Abstract
CYP119 from Sulfolobus solfataricus, the first thermophilic cytochrome P450, is stable at up to 85 °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 T1 NMR studies confirm that styrene is bound in the active site. CYP119 catalyzes the H2O2-dependent epoxidation of styrene, cis-β-methylstyrene, and cis-stilbene with retention of stereochemistry. This catalytic activity is stable to preincubation at 80 °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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 14112-14123 |
| Number of pages | 12 |
| Journal | Journal of Biological Chemistry |
| Volume | 275 |
| Issue number | 19 |
| DOIs | |
| State | Published - May 12 2000 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
The active site of the thermophilic CYP119 from Sulfolobus solfataricus. / Koo, Laura S.; Tschirret-Guth, Richard A.; Straub, Wesley E.; Moenne-Loccoz, Pierre; Loehr, Thomas M.; Ortiz De Montellano, Paul R.
In: Journal of Biological Chemistry, Vol. 275, No. 19, 12.05.2000, p. 14112-14123.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - The active site of the thermophilic CYP119 from Sulfolobus solfataricus
AU - Koo, Laura S.
AU - Tschirret-Guth, Richard A.
AU - Straub, Wesley E.
AU - Moenne-Loccoz, Pierre
AU - Loehr, Thomas M.
AU - Ortiz De Montellano, Paul R.
PY - 2000/5/12
Y1 - 2000/5/12
N2 - CYP119 from Sulfolobus solfataricus, the first thermophilic cytochrome P450, is stable at up to 85 °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 T1 NMR studies confirm that styrene is bound in the active site. CYP119 catalyzes the H2O2-dependent epoxidation of styrene, cis-β-methylstyrene, and cis-stilbene with retention of stereochemistry. This catalytic activity is stable to preincubation at 80 °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.
AB - CYP119 from Sulfolobus solfataricus, the first thermophilic cytochrome P450, is stable at up to 85 °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 T1 NMR studies confirm that styrene is bound in the active site. CYP119 catalyzes the H2O2-dependent epoxidation of styrene, cis-β-methylstyrene, and cis-stilbene with retention of stereochemistry. This catalytic activity is stable to preincubation at 80 °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.
UR - http://www.scopus.com/inward/record.url?scp=0034640268&partnerID=8YFLogxK
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U2 - 10.1074/jbc.275.19.14112
DO - 10.1074/jbc.275.19.14112
M3 - Article
C2 - 10799487
AN - SCOPUS:0034640268
VL - 275
SP - 14112
EP - 14123
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 19
ER -