Biochemical and structural studies of hepatitis B virus reverse transcriptase and glycosylasparaginase

This dissertation is composed of two parts. The first part describes the major project on purification and characterization of structural and functional domains derived from duck and human HBV reverse transcriptases. The second part discusses a project on biochemical and crystallographic studies of a glycosylasparaginase (GA) mutant.; Part I. Hepatitis B virus (HBV) infection is a serious public health problem with more than 2 billion people infected worldwide. The reverse transcriptases of the viruses in the HBV family share a highly conserved domain structure and play key roles in viral replication. Thus, the HBV reverse transcriptase (RT) is an ideal target for developing effective anti-HBV agents to prevent the development of HBV-induced liver disease.; With the help of mild detergents, we successfully reconstituted the protein priming activity of duck HBV RT using a recombinant RT protein expressed and refolded from bacterial inclusion bodies. Furthermore, the unique N-terminal domain of the duck HBV reverse transcriptase has been subcloned and expressed in E. coli. In the presence of the mild detergent, this domain was purified and refolded from inclusion bodies to a relatively stable state at high concentration. In addition, we have initiated studies on human HBV RT and designed some constructs to test their stability and functions.; Part II. Glycosylasparaginase (GA) uses an autoproteolytic processing mechanism, through an N-O acyl shift, to generate a mature/active enzyme from a single-chain precursor. Structures of GA precursors complexed with a glycine inhibitor have revealed that the backbone in the immediate vicinity of the scissile peptide bond is in a distorted trans conformation. This is believed to be the driving force for the N-O acyl shift to break the peptide bond. Here we report x-ray crystallographic structural studies of the effects of the designed point mutation D151N. In addition to the loss of the base essential in autoproteolysis, this mutation also eradicates the backbone distortion near the scissile peptide bond. Binding of glycine inhibitor to the autoproteolytic site of the D151N mutant does not restore the backbone distortion. Therefore, Asp151 plays a dual role, acting as the general base to activate the nucleophile and holding the distorted trans conformation that is critical for initiating an N-O acyl shift.