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ATP-binding cassette (ABC) transporters are one of the largest families of membrane proteins and are ATP-driven transport systems, which are found universally in all pro-and eukaryotes. Human ABC transporters have key roles in many physiological processes in the plasma membrane and within membranes of cell organelles, such as peptide transport, lipid flipping and multi-drug resistance. ABCB8, a sub-family B ABC transporter of the inner mitochondrial membrane, was found to be crucial for normal cardiac function, regulation of mitochondrial iron levels and cytosolic iron-sulphur cluster enzyme function and oxidative stress. Impaired function of ABCB8 resulted in mitochondrial iron accumulation, oxidative stress and cell death. In this thesis I developed a protocol for the purification of human ABCB8 pro¬viding yields and stable protein sufficient for structural studies using X-ray crystallography. Strategic optimisation of the purification protocol and crystallisation methods, resulted in crystals that diffracted to a resolution of 3.8 Å and allowed me to solve the first structure of human ABCB8. The ABCB8 structure adopted an outward-facing conformation that resembles a post-ATP-hydrolysis state in the alternating access mechanism, that was not yet observed in type I ABC exporters. In addition to the structural studies on ABCB8, I set out to explore the effects of ATP analogues, ATP transition state analogues and ADP on purified ABCB8 with biochemical and biophysical methods. Furthermore, I find that ATPase activity of ABCB8 in proteoliposomes is dependent on cardiolipin (CDL). The first structure of ABCB8 in an outward-facing conformation gives new insights into the alternating-access mechanism of subfamily B ABC transporters. Moreover, the purification and reconstitution protocol provided in this thesis is the foundation for further studies aiming to understand ABCB8’s function in more detail.

This study aimed to explore poverty among ASEAN member nations by introducing a multidimensional poverty index that can be used to determine the relative success of a country in deterring several deprivations that are identified and measured. It utilized multiple factors that can be useful for policy and investment decisions and are intended to complement analyses using financial poverty indicators. Using exploratory data analysis, several indicators were selected to represent the different dimensions of poverty. Said indicators were then subjected to factor analysis which yielded four multidimensional indices namely: General Welfare Index, Governance and Emotional Landscape Index, and Social Climate Index. Singapore, Brunei, and Malaysia ranked 1st, 2nd, and 3rd in all three indices, respectively. In addition, multivariate cluster analysis was also performed. It revealed that Singapore exhibited exceptional performance in all indices, thereby earning it a cluster all to its own. Similarities were observed between Brunei and Malaysia while Thailand, Vietnam, Philippines, and Indonesia were clustered together. Cambodia, Lao PDR, and Myanmar belonged to another cluster, indicating similarities among them. The study concluded that a wide gap exists among ASEAN countries in terms of the various deprivations measured. It also identified several problem areas and strengths of the ASEAN member states. This information can be helpful in making sound judgments especially in the delicate circumstances that surround economic integration.

Background: Streptomyces fradiae is the principal producer of urdamycin A. The antibiotic consists of a polyketide-derived aglycone, which is glycosylated with four sugar components, 2× D-olivose (first and last sugar of a C-glycosidically bound trisaccharide chain at the 9-position), and 2× L-rhodinose (in the middle of the trisaccharide chain and at the 12b-position). Limited information is available about both the biosynthesis of D-olivose and L-rhodinose and the influence of the concentration of both sugars on urdamycin biosynthesis. Results: To further investigate urdamycin biosynthesis, a 5.4 kb section of the urdamycin biosynthetic gene cluster was sequenced. Five new open reading frames (ORFs) ( urdZ3 , urdQ , urdR , urdS , urdT ) could be identified each one showing significant homology to deoxysugar biosynthetic genes. We inactivated four of these newly allocated ORFs ( urdZ3 , urdQ , urdR , urdS ) as well as urdZ1 , a previously found putative deoxysugar biosynthetic gene. Inactivation of urdZ3 , urdQ and urdZ1 prevented the mutant strains from producing L-rhodinose resulting in the accumulation of mainly urdamycinone B. Inactivation of urdR led to the formation of the novel urdamycin M, which carries a C-glycosidically attached D-rhodinose at the 9-position. The novel urdamycins N and O were detected after overexpression of urdGT1c in two different chromosomal urdGT1c deletion mutants. The mutants lacking urdS and urdQ accumulated various known diketopiperazines. Conclusions: Analysis of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster revealed a widely common biosynthetic pathway leading to D-olivose and L-rhodinose. Several enzymes responsible for specific steps of this pathway could be assigned. The pathway had to be modified compared to earlier suggestions. Two glycosyltransferases normally involved in the C-glycosyltransfer of D-olivose at the 9-position (UrdGT2) and in conversion of 100-2 to urdamycin G (UrdGT1c) show relaxed substrate specificity for their activated deoxysugar co-substrate and their alcohol substrate, respectively. They can transfer activated D-rhodinose (instead of D-olivose) to the 9-position, and attach L-rhodinose to the 4A-position normally occupied by a D-olivose unit, respectively.

Urdamycin A, the principal product of Streptomyces fradiae Tu2717, is an angucycline-type antibiotic and anticancer agent containing C-glycosidically linked d-olivose. To extend knowledge of the biosynthesis of urdamycin A the authors have cloned further parts of the urdamycin biosynthetic gene cluster. Three new ORFs (urdK, urdJ and urdO) were identified on a 3·35 kb fragment, and seven new ORFs (urdL, urdM, urdJ2, urdZ1, urdGT2, urdG and urdH) on an 8·05 kb fragment. The deduced products of these genes show similarities to transporters (urdJ and urdJ2), regulatory genes (urdK), reductases (urdO), cyclases (urdL) and deoxysugar biosynthetic genes (urdG, urdH and urdZ1). The product of urdM shows striking sequence similarity to oxygenases (N-terminal sequence) as well as reductases (C-terminal sequence), and the deduced amino acid sequence of urdGT2 resembles those of glycosyltransferases. To determine the function of urdM and urdGT2, targeted gene inactivation experiments were performed. The resulting urdM deletion mutant strains accumulated predominantly rabelomycin, indicating that UrdM is involved in oxygenation at position 12b of urdamycin A. A mutant in which urdGT2 had been deleted produced urdamycin I, urdamycin J and urdamycin K instead of urdamycin A. Urdamycins I, J and K are tetracyclic angucyclinones lacking a C–C connected deoxysugar moiety. Therefore UrdGT2 must catalyse the earliest glycosyltransfer step in the urdamycin biosynthetic pathway, theC-glycosyltransfer of one NDP-d-olivose.

From a cosmid library of Streptomyces cyanogenus S136, DNA fragments encompassing approximately 35 kb of the presumed landomycin biosynthetic gene cluster were identified and sequenced, revealing 32 open reading frames most of which could be assigned through data base comparison.

In the present study S-adenosylhomocysteine (SAH) hydrolase from the bovine kidney has been purified to apparent homogeneity by standard chromatographic procedures. The purified enzyme was free from adenosine deaminase activity and showed a one-banded pattern in SDS-PAGE with a monomer molecular mass of 47,500. The molecular mass of the native enzyme estimated by gel filtration was about 190,000. The pI was 5.5. For hydrolysis of SAH we found a Km of 5.0 +/- 1.2 microM and a V of 0.25 mumol/min/mg. In the direction of synthesis the Km for adenosine was 5.6 microM and V 0.53 mumol/min/mg. The enzyme activity was inhibited in the presence of adenosine with a Ki = 3 microM. In a second set of experiments we determined the binding characteristics of [3H]-adenosine to purified enzyme. The enzyme bound [3H]-adenosine with three apparent affinities: Kd1 = 6.8 +/- 0.7 nM and Bmax1 = 0.24 +/- 0.04 nmol/mg protein; Kd2 = 387 +/- 41 nM and Bmax2 = 1.4 nmol/mg protein, and Kd3 = 7.05 +/- 0.9 microM and Bmax3 = 9 nmol/mg protein. Binding of 25 nM [3H]-adenosine obeyed a monophasic reaction with a k+1 value of 0.025 min/nM. Dissociation of [3H]-adenosine-SAH hydrolase complex was markedly temperature dependent. After a 240-min incubation at 0 degrees C only 5-10% and at 20 degrees C 75% were displaceable. A fraction of 25% bound [3H]-adenosine was not displaceable by unlabeled adenosine. Our data show that the renal SAH hydrolase exhibits similar enzyme kinetics as the well-characterized SAH hydrolase from liver. The amount of SAH hydrolase present in renal tissues (1.4 nmol/g wet weight) could account almost entirely for the binding of renal tissue adenosine. Finally, we report for the first time a high affinity binding site of SAH hydrolase for adenosine, which remains unexplained at present.