Crystal Structures of a Bacterial Isocitrate Dehydrogenase and the Human Sulfamidase
Pushing the Limits of Molecular Replacement
Sidhu, Navdeep Singh
Dissertation
Angenommen am:
2014-01-09
Erschienen:
2015-01-09
Betreuer:
Sheldrick, George M. Prof. Dr.
Gutachter:
Ficner, Ralf Prof. Dr.
Gutachter:
Tittmann, Kai Prof. Dr.
Gutachter:
Laatsch, Hartmut Prof. Dr.
Gutachter:
Dittrich, Birger PD Dr.
Gutachter:
Fabbiani, Francesca Dr.
Zum Verlinken/Zitieren: http://hdl.handle.net/11858/00-1735-0000-0015-A383-5
Dateien
Name:
thesis_online_2.pdf
Größe:
107,9 MB
Format:
PDF
Beschreibung:
Dissertation copy ...
Lizenzbestimmungen:
Zusammenfassung
Englisch
In an X-ray crystallographic experiment, intensities and directions of X-rays scattered by electrons in a crystal are measured. However, the relative phase information in the interference pattern is lost. If we knew the phases, we could calculate the electron density in the crystal, fit an atomic model to the density and thus solve the three-dimensional structure of the substance forming the crystal. One method of deriving the phase information is called molecular replacement (MR). In this method, the diffraction data are used to correctly position in the crystal a search model that is derived from a known atomic structure believed to be similar to the unknown target structure. Phases calculated for the correctly positioned model can then be combined with X-ray intensity data for the unknown structure to calculate a more or less hybrid electron density map. A crucial test is whether we are able to identify features corresponding to the target structure in this map as the phases come from a different structure. For proteins, if the sequence identity between the search and target structure is below 30%, the method is often difficult or impossible to use and has typically failed. However, recent research is expanding this threshold and it continues to be an area of active research. Here, the method was used to attempt to solve crystal structures of two protein enzymes. In the first, the shared sequence identity between the search and target structures was 100%, making the method straightforward to apply. In the second, the sequence identity was approximately 22%. The method was successfully applied also in this case. The first enzyme was the Krebs cycle enzyme isocitrate dehydrogenase from the bacterium Corynebacterium glutamicum. It is of special interest as it has been shown to recognize with high specificity its coenzyme nicotinamide adenine dinucleotide phosphate from the closely similar coenzyme nicotinamide adenine dinucleotide, which lacks a phosphate group that the former has, and has a hydroxyl group instead. Crystals of the enzyme were grown in the presence of the coenzyme. The crystal structure was solved to attempt to better understand how the high-specificity recognition is achieved at the atomic level. Heparin and heparan sulfate are glycosaminoglycans (or mucopolysaccharides)–polysaccharides made up of a repeating disaccharide structural unit with many covalently bound sulfate groups. Their degradation in the body occurs in the cell organelles lysosomes. The deficient activity of one of the enzymes involved causes a progressive neurodegenerative disease known as mucopolysaccharidosis IIIA. It typically manifests itself in childhood, often leading to premature death in the second or third decade of life. Many mutations associated with the disease have been described. However, our understanding of how these mutations affect enzyme structure and function has been limited, one of the major hurdles being that the three-dimensional structure of the enzyme was unknown. Here, crystals of the enzyme were grown. The first structure of a sulfamidase, specifically the human enzyme, was solved using an MR search model that shared a low sequence identity with the target enzyme. The structural effects of the mutations are described, and an enzyme mechanism proposed.
Keywords: sulfamidase; crystal structure; isocitrate dehydrogenase; molecular replacement; nicotinamide adenine dinucleotide phosphate; sulfamate sulfohydrolase; heparan N-sulfatase; N-sulfoglucosamine sulfohydrolase
