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Study of the key points of lincomycin and celesticetin biosynthesis
Vobruba, Šimon ; Janata, Jiří (advisor) ; Bobek, Jan (referee) ; Kutejová, Eva (referee)
Lincosamides form a small but important group of specialized microbial metabolites with antibiotic activity. The most important members of this group are celesticetin and clinically used lincomycin. Structurally, lincosamides are composed of an amino sugar and an amino acid connected by an amide bond. The amino acid precursors of both lincosamides remarkably differ. Proteinogenic L-proline is the precursor of celesticetin, while an unusual amino acid (2S,4R)-4-propyl- L-proline (PPL) is incorporated in the more efficient compound lincomycin. Surprisingly, both these precursors are recognized and activated for further biosynthetic steps by homologous adenylation domains CcbC and LmbC, respectively. The detailed description of this amino acid recognition and activation step, which is critical for the biological activity of the resulting compound, was the aim of the first part of this thesis. The site-directed mutagenesis of the LmbC substrate binding pocket and biochemical characterization of resulting mutants were employed to identify the residues crucial for the activation of PPL. Subsequently, we experimentally simulated the molecular evolution leading from L-proline-specific substrate binding pocket (like in CcbC) to the PPL-specific enzyme (LmbC). The substitution of only three amino acid...
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Genetic and functional characterisation of mitochondrial diseases caused by ATP synthase defects
Tauchmannová, Kateřina ; Houštěk, Josef (advisor) ; Flachs, Pavel (referee) ; Kutejová, Eva (referee)
Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting mostly as early-onset mitochondrial encephalo-cardio-myopathies. Mutations in four nuclear genes can result in isolated deficiency of ATP synthase, all sharing a similar biochemical phenotype - pronounced decrease in the content of fully assembled and functional ATP synthase complex. The thesis summarises studies on two distinct causes of ATP synthase deficiency. First is TMEM70 protein, a novel ancillary factor of ATP synthase, which represents most frequent determinant of severe inborn deficiency of ATP synthase. TMEM70 is a 21 kDa protein of the inner mitochondrial membrane, facilitating the biogenesis of mitochondrial ATP synthase, possibly through TMEM70 protein region exposed to the mitochondrial matrix, but the proper regulatory mechanism remains to be elucidated. In TMEM70-lacking patient fibroblasts the low content of ATP synthase induces compensatory adaptive upregulation of mitochondrial respiratory chain complexes III and IV, interestingly by a posttranscriptional mechanisms. The second type of ATP synthase deficiency studied was mtDNA m.9205delTA mutation affecting maturation of MT-ATP8/MT-ATP6/MT-CO3 mRNA and thus biosynthesis of Atp6 (subunit a) and Cox3...
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Genetic and functional characterisation of mitochondrial diseases caused by ATP synthase defects
Tauchmannová, Kateřina ; Houštěk, Josef (advisor) ; Flachs, Pavel (referee) ; Kutejová, Eva (referee)
Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting mostly as early-onset mitochondrial encephalo-cardio-myopathies. Mutations in four nuclear genes can result in isolated deficiency of ATP synthase, all sharing a similar biochemical phenotype - pronounced decrease in the content of fully assembled and functional ATP synthase complex. The thesis summarises studies on two distinct causes of ATP synthase deficiency. First is TMEM70 protein, a novel ancillary factor of ATP synthase, which represents most frequent determinant of severe inborn deficiency of ATP synthase. TMEM70 is a 21 kDa protein of the inner mitochondrial membrane, facilitating the biogenesis of mitochondrial ATP synthase, possibly through TMEM70 protein region exposed to the mitochondrial matrix, but the proper regulatory mechanism remains to be elucidated. In TMEM70-lacking patient fibroblasts the low content of ATP synthase induces compensatory adaptive upregulation of mitochondrial respiratory chain complexes III and IV, interestingly by a posttranscriptional mechanisms. The second type of ATP synthase deficiency studied was mtDNA m.9205delTA mutation affecting maturation of MT-ATP8/MT-ATP6/MT-CO3 mRNA and thus biosynthesis of Atp6 (subunit a) and Cox3...
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