Structural Studies of Enzymes Important in RNA-Editing and in Chemoenzymatic Synthesis of Carbohydrate

The most effective approaches in developing biochemical and pharmaceutical technologies include an understanding of the structure-function relationship in the enzymes involved. Presented here are structural studies of three different enzymes. Protein crystallography and X-ray diffraction has been the main method of study, but biochemical approaches have also been used. The first project investigates an essential human RNA-editing enzyme, and the other two are related to the synthesis of carbohydrate structures.;The process of A-to-I RNA editing is present in both plant and animal species. The human A-to-I RNA editing enzyme adenosine deaminase acting on double-stranded RNA (ADAR), has been implicated in many types of cancers and neurological disorders and is a known cause of Aicardi-Goutieres Syndrome. Crystallographic studies of human ADAR2 deaminase domain (ADAR2d) in this work have captured several complexes of ADAR2d and mutants of ADAR2d with various double-stranded RNAs (dsRNAs), which reveal a base-flipping mechanism and the basis for both 5'- and 3'-nucleotide preferences. Further studies which include the ADAR2 deaminase domain and one of the enzyme's double-stranded RNA-binding domains (DSRBDs) are now being conducted, and a crystal structure of a mutant of ADAR2d with application to site-specific RNA-editing technologies is also described. Overall, this project has made considerable progress toward an understanding of the mechanism and selectivity of human ADARs, giving us a better understanding of how mutation in ADAR leads to disease, and enabling us to develop technologies which can treat those diseases.;The nine-carbon alpha-keto acids called sialic acids are essential and diverse carbohydrate building blocks for cell-cell communication as well as pathogenic infection. Chemoenzymatic synthesis using bacterial enzymes has the potential to generate a library of sialic acid-containing structures. The CMP-sialic acid synthetase from Neisseria meningitidis (NmCSS) is one of these enzymes, and I present four crystal structures of NmCSS along the various stages of its catalytic cycle. These structures suggest a mechanism for an "open" to "closed" conformational transition and a catalytic mechanism for the enzyme. Knowledge gained from these structures was used to carry out an initial round of structure-guided enzyme engineering aimed at increasing or altering the substrate tolerance of NmCSS. Ongoing work continues to use the crystal structures to create mutants of NmCSS which will increases its versatility in chemoenzymatic methods.;The last project is a structural investigation of the bacterial glycosyltransferase Pasteurella multocida heparosan synthase 2 (PmHS2). PmHS2 catalyzes the synthesis of heparosan, a precursor to the anticoagulant heparin. Heparin has been used by doctors for over half a century, but currently the main source of heparin is animal tissue extract. Chemoenzymatic synthesis of this molecule would allow for the mass production of highly pure and structurally specific heparin for medical use. In this work, crystallographic studies of full length and truncated PmHS2 are described. In an effort to produce constructs which will be suitable for protein crystallography, structural comparison with a similar enzyme of known structure and secondary structure prediction suggest the presence of a disordered N-terminal region and serve as a guide for domain separation. Finally, an enzyme-coupled continuous UV-Vis assay is developed to screen constructs for enzymatic activity.