ABSTRACT
The cytochromes P450 are an important class of mono-oxygenases involved in xenobiotic metabolism and steroid biosynthesis in a diverse set of life forms. Discovery of CYP-119, a P450 from the archea Sulfolobus solfataricus has provided a means for understanding nature's method of stabilizing this important protein superfamily. To identify classes of stabilizing interactions used by CYP-119, we have generated a randomized library of point mutants and screened for mutants that are less thermostable than the wild type by monitoring the characteristic Soret band in the visible region of the cell lysis. The selected mutants were characterized by differential scanning calorimetry to compare the temperatures of the melting transitions of the various mutants. The identified mutations suggested that electrostatic interactions involving salt links and charge-charge interactions, as well as contributions from other interactions such as aromatic stacking, and side chain volume of hydrophobic residues contribute to enhanced thermostability in this cytochrome P450.
Subject(s)
Cytochrome P-450 Enzyme System/chemistry , Sulfolobus/enzymology , Cytochrome P-450 Enzyme System/genetics , Enzyme Stability , Models, Molecular , Mutagenesis , Protein Engineering , ThermodynamicsABSTRACT
Members of the cytochrome P450 superfamily catalyze the addition of molecular oxygen to nonactivated hydrocarbons at physiological temperature-a reaction that requires high temperature to proceed in the absence of a catalyst. Structures were obtained for three intermediates in the hydroxylation reaction of camphor by P450cam with trapping techniques and cryocrystallography. The structure of the ferrous dioxygen adduct of P450cam was determined with 0.91 angstrom wavelength x-rays; irradiation with 1.5 angstrom x-rays results in breakdown of the dioxygen molecule to an intermediate that would be consistent with an oxyferryl species. The structures show conformational changes in several important residues and reveal a network of bound water molecules that may provide the protons needed for the reaction.
Subject(s)
Camphor 5-Monooxygenase/chemistry , Camphor 5-Monooxygenase/metabolism , Camphor/chemistry , Camphor/metabolism , Catalysis , Crystallization , Crystallography, X-Ray , Electrons , Ferric Compounds/chemistry , Ferric Compounds/metabolism , Ferrous Compounds/chemistry , Ferrous Compounds/metabolism , Hydrogen Bonding , Hydroxylation , Ligands , Models, Molecular , Molecular Conformation , Oxygen/chemistry , Oxygen/metabolism , Protein Conformation , Protein Structure, Secondary , Protons , Pseudomonas putida/enzymology , Water/chemistry , Water/metabolismABSTRACT
We report the cloning, expression, purification, and molecular characterization of a cytochrome P450 (CYP119) from the thermophilic archaea Sulfolobus solfataricus. This protein displays an absorption spectra in the reduced, oxidized, and carbonyl adduct analogous to those of other P450 enzymes. We demonstrate that P450 (CYP119) exhibits remarkable thermo- and pressure stability, with a melting temperature 40 degrees higher than that of the extensively studied cytochrome P450cam (CYP101) and an optical spectra completely resistant to the formation of the inactive P420 by hydrostatic pressure up to 2 kbar. CO flash photolysis experiments, as well as construction of a CYP119 homology model, suggest an open active site with greater solvent access than P450 (CYP101) and similar to that of P450 (CYP102). This communication represents the first molecular characterization of an extremophilic cytochrome P450.
Subject(s)
Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Oxygenases/chemistry , Oxygenases/metabolism , Sulfolobus/enzymology , Amino Acid Sequence , Archaeal Proteins , Base Sequence , Calorimetry, Differential Scanning , Carbon Dioxide/metabolism , Cloning, Organism , Cytochrome P-450 Enzyme System/genetics , DNA Primers , Escherichia coli , Kinetics , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Oxygenases/genetics , Photolysis , Polymerase Chain Reaction , Protein Conformation , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrophotometry , Sulfolobus/geneticsABSTRACT
A mutation at the surface of the substrate access channel which dramatically decreases the affinity for some fatty acids in P450(BM-3) was discovered by random mutagenesis. The mutation introduced, proline-25 to glutamine, is in close proximity to the arginine-47 residue thought to be responsible for the initial docking of fatty acid substrates. The P25Q mutant displays an affinity for palmitate which is approximately 100-fold weaker than the wild-type enzyme. In addition to its altered substrate affinity, P25Q also exhibits altered hydroxylation specificity and carbon monoxide recombination kinetics in the substrate-free form.
Subject(s)
Bacterial Proteins , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/metabolism , Protein Structure, Secondary , Arginine , Binding Sites , Cloning, Molecular , Escherichia coli , Glutamine , Kinetics , Lauric Acids/metabolism , Models, Structural , Mutagenesis, Site-Directed , Myristic Acid , Myristic Acids/metabolism , NADPH-Ferrihemoprotein Reductase , Palmitic Acid/metabolism , Point Mutation , Proline , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate Specificity , ThermodynamicsABSTRACT
Building on the vast knowledge of active site structure and catalytic mechanisms of the P450 monooxygenase systems, significant efforts to utilize the rational design of engineered P450s are emerging as an approach to solve the problems of bioremediation. P450 enzymes are being designed to alter substrate specificities and catalytic efficiency in predefined ways. In addition, random mutagenesis and in vitro evolution are being considered as exciting methods for generating mutant P450s with increased bioremediation abilities.
