Characterization of a cytochrome P450 from the acidothermophilic archaea Sulfolobus solfataricus.
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.
McLean
M A
MA
Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana, Illinois, 61801, USA.
Maves
S A
SA
Weiss
K E
KE
Krepich
S
S
Sligar
S G
SG
D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002245:Carbon Dioxide / Q000378:metabolism; D019976:Cloning, Organism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010782:Photolysis; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D002245:Carbon Dioxide; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C408874:CYP119 protein, Sulfolobus solfataricus