Browsing by Person "Ilg, Thomas"

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    Identifizierung und Charakterisierung der Succinsemialdehyd-Dehydrogenase aus parasitischen und nichtparasitischen Arthropoden
    (2008) Rothacker, Boris; Ilg, Thomas
    The objective of the PhD project was the molecular and enzymological characterization of SSADH in parasitic and nonparasitic insects and acarids, organism groups where this enzyme was virtually unexplored. In this project, the list of investigated non-arthropod organisms included the bacterium Escherichia coli (Ec) and the mammal Mus musculus (mouse). Their SSADH served as reference enzymes or were used for biophysical experiments. Amongst the arthropods, Drosophila melanogaster (Dm) (fruit fly) was chosen as nonparasitic model insect. Lucilia cuprina (Lc) (sheep blowfly) and Ctenocephalides felis (Cf) (cat flea) were included as important parasitic insects, as was the acarid Rhipicephalus microplus (Rm) as the economically most important tick species. At the start of this thesis, expressed sequence tag studies and whole genome sequencing on Dm suggested the existence of a single copy gene candidate that, based on amino acid sequence homology, was considered to be a candidate for a SSADH ortholog in this species (DmSSADH). The Dm SSADH gene candidate was cloned and expressed in Ec as a soluble protein. To compare the enzymological properties of DmSSADH, another so far uncharacterized Dm gene candidate for an acetaldehyde dehydrogenase (DmALDH) was cloned and expressed in Ec as a soluble protein. Both expression products showed the expected enzymological properties: a NAD+-dependent ssa-oxidizing activity for DmSSADH and a NAD+-dependent acetaldehyde-oxidizing activity for Dm-ALDH. Site-directed mutageneses on DmSSADH performed in this study demonstrated that two residues essential for catalysis are glutamate 277 and cysteine 311. The second part of the thesis encompassed the gene identification, full length gene cloning of SSADH in Lc (LcSSADH) and Cf (CfSSADH), as well as functional expression of one gene version, respectively. Substrate/cosubstrate specificity determinations combined with enzyme kinetics studies showed that both enzymes are predominantly NAD+-dependent SSADHs. Bioinformatics analyses detected N-terminal mitochondrial import sequences in both Lc and Cf SSADH suggesting that these enzymes are localized in vivo in the mitochondrial matrix. The investigation of the genomic structure of the LcSSADH and CfSSADH genes revealed significant differences to the previously known gene organizations: firstly, different to the single copy SSADH gene situation in Ec, Dm, Mus musculus and Homo sapiens, Lc appears to possess 2-3 SSADH genes, while in Cf up to 8 gene copies may be present. Secondly, compared to Dm (2 exons, 1 intron), the exon/intron structure of the SSADH genes in Lc and Cf is not conserved: the one SSADH genomic gene version investigated in detail in Lc contained 3 exons and 2 introns, while the genomic gene version of Cf analysed in this study was devoid of any introns. The central topic of the third part of this thesis was the gene identification and biochemical characterization of SSADH from the tick Rm. By a combination of different PCR methods a tick gene orthologous to insect and mammalian SSADH (RmSSADH) could be identified and isolated. The results of Southern blot analyses of Rm DNA are incompatible with a single copy situation and suggest the presence of 2-3 RmSSADH genes. To compare RmSSADH with a mammalian SSADH, the respective gene was isolated from mouse. Both the tick and the mouse SSADH genes were then expressed as soluble functional proteins in Ec. The initial comparison showed that both proteins are potent NAD+-dependent ssa-oxidizing enzymes with very similar enzyme kinetics. A more detailed comparison of both enzymes suggests that in general, the mouse enzyme appears to be more specific for succinic semialdehyde than the tick enzyme. In the fourth section of the experimental part of this thesis, saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR) experiments were performed on three of the above SSADH preparations, to answer questions on the aldehyde substrate and cosubstrate binding to these enzymes. Importantly, the long-standing question whether the free aldehyde of succinic semialdehyde or its hydrated gem-diol form (present in aqueous solution in equimolar amounts) is the binding substrates was answered conclusively in favour of the aldehyde form for both the Ec and the Dm enzyme. Most remarkably, STD-NMR experimental investigation of the ssa interaction with the Dm SSADH cysteine311alanine mutant enzyme demonstrated binding of both the aldehyde and the gem-diol form. This experiment strongly suggests that cysteine311 is acting as an aldehyde versus gem-diol selectivity filter in the active site of the enzyme. Furthermore, STD-NMR epitope mapping of the NAD+/NADP+ binding to Ec SSADH and Dm SSADH was performed. In both cases, the data suggested that the dominant protein-ligand interactions are via the adenine and the nicotinamide ring systems, while the ribose moieties interact much less intensely with the enzymes.