title: Purification and characterization of cytochrome P450RR1 from Rhodococcus rhodochrous.
abstract: A soluble cytochrome P450 whose synthesis is induced by and that binds 2-ethoxyphenol was purified to apparent homogeneity from Rhodococcus rhodochrous strain 116. The enzyme had a subunit molecular mass of 44.5 kDa as determined by SDS/PAGE and a pI of 5.2. The electronic absorption spectrum indicates that the native cytochrome in the absence of substrate is predominantly in the low-spin state (13% high-spin state in 50 mM Mops, pH 7.0 25 degrees C). 2-Methoxyphenol binds to the cytochrome with a macroscopic dissociation constant of 0.53 +/- 0.03 microM (50 mM Mops, pH 7.0, 25 degrees C) and induces a 99.7% transition of the heme iron to the pentacoordinate high-spin form. Using a reconstituted in-vitro activity assay, it was demonstrated that P450RR1 catalyzed the O-dealkylation of 2-ethoxyphenol and 2-methoxyphenol to produce catechol. The cytochrome binds other ortho-substituted phenols, including 2-ethoxyphenol, 2-methylphenol (o-cresol) and 2-chlorophenol. The affinity of P450RR1 for these compounds is lower than that of 2-methoxyphenol and they are less effective than 2-methoxyphenol at inducing a transition in the heme iron to the high-spin state. Para-substituted and meta-substituted ether phenols did not induce a spin transition.
authors: Department of Microbiology, Gesellschaft für Biotechnologische Forschung, National Research Centre for Biotechnology, Braunschweig, Federal Republic of Germany.
authors: L D
authors: LD
authors: Eltis
authors: U
authors: U
authors: Karlson
authors: K N
authors: KN
authors: Timmis
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D012240:Rhodococcus / Q000201:enzymology*
chemical_list: D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System