Structural and energetic studies on the enzymes 5-keto-4-deoxyuronate isomerase of Escherichia coli and reverse transcriptase of Moloney murine leukemia virus

Structure-function relationships in proteins have long been of interest and provided the basis of our current understanding of many biological systems. This dissertation comprises two parts that are related to each other by the use of X-ray crystallography to study the structure and function of proteins. In the first part, the crystal structure of the enzyme, 5-keto-4-deoxyuronate isomerase (KduI), from Escherichia coli is presented. The structure was solved at 1.94 A resolution using multiwavelength anomalous diffraction. The monomer of KduI is comprised of two similar beta-barrels. The C-terminal half of the monomer contains a bound metal atom tentatively identified as zinc. There is a region of unexplained electron density surrounding the Nzeta atom of Lysine 165 that is likely to be a bound molecule of polyethylene glycol, which may possibly mimic the binding of substrate or product. KduI is found to be structurally similar to members of the cupin superfamily, which includes the seed storage proteins of plants, human pirin, phosphomannose isomerase in yeast, and glucose-6-phosphate isomerase in archaebacteria. Like some members of the cupin family, KduI most likely forms hexamers in solution. In the second part of the dissertation I present the structure of the R116A mutant of the N-terminal fragment of reverse transcriptase from Moloney Murine Leukemia Virus (MMLV-RT). Arginine 116 is known to form hydrogen bonds with DNA bound to the fingers domain of MMLV-RT. The importance of R116 to the structure and energetics of DNA binding was studied by X-ray crystallography and by isothermal titration calorimetry using two distinct oligonucleotides. I find that the R116A mutation has virtually no effect on the protein structure of the MMLV-RT N-terminal fragment compared to wild type protein. Even though the structure is preserved, there is no detectable binding of oligonucleotides to the R116A mutant protein. This suggests that the loss of hydrogen bonding potential with R116 is primarily responsible for the lack of DNA binding to the R116A mutant.