Characterization of a novel metalloenzyme OsARD1 and structural, functional and dynamic studies of cytochrome P450cam

Acireductone dioxygenases (ARD) involved in the methionine salvage pathway are unique proteins that can acquire two different activities depending on the metal ion bound. OsARD1 from deepwater rice (Oryza sativa L.) was putatively assigned as an ARD based on sequence similarity to an ARD enzyme from K. oxytoca. OsARD1 has been expressed, purified and characterized in terms of structure and function. Ectopically expressed apo-OsARD1 preferentially binds Fe2+ and reconstituted Fe-OsARD1 catalyzes the formation of 2-keto-pentanoate and formate from the model substrate and dioxygen, indicating that OsARD1 is capable of catalyzing the penultimate step in the methionine cycle. The highly conserved overall chemistry of this enzyme confirmed that OsARD1 belongs to the ARD family.;Cytochrome P450cam (CYP101) from soil bacterium Pseudomonas putida catalyzes the 5-exo hydroxylation of camphor. The presence of an effector is required in the catalytic cycle in order to drive product formation from the reduced O2- and substrate-bound intermediate form of CYP101. With the recent technical and methodological advances available to investigate this large protein, a significant number of backbone resonances have been additionally assigned in the camphor- and CO-bound reduced CYP101 (CYP-S-CO) spectra. Using these sequential assignments, we have investigated the effects of potassium on enzyme-substrate interactions in P450cam by multiple methods. The spectral effects of K+ binding in CYP-S-CO oppose those observed upon Pdxr titration. Evidence was also found from nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis and simulations suggesting that binding of Pdx converts a single X-proline amide bond in CYP101 from trans or distorted trans to cis. Backbone dynamics of CYP101 as a function of oxidation/ligation state of the heme iron were investigated via hydrogen/deuterium exchange (H/D exchange) as monitored by mass spectrometry and nuclear magnetic resonance spectroscopy. The reduced enzyme CYP-S-CO shows significantly slower exchange rates than the oxidized CYP-S.