Structures and function of 5'-fluorodeoxyadenosine synthase

Abstract

Halogenation in nature is becoming better understood and in recent studies (Harper etal., 2003; Murphy, 2003) there appears to be several mechanisms available to nature for halogenation. 5'-Fluorodeoxyadenosine synthase (FDAS) is the only enzyme identified in nature that is capable of formation of a C-F bond. FDAS does this by catalysing the transformation of S-adenosyl-L-methionine (SAM) and inorganic fluoride to 5'-fluoro-5'-deoxyadenosine (FDA). It is the essential enzyme in the fluorinase pathway in Streptomyces cattleya which results in the production of 4-fluorothreonine and fluoroacetate. This thesis describes a structural investigation into FDAS. Adenosine was identified as a contaminant and removed, permitting the X-ray crystal structures of FDAS in apo-form and in complex with several important substrates and substrate analogues. FDAS was identified to catalyse incorporation of chlorine by a nucleophilic mechanism. FDAS is also capable of utilising 2'-deoxy substrate analogues but not 3'- deoxy ones. The structural data allow both substrate specificity and chlorine incorporation to be rationalised. Thermodynamic studies were carried out on FDAS using isothermal calorimetry and contribute to improving our understanding of the enzyme. Sso6206 is a small acidic protein with unknown function from Sulfolobus solfataricus (Sso). The protein was overexpressed, purified and crystallized in native and selenomethionine derivative forms. Data were measured to 2.4 A indicating that the asymmetric unit contains between 15 and 30 monomers. This could be a result of a large protomer complex formation, suggesting an unusual function. Attempts to obtain phases using several methods are described. 2-Keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase was identified in E. coli as responsible for the reversible conversion of KDPGal to glyceraldehyde-3- phosphate (G3P) and pyruvate. The protein is homologous to the 2-keto-3-deoxy-6- phosphogluconte (KDPG) aldolase which performs a similar reaction with KDPG. These protein are important biosynthetic tools. The apo-stmcture was solved in a previous study, however the reasons for the observed stereo-specificity were not clear. This thesis reports several complex structures and permits a rationalisation of the different stereochemistry between the enzymes.