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Purification and characterization of cytochrome P450RR1 from Rhodococcus rhodochrous.
Eltis, L D; Karlson, U; Timmis, K N.
Afiliação
  • Eltis LD; Department of Microbiology, Gesellschaft für Biotechnologische Forschung, National Research Centre for Biotechnology, Braunschweig, Federal Republic of Germany.
Eur J Biochem ; 213(1): 211-6, 1993 Apr 01.
Article em En | MEDLINE | ID: mdl-8477696
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.
Assuntos
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Rhodococcus / Sistema Enzimático do Citocromo P-450 / Isoenzimas Idioma: En Revista: Eur J Biochem Ano de publicação: 1993 Tipo de documento: Article
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Rhodococcus / Sistema Enzimático do Citocromo P-450 / Isoenzimas Idioma: En Revista: Eur J Biochem Ano de publicação: 1993 Tipo de documento: Article