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 P450: cytochrome P-450O1 was induced by 2-ethoxyphenol, and cytochrome P-450R= was induced by 4-methoxybenzoate. A type I difference spectrum typical of substrate binding was induced in cytochrome P-450RR1 by both 2-ethoxyphenol (K, = 4.2 0.3 pM) and 2-methoxyphenol (K, = 2.0 + 0.1 pM), but not 4-methoxybenzoate or 4-ethoxybenzoate. Similarly, a type I difference spectrum was induced in cytochrome P-450Rm by both 4-methoxybenzoate (K, = 2.1 0.1 FM) and 4-ethoxybenzoate (K, = 1.6 0.1 RM), but not 2-methoxyphenol or 2-ethoxyphenol. A purified polyclonal antiserum prepared against cytochrome P-450RR1 did not cross-react with cytochrome P-45Rm, indicating that the proteins are immunologically distinct. The cytochromes appear to catalyze the 0-dealkylation of their respective substrates. The respective products of the 0-dealkylation are further metabolized via ortho cleavage enzymes, whose expression is also regulated by the respective aromatic ethers.The design of novel efficient microbial catabolic pathways to degrade otherwise recalcitrant environmental compounds involves not only the recruitment of enzymes with desirable catalytic activities from different organisms but also the modification of such enzymes to improve their catalytic abilities. Cytochromes P-450 are good candidates for such recruitment, as this class of heme monooxygenases catalyze a remarkably broad range of chemical reactions on an equally broad range of substrates (29). In bacterial systems, P-450s generally occur as part of a soluble three-component system, consisting of a flavin-containing reductase and an iron-sulfur electron transfer protein in addition to the cytochrome (37). Bacterial P-450s often occur in catabolic pathways, frequently catalyzing an initial hydroxylation reaction of the growth substrate, as in the case of P-450cam (21) and P-4501in (42). P-450's have also been reported to catalyze the O-dealkylation of para-substituted benzoates (3), veratrole (41), and ortho-substituted alkoxyphenols (5). The characteristic absorbance peak at approximately 450 nm of the CO-difference spectrum of the reduced P-450 provides a ready means to screen for the presence of these enzymes in whole cells and crude extracts (31 interest in recruiting P-450's into both biotransformation and biodegradation processes has increased rapidly (6, 29, 37).A structural element that contributes to both the recalcitrance and the toxicity of such pollutants as 2,3,7,8-tetrachlorodibenzo-p-dioxin is the ether bond. To investigate the mechanism of ether bond cleavage in aromatic compounds, microorganisms were sc...

A re-evaluation of the taxonomy of the genus Anaerovibrio, with the reclassification of Anaerovibrio glycerini as Anaerosinus glycerini gen. nov., comb. nov., and Anaerovibrio burkinabensis as Anaeroarcus burkinensis [corrig.] gen. nov., comb. nov.

“…Nearly complete 16s rRNA genes were amplified from chromosomal DNA by using the PCR (Mullis & Faloona, 1987) using a forward primer hybridizing at positions 8-27 (Escherichia coli rRNA gene numbering, primer sequence 5'-GAGTTTGATCMTGGCTCAG-3') and a reverse primer hybridizing at the complement of positions 1492-1 5 12 (5'-CGGTTACCTTGTTACGGAC-TT-3'). The PCR was carried out using a GeneAmp 9600 thermocycler (Perkin-Elmer) and conditions described previously (Karlson et al, 1993). Amplified DNA was purified with Microcon 100 microconcentrators (Amicon) and the quality was checked using gel electrophoresis on a 1 YO (w/v) agarose gel with Tris-acetate/EDTA (TAE) buffer and subsequent ethidium bromide staining.…”

Roles of key active-site residues in flavocytochrome P450 BM3

“…Generally, fewer P450s are found in lower eukaryotes and bacteria, although the recent determination of the entire genome sequence of Mycobacterium tuberculosis indicated the presence of 21 P450 and P450-like proteins [6]. In bacteria, the P450s have frequently evolved to catalyse the oxidation of environmental organic compounds, such as camphor, alkoxyphenols and herbicides, allowing these materials to be used as growth substrates [2,7,8].…”

Rubrobacter xylanophilus sp. nov., a New Thermophilic Species Isolated from a Thermally Polluted Effluent

“…Nearly complete 16s rRNA genes were amplified by PCR (22,30) by using forward primer 16F27 (5'-AGAGTTTGATCMTGGCTCAG-3' [M = A or C]; hybridizing at positions 8 to 27 [E. coli 16s rRNA gene sequence numbering]) and reverse primer 16R1525 (5'-AAGGAGGTGWTCCAGCC-3' [W = A or TI; hybridizing at the complement of positions 1525 to 1541). PCR amplification was performed by using a GeneAmp 9600 apparatus (Perkin-Elmer Corp., Nonvalk, Conn.) and the reaction conditions described previously (14).…”