Structure-function studies of metallo-enzymes acireductone dioxygenase and cytochrome P450cam.

摘要

Located at a branching point in the methionine salvage pathway, acireductone dioxygenase (ARD) represents an unusual case where two functional enzymes with different chemistries are derived from the same polypeptide chain. When Fe2+ is bound in the active site, the enzyme catalyzes the on-pathway reaction that leads to methionine recovery. However, Ni 2+-bound ARD, which targets the same substrate, leads to an off-pathway shunt. The active site characterization of these two isozymes in Klebsiella oxytoca is described in the first part of this thesis. This first part also deals with structural and mechanistic studies of bacterial E1 enolase-phosphatase, the bifunctional enzyme that catalyzes the formation of ARD substrate.;Part II is directed toward the structural characterization of the human ARD homolog (HsARD). Just like its bacterial counterpart, Fe2+ in the active site leads to the on-pathway reaction, but Mn2+-containing HsARD is the form responsible for the off-pathway reaction. The implications of Mn2+ paramagnetism on NMR signals are discussed. For structural studies, HsARD expression in a bacterial system was improved by a factor of 4 in minimal growth medium supplied with isotopic labels and trace metals. Rapid loss of enzyme activity was successfully suppressed, although the issue of protein aggregation remains to be addressed.;Part III of this thesis concerns structure-function studies of a bacterial P450 enzyme. Cytochrome P450s constitute a large superfamily of heme-thiolate proteins that catalyze a wide variety of reactions. Although they share similar structural folds, some P450s are highly substrate specific, while others are promiscuous. P450cam, or CYP101, from Pseudomonas putida catalyzes the 5-exo-hydroxylation of camphor and requires the presence of redox partner putidaredoxin (Pdx). High-resolution solution NMR was used to investigate the effects of substrate replacement in CYP101 on regions that are remote from the active site. Finally, we used residual dipolar couplings along with molecular dynamics to generate a preliminary structure of Pdx-free CYP101 in solution.