Structure and function of tetrapyrrole biosynthesis enzymes: ALA synthase and cobalt chelatase

Modified tetrapyrroles are versatile compounds that are universally utilized by enzymes as co-factors in a myriad of enzyme cata lyzed reaction s. Examples include heme, the cofactor of hemoglobin and myoglobin, that carries and stores molecular oxygen in the blood and muscle respectively, and cobalamin a cofactor that is synthesized by prokaryotes and which is an important nutrient for humans. This work focuses on the enzymatic mechanism of metal chelation and in particular on the structural characterization of the cobalt-chelatases CbiK from Salmonella enterica and CbiX from Archaeoglobus /u/gidus that take part in the biosynthesis of cobalamin. Crystal structures of the enzyme-tetrapyrrole complex were obtained that reveal radically different modes of binding compared to the well characterized ferrochelatases. Furthermore protein structures reveal the evolutionary re lationships between cobaltochelatases from different organisms. The second part of this thesis is focusing on 5-aminolevulinic acid synthase (ALAS), an enzyme that catalyzes the synthesis of the first common precursor in t he biosynthesis of all tetrapyrroles, 5-aminolevulinic acid (ALA). Mutations in the C-terminal region of the human erythroid specific ALAS (ALAS2) lead to X-linked dominant protoporphyria a condition that is characterized by the accumulation of protoporphyrin in red blood cells leading to photosensitivity and liver disease. The erythroid specific ALAS2 and its disease - causing variants have been characterized using steady state kinetic methods to investigate the molecular basis of the disorder. Furthermore the "house-keeping" ALASl isoenzyme was characterized as well and the effect of mutations equivalent to the disease causing ALAS2 mutations have been tested on the enzyme. Chimeric bacterial enzymes that have been engineered to contain the human erythroid specific C-terminal extension were characterized as an alternative to understanding the role of the C-terminal extension in regulating the activity of the human ALASl and ALAS2EThOS - Electronic Theses Online ServiceGBUnited Kingdo