The active site of the thermophilic CYP119 from Sulfolobus solfataricus.
CYP119 from Sulfolobus solfataricus, the first thermophilic cytochrome P450, is stable at up to 85 degrees C. UV-visible and resonance Raman show the enzyme is in the low spin state and only modestly shifts to the high spin state at higher temperatures. Styrene only causes a small spin state shift, but T(1) NMR studies confirm that styrene is bound in the active site. CYP119 catalyzes the H(2)O(2)-dependent epoxidation of styrene, cis-beta-methylstyrene, and cis-stilbene with retention of stereochemistry. This catalytic activity is stable to preincubation at 80 degrees C for 90 min. Site-specific mutagenesis shows that Thr-213 is catalytically important and Thr-214 helps to control the iron spin state. Topological analysis by reaction with aryldiazenes shows that Thr-213 lies above pyrrole rings A and B and is close to the iron atom, whereas Thr-214 is some distance away. CYP119 is very slowly reduced by putidaredoxin and putidaredoxin reductase, but these proteins support catalytic turnover of the Thr-214 mutants. Protein melting curves indicate that the thermal stability of CYP119 does not depend on the iron spin state or the active site architecture defined by the threonine residues. Independence of thermal stability from active site structural factors should facilitate the engineering of novel thermostable catalysts.
Koo
L S
LS
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.
Tschirret-Guth
R A
RA
Straub
W E
WE
Moënne-Loccoz
P
P
Loehr
T M
TM
Ortiz de Montellano
P R
PR
D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002849:Chromatography, Gas; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D016944:Sulfolobus / Q000201:enzymology*; D013912:Threonine / Q000235:genetics / Q000378:metabolism
D019843:Archaeal Proteins; D017931:DNA Primers; D013912:Threonine; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C408874:CYP119 protein, Sulfolobus solfataricus