Purification and characterization of cytochrome P450_RR1 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 SDSRAGE 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°C). 2-Methoxyphenol binds to the cytochrome with a macroscopic dissociation constant of 0.53 +/-0.03 pM (50 mM Mops, pH 7.0, 25OC) and induces a 99.7% transition of the heme iron to the pentacoordinate highspin form. Using a reconstituted in-vitro activity assay, it was demonstrated that P450,, catalyzed the 0-dealkylation of 2-ethoxyphenol and 2-methoxyphenol to produce catechol. The cytochrome binds other ortho-substituted phenols, including 2-ethoxyphenol, 2-methylphenol (0-cresol) and 2-chlorophenol. The affinity of P450RR, 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 rnetu-substituted ether phenols did not induce a spin transition.

Two independently regulated cytochromes P-450 in a Rhodococcus rhodochrous strain that degrades 2-ethoxyphenol and 4-methoxybenzoate

“…Third, we have identified enzymatic activities in the cell extracts that catalyze the ring cleavage of the expected products of the postulated O-dealkylation reactions. Finally, in an in vitro system consisting of purified P-450RR1 and chemical reductants, we have demonstrated that P-450RR1 catalyzes the O-dealkylation of 2-ethoxyphenol to produce catechol (10).…”

“…The presence of P-450 in extracts of cells grown on 4-ethoxybenzoate was confirmed by CO difference spectra. Coomassie blue staining of the lanes of the same In experiments performed with the purified cytochrome, it has been shown that P-450RR1 catalyzes the O-dealkylation of 2-ethoxyphenol and 2-methoxyphenol to form catechol (10). It was conjectured that P-450RR2 catalyzes the 0-dealkylation of 4-ethoxybenzoate and 4-methoxybenzoate, producingp-hydroxybenzoate.…”

“…As part of a study to characterize and modify the enzymatic activities of these ether bond-cleaving cytochromes, we have purified both proteins to apparent homogeneity (10,12). The polyclonal antibodies described in this study are being used to screen an expression library in order to clone the structural gene of P-450RR1.…”

“…P-450RR1 was purified to homogeneity from R. rhodochrous grown on 5 mM 2-ethoxyphenol as described elsewhere (10). The concentration of the cytochrome was determined spectrophotometrically, using a difference in extinction coefficients De45490 of 92.8 cm-' mM-1 for the reduced CO-bound species (14).…”

Degradation of the thiocarbamate herbicide EPTC (S-ethyl dipropylcarbamothioate) and biosafening by Rhodococcus sp. strain NI86/21 involve an inducible cytochrome P-450 system and aldehyde dehydrogenase

“…strain NI86/21. On the basis of the lack of N-terminal sequence homology, the 2-ethoxyphenolinduced cytochrome P-450 RR1 from Rhodococcus rhodochrous (18) and the enzyme of Rhodococcus sp. strain NI86/21 also appear not to be related.…”