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

Abstract

A red-pigmented coryneform bacterium, identified as Rhodococcus rhodochrous strain 116, that grew on 2-ethoxyphenol and 4-methoxybenzoate as sole carbon and energy sources was isolated. Phylogenetic analysis based on the 16S rDNA sequences indicates that the strain clusters more closely to other rhodococci than to other gram-positive organisms with a high G+C content. Each of the abovementioned growth substrates was shown to induce a distinct cytochrome P-450: cytochrome P-450(RR1) was induced by 2- ethoxyphenol, and cytochrome P-450(RR2) was induced by 4-methoxybenzoate. A type I difference spectrum typical of substrate binding was induced in cytochrome P-450(RR1) by both 2-ethoxyphenol (K(s) = 4.2 ± 0.3 μM) and 2- methoxyphenol (K(s) = 2.0 ± 0.1 μM), but not 4-methoxybenzoate or 4- ethoxybenzoate. Similarly, a type I difference spectrum was induced in cytochrome P-450(RR2) by both 4-methoxybenzoate (K(s) = 2.1 ± 0.1 μM) and 4-ethoxybenzoate (K(s) = 1.6 ± 0.1 μM), but not 2-methoxyphenol or 2- ethoxyphenol. A purified polyclonal antiserum prepared against cytochrome P- 450(RR1) did not cross-react with cytochrome P-450(RR2), indicating that the proteins are immunologically distinct. The cytochromes appear to catalyze the O-dealkylation of their respective substrates. The respective products of the O-dealkylation are further metabolized via ortho cleavage enzymes, whose expression is also regulated by the respective aromatic ethers.