Characterization of aromatic dehalogenases of Mycobacterium fortuitum CG-2

Abstract

Two different dehalogenation enzymes were found in cell extracts ofMycobacteriumfortuitum CG-2. The first enzyme was a halophenol para-hydroxylase, a membrane-associated monooxygenase that required molecular oxygen and catalyzed the para-hydroxylation and dehalogenation of chlorinated, fluorinated, and brominated phenols to the corresponding halogenated hydroquinones. The membrane preparation with this activity was inhibited by cytochrome P-450 inhibitors and also showed an increase in the A4,, caused by CO. The second enzyme hydroxylated and reductively dehalogenated tetrahalohydroquinones to 1,2,4-trihydroxybenzene. This halohydroquinone-dehalogenating enzyme was soluble, did not require oxygen, and was not inhibited by cytochrome P-450 inhibitors.Actinomycetes are a large and variable group of microorganisms occurring in many habitats, where they take part in the degradation of organic material. Many microbial strains degrading various aromatic pollutants have been isolated from this group. Enzymes so far characterized have been reported to be monooxygenases, hydroxylases, or dioxygenases (for a review, see reference 31). Actinomycetes, including those degrading xenobiotic compounds, have also been shown to be environmentally tenacious, making them a potential source of microorganisms for bioremediation (6,18,29,30).Mycobacterium fortuitum CG-2 was isolated from a tetrachloroguaiacol enrichment culture and shown to degrade several chlorinated phenols, guaiacols, and syringols at micromolar levels (15). Pentachlorophenol (PCP) was shown to be mineralized to CO2, and tetrachloro-para-hydroquinone (TeCH) was detected as an initial intermediate. We have shown that the degradation of polychlorophenols, guaiacols, and syringols proceeds through chlorinatedparahydroquinones in three different Rhodococcus chlorophenolicus strains (1,3,12,13,15) and is mediated by a membrane-associated cytochrome P-450 enzyme (28). The product of this para-hydroxylation, TeCH, is further orthohydroxylated by a soluble halohydroquinone dehalogenase (4,14,28).In this paper, we report on the enzymatic dehalogenation of chlorinated, brominated, and fluorinated phenols and hydroquinones by M. fortuitum CG-2 cell fractions. We show that the halogenated phenols were converted to halogenated para-hydroquinones by a membrane-associated cytochrome P-450 enzyme. The halohydroquinones formed by the para-hydroxylation were further dehalogenated by a soluble halohydroquinone dehalogenase. The results show that M. fortuitum CG-2 dehalogenases are similar to those previously described by us for R. chlorophenolicus PCP-1 and CP-2 (3, 4, 14, 15, 28). MATERIALS AND METHODSBacterial strains and culture conditions. M. fortuitum CG-2 (12, 16) was grown in nutrient broth-yeast extract medium (21) in a gyratory shaker at 28°C. Two-day-old cultures were harvested or induced by the addition of increasing amounts (10 to 50 FtM) of PCP at 24-h intervals and then harvested.Cell fractionation. Cells were harvested by centrifugation (3,000 x g, 10 min, 4°C), resus...

Metabolism of polychlorinated phenols by Pseudomonas cepacia AC1100: determination of the first two steps and specific inhibitory effect of methimazole

“…Recently, several kinds of bacterial monooxygenases involved in the hydroxylation of halophenols have been characterized. Two cytochrome P-450 membranebound enzymes have been identified from Rhodococcus chlorophenolicus (19) and Mycobacterium fortuitum (18). Two soluble flavin-containing monooxygenases have also been isolated and purified: (i) a 2,4-dichlorophenol hydroxylase which catalyzes the ortho hydroxylation of 2,4-DCP (14) and (ii) a pentachlorophenol hydroxylase from a Flavobacterium sp.…”

Multicenter Study of Prevalence of Nontuberculous Mycobacteria in Patients with Cystic Fibrosis in France

“…Previous studies involving non-CF patients have reported other NTM species, such as Mycobacterium kansasii, to be associated with residence in nonrural areas (20). Moreover, several studies have suggested that certain chemical compounds present in polluted water distribution systems stimulate the growth of NTM (28). Air pollution in cities may also contribute to NTM lung disease by interfering with the mucosal immune system.…”

Transfer of Polychlorophenol-Degrading Rhodococcus chlorophenolicus (Apajalahti et al. 1986) to the Genus Mycobacterium as Mycobacterium chlorophenolicum comb. nov.

“…The strains which we studied are able to degrade several chlorinated phenols, guaiacols, and syringols (2,19,20,24), and they also 0-methylate various chlorinated phenolic compounds (24); however, there are some differences in substrate specificity. The M. chlorophenolicum strains, as well as a strain of M. fortuitum (35), degrade pentachlorophenol via two hydroxylations, followed by reductive dechlorinations, producing 1,2,4-trihydroxybenzene, which is mineralized to CO, (3,4,20,22,24,45,46). Interestingly, these polychlorophenol-degrading strains did not utilize most of the nonhalogenated aromatic compounds which we tested ( Table 4).…”