Abstract
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.
Highlights
CYP119 from Sulfolobus solfataricus, the first thermophilic cytochrome P450, is stable at up to 85 °C
CYP119, which was first identified as a possible P450 enzyme from the S. solfataricus genome sequence, has the general properties characteristic of a cytochrome P450 enzyme
CYP119, the first P450 enzyme from a thermophilic organism, withstands prolonged incubation at high temperatures at a pH of 6.0. This is clearly demonstrated by the finding that the absorbance at 450 nm of the ferrous CO complex remains unchanged after preincubation of the protein at a temperature of up to 80 °C for 90 min (Fig. 4) and by the finding that the catalytic oxidation of styrene is unimpaired by the same preincubation (Fig. 4)
Summary
P450, cytochrome P450; heme, iron protoporphyrin IX independent of the iron ligation and oxidation state; PPIX, protoporphyrin IX; RR, resonance Raman; LS, low spin; HS, high spin; Pd, putidaredoxin; PdR, putidaredoxin reductase; HPLC, high pressure liquid chromatography; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. Mutation of the conserved threonine to a large hydrophobic residue can increase the fraction of the protein in the high spin state due to displacement of the water ligand by the amino acid side chain (8 –10). We describe the construction, expression, and characterization of its Thr-213 and Thr-214 mutants These studies show that the threonine residues in CYP119 play an important role in controlling the active site structure, spin state, and catalytic activity of the enzyme. They are not, critical for its thermal stability. The basic properties of CYP119 has been published [15], as has a communication on the same enzyme by another laboratory [16]
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