial natural product engineering was also explored with the characterisation of two relaxed specificity adenylation domains (A-domains) from the biosynthetic pathway of the toxin microcystin. The wide variety of microcystin compounds produced by cyanobacterial species suggests that multiple amino acids can be activated by the same A-domain. This was supported by preliminary ATP-[32P]PPi exchange assays and was subsequently harnessed in the production of a variety of dipeptides using two reconstituted modules in vitro. Transposition was investigated as a potential mechanism for the transfer of nonribosomal peptide synthetase gene clusters to heterologous hosts. This was performed via the characterisation of the putative transposase, Mat, physically linked with the microcystin synthetase gene cluster (mcyS). PCR screening, in silico analysis and nitrocellulose filter binding assays indicated that this transposase may have mediated mcyS gene cluster rearrangements but not entire gene cluster mobilisation between species. The potential role of transposases in the natural combinatorial biosynthesis of microcystin has evolutionary implications for the dynamic nature of cyanobacterial genomes and applications for use in the engineering of novel bioactive compounds. Therefore, the results from this study may provide a biotechnological platform for the transfer, expression and combinatorial biosynthesis of novel cyanobacterial-derived natural products.

AIM2 (absence in melanoma 2) can sense foreign cytosolic double-stranded DNA (dsDNA) through the dsDNA binding HIN-200 domain at the C-terminus. Once dsDNA is bound to HIN-200 domain, AIM2 can activate the AIM2 inflammasome, resulting in maturation of pro-interleukin-1β by activation of caspase-1. To investigate the mechanism of DNA binding, HIN-200 domain of mouse AIM2 bound to 15bp, 18bp, 20bp and 30bp dsDNAs were purified and crystallized. Diffraction data for four different crystals were collected to about 4Å resolution. Interestingly, all the crystals were in the same cubic space group I23 or I213 with almost same unit-cell parameters. Mutagenesis of HIN-200 domain of mouse AIM2 and DNA binding studies revealed amino acid residues involved in DNA binding. These residues were compared with dsDNA binding residues identified from the structure of HIN-200 domains of human AIM2, which suggested mouse AIM2 uses a similar dsDNA binding surface as human AIM2. Polynucleotidyl transferase (PNT1) was discovered in the proteomics study of Argonaute10 (AGO10). AGO10 was known as a key regulator of shoot apical meristem (SAM) maintenance in Arabidopsis. A recent study reported that AGO10 can regulate the developmental processes in SAM by repressing two miRNAs, miR166/165, and bind to these two miRNAs. Sequence analysis of PNT1 showed that it belongs to DnaQ-like 3'-5' exonuclease family, and hypothesized to be involved in RNA regulation with AGO10. PNT1 was purified and strong 3´-5´ exonuclease activity was revealed by activity test. Purified protein was crystallized, and the solved structure showed hexameric ring formation. Investigation of the crystal structure of PNT1 revealed presumed active site which was found from other 3´-5´ exonuclease homologues. Through mutagenesis of four residues in the presumed active site, Glu52 was identified as a key residue for its 3´-5´ exonuclease activity.

Cyanobacteria produce a myriad of structurally unique secondary metabolites with useful bioactive properties. Heterologous expression of a variety of microbial natural compounds has been used to harness their diversity and facilitate their combinatorial biosynthesis. However, these genetic techniques have not been developed for secondary metabolite-producing cyanobacteria. Therefore the genetically manipulable Escherichia coli and Synechocystis sp. PCC6803 were engineered in order to develop effective heterologous hosts and promoters for the expression of cyanobacterial-derived compounds. The phosphopanthetheinyl transferase (PPT), Sppt, from Synechocystis sp. PCC6803 was characterised to determine its ability to activate carrier proteins from secondary metabolite pathways. Despite in silico evidence which suggested Sppt was able to activate a wide range of carrier proteins, biochemical analysis revealed that it is dedicated for fatty acid synthesis. Consequently, E. coli and Synechocystis sp. PCC6803 were engineered to encode a broad-range PPT, from the filamentous cyanobacteria Nodularia spumigena NSOR10, for the activation of carrier proteins from nonribosomal peptide synthesis. Cyanobacterial natural product engineering was also explored with the characterisation of two relaxed specificity adenylation domains (A-domains) from the biosynthetic pathway of the toxin microcystin. The wide variety of microcystin compounds produced by cyanobacterial species suggests that multiple amino acids can be activated by the same A-domain. This was supported by preliminary ATP-[32P]PPi exchange assays and was subsequently harnessed in the production of a variety of dipeptides using two reconstituted modules in vitro. Transposition was investigated as a potential mechanism for the transfer of nonribosomal peptide synthetase gene clusters to heterologous hosts. This was performed via the characterisation of the putative transposase, Mat, physically linked with the microcystin synthetase gene cluster (mcyS). PCR screening, in silico analysis and nitrocellulose filter binding assays indicated that this transposase may have mediated mcyS gene cluster rearrangements but not entire gene cluster mobilisation between species. The potential role of transposases in the natural combinatorial biosynthesis of microcystin has evolutionary implications for the dynamic nature of cyanobacterial genomes and applications for use in the engineering of novel bioactive compounds. Therefore, the results from this study may provide a biotechnological platform for the transfer, expression and combinatorial biosynthesis of novel cyanobacterial-derived natural products.

Cyanobacteria produce a myriad of structurally unique secondary metabolites with useful bioactive properties. Heterologous expression of a variety of microbial natural compounds has been used to harness their diversity and facilitate their combinatorial biosynthesis. However, these genetic techniques have not been developed for secondary metabolite-producing cyanobacteria. Therefore the genetically manipulable Escherichia coli and Synechocystis sp. PCC6803 were engineered in order to develop effective heterologous hosts and promoters for the expression of cyanobacterial-derived compounds. The phosphopanthetheinyl transferase (PPT), Sppt, from Synechocystis sp. PCC6803 was characterised to determine its ability to activate carrier proteins from secondary metabolite pathways. Despite in silico evidence which suggested Sppt was able to activate a wide range of carrier proteins, biochemical analysis revealed that it is dedicated for fatty acid synthesis. Consequently, E. coli and Synechocystis sp. PCC6803 were engineered to encode a broad-range PPT, from the filamentous cyanobacteria Nodularia spumigena NSOR10, for the activation of carrier proteins from nonribosomal peptide synthesis. Cyanobacterial natural product engineering was also explored with the characterisation of two relaxed specificity adenylation domains (A-domains) from the biosynthetic pathway of the toxin microcystin. The wide variety of microcystin compounds produced by cyanobacterial species suggests that multiple amino acids can be activated by the same A-domain. This was supported by preliminary ATP-[32P]PPi exchange assays and was subsequently harnessed in the production of a variety of dipeptides using two reconstituted modules in vitro. Transposition was investigated as a potential mechanism for the transfer of nonribosomal peptide synthetase gene clusters to heterologous hosts. This was performed via the characterisation of the putative transposase, Mat, physically linked with the microcystin synthetase gene cluster (mcyS). PCR screening, in silico analysis and nitrocellulose filter binding assays indicated that this transposase may have mediated mcyS gene cluster rearrangements but not entire gene cluster mobilisation between species. The potential role of transposases in the natural combinatorial biosynthesis of microcystin has evolutionary implications for the dynamic nature of cyanobacterial genomes and applications for use in the engineering of novel bioactive compounds. Therefore, the results from this study may provide a biotechnological platform for the transfer, expression and combinatorial biosynthesis of novel cyanobacterial-derived natural products.

Cyanobacteria produce a myriad of structurally unique secondary metabolites with useful bioactive properties. Heterologous expression of a variety of microbial natural compounds has been used to harness their diversity and facilitate their combinatorial biosynthesis. However, these genetic techniques have not been developed for secondary metabolite-producing cyanobacteria. Therefore the genetically manipulable Escherichia coli and Synechocystis sp. PCC6803 were engineered in order to develop effective heterologous hosts and promoters for the expression of cyanobacterial-derived compounds. The phosphopanthetheinyl transferase (PPT), Sppt, from Synechocystis sp. PCC6803 was characterised to determine its ability to activate carrier proteins from secondary metabolite pathways. Despite in silico evidence which suggested Sppt was able to activate a wide range of carrier proteins, biochemical analysis revealed that it is dedicated for fatty acid synthesis. Consequently, E. coli and Synechocystis sp. PCC6803 were engineered to encode a broad-range PPT, from the filamentous cyanobacteria Nodularia spumigena NSOR10, for the activation of carrier proteins from nonribosomal peptide synthesis. Cyanobacterial natural product engineering was also explored with the characterisation of two relaxed specificity adenylation domains (A-domains) from the biosynthetic pathway of the toxin microcystin. The wide variety of microcystin compounds produced by cyanobacterial species suggests that multiple amino acids can be activated by the same A-domain. This was supported by preliminary ATP-[32P]PPi exchange assays and was subsequently harnessed in the production of a variety of dipeptides using two reconstituted modules in vitro. Transposition was investigated as a potential mechanism for the transfer of nonribosomal peptide synthetase gene clusters to heterologous hosts. This was performed via the characterisation of the putative transposase, Mat, physically linked with the microcystin synthetase gene cluster (mcyS). PCR screening, in silico analysis and nitrocellulose filter binding assays indicated that this transposase may have mediated mcyS gene cluster rearrangements but not entire gene cluster mobilisation between species. The potential role of transposases in the natural combinatorial biosynthesis of microcystin has evolutionary implications for the dynamic nature of cyanobacterial genomes and applications for use in the engineering of novel bioactive compounds. Therefore, the results from this study may provide a biotechnological platform for the transfer, expression and combinatorial biosynthesis of novel cyanobacterial-derived natural products.

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser. Med sammanfattning på svenska.