The Active Site of the Thermophilic CYP119 from Sulfolobus solfataricus

Abstract

CYP119 from Sulfolobus solfataricus, the first thermophilic cytochrome P450, is stable at up to 85°C. UVvisible 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-␤-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.

Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

“…Moving the phenyl group of 4-PI to the nitrogen, as in 1-phenylimidazole (1-PI), resulted in a 2-fold decrease in binding affinity, in agreement with data reported for CYP2B4 (45). Lauric acid and stearic acid, two substrates, had similar binding constants with CYP119, whereas styrene is only a weak substrate of CYP119 (46 17 Phe residues in CYP119, the signals for four Phe residues were absent or unresolved in the spectrum. Signals from these Phe residues may be unobservable due to resonance overlap, H-D exchange, line broadening caused by variable dynamics of these particular regions, or ferric iron paramagnetic effects.…”

Crystal Structure of a Thermophilic Cytochrome P450 from the Archaeon Sulfolobus solfataricus

“…3), which is present in most P450s. The positioning of Thr-213 close to the heme surface relative to Thr-214 and Thr-215 was correctly predicted based on chemical modification data (12). This region has been postulated to be involved in a proton shuttle network considered important for delivering solvent protons required for the activation of O 2 during the catalytic cycle (28).…”

“…This is very similar to the H-bonding pattern found in P450BM-3 that also has a Thr corresponding to Thr-214 in CYP119 (29). Interestingly, the T214A (but not T213A) mutant results in a large increase in the rate at which CYP119 catalyzes H 2 O 2 -supported styrene epoxidation (12). Because Thr-214 but not Thr-213 H-bonds with the backbone, it might be expected that mutation of Thr-214 would cause a greater perturbation in the I helix thereby leading to a greater change in reactivity toward styrene.…”

“…As expected, CYP119 exhibits unusual thermal stability with a melting temperature of ϳ90°C compared with ϳ55°C for bacterial P450cam (12,13). The function of CYP119 remains unknown, but considering that S. solfataricus can utilize elemental sulfur as an energy source (11) and that P450s are known to participate in sulfoxide formation (14), the oxidative metabolism of sulfur compounds is a possibility.…”

“…cells were transformed with a pCWori plasmid containing the gene for CYP119 (12). Single colonies were selected for overnight growth in 5-ml cultures, which were used to inoculate 1-liter cultures.…”