Purification and characterization of 6-chlorohydroxyquinol 1,2-dioxygenase from Streptomyces rochei 303: comparison with an analogous enzyme from Azotobacter sp. strain GP1

Abstract

The enzyme which cleaves the benzene ring of 6-chlorohydroxyquinol was purified to apparent homogeneity from an extract of 2,4,6-trichlorophenol-grown cells of Streptomyces rochei 303. Like the analogous enzyme from Azotobacter sp. strain GP1, it exhibited a highly restricted substrate specificity and was able to cleave only 6-chlorohydroxyquinol and hydroxyquinol and not catechol, chlorinated catechols, or pyrogallol. No extradiolcleaving activity was observed. In contrast to 6-chlorohydroxyquinol 1,2-dioxygenase from Azotobacter sp. strain GP1, the S. rochei enzyme had a distinct preference for 6-chlorohydroxyquinol over hydroxyquinol (k cat /K m ‫؍‬ 1.2 and 0.57 s ؊1 ⅐ M ؊1 , respectively). The enzyme from S. rochei appears to be a dimer of two identical 31-kDa subunits. It is a colored protein and was found to contain 1 mol of iron per mol of enzyme. The NH 2 -terminal amino acid sequences of 6-chlorohydroxyquinol 1,2-dioxygenase from S. rochei 303 and from Azotobacter sp. strain GP1 showed a high degree of similarity.Two pathways for the aerobic degradation of chlorophenols have been described so far: one via chlorocatechols and the other via chlorohydroquinones (2, 9, 10, 15). A predominant catabolic route for the compounds carrying one or two chlorine substituents was shown to be the modified ortho-cleavage pathway (14,18,24). In this pathway, chlorocatechols formed by introduction of a second hydroxy group are subjected to intradiol cleavage. In the case of chlorophenol degradation through the chlorohydroquinone pathway, the introduction of a third hydroxy group leads to the formation of hydroxyquinol or chlorohydroxyquinol (3, 13). Both unsubstituted and chlorinated 1,2,4-trihydroxybenzene were shown to be subject to ortho cleavage in Streptomyces rochei 303 (13). Extracts from cells grown in the presence of 2-chloro-or 2,4-dichlorophenol were found to prefer hydroxyquinol over 6-chlorohydroxyquinol as a ring fission substrate, whereas extracts from cells grown on 2,6-dichloro-or 2,4,6-trichlorophenol showed a preference for the chlorinated substrate (13). These results led us to suggest that two different dioxygenases play a role in the cleavage of the trihydroxylated aromatic ring: 6-chlorohydroxyquinol 1,2-dioxygenase in the ring fission of 6-chlorohydroxyquinol, the proposed intermediate of 2,4,6-trichloro-and 2,6-dichlorophenol degradation, and a second dioxygenase in the cleavage of hydroxyquinol, the proposed intermediate of 2,4-dichloro-and 2-chlorophenol degradation (Fig. 1) (13).In this paper, we describe the purification and characterization of 6-chlorohydroxyquinol 1,2-dioxygenase. Three other hydroxyquinol 1,2-dioxygenases have been purified and characterized so far (21,25,34). One of them takes part in the cleavage of hydroxyquinol, found as an intermediate of 4-hydroxybenzoate degradation in Trichosporon cutaneum (34).The second dioxygenase, from the basidiomycete Phanerochaete chrysosporium, catalyzes a key step in the degradation of vanillate, an intermediate in lignin degradation (25)....

Characterization of a gene cluster encoding the maleylacetate reductase from Ralstonia eutropha 335T, an enzyme recruited for growth with 4-fluorobenzoate

“…For bacteria, it has been shown that maleylacetate reductases are involved in the degradation of resorcinol and 2,4-dihydroxybenzoate via hydroxyquinol (Larway & Evans, 1965;Chapman & Ribbons, 1976;Stolz & Knackmuss, 1993). Other substrates whose catabolic routes comprise this activity have a more complex structure, such as 2-hydroxydibenzo-p-dioxin and 3-hydroxydibenzofuran (Armengaud et al, 1999), or carry unusual substituents, such as nitro or sulfo groups (Spain & Gibson, 1991;Feigel & Knackmuss, 1993;Jain et al, 1994;Rani & Lalithakumari, 1994), fluorine or chlorine atoms (Duxbury et al, 1970; Schlömann et al, 1990a;Latus et al, 1995;Zaborina et al, 1995;Daubaras et al, 1996;Miyauchi et al, 1999).The maleylacetate reductase genes characterized so far tend to belong to specialized gene clusters for the degradation of aromatic compounds. Thus, 3-hydroxydibenzofuran and 2-hydroxydibenzo-p-dioxin degradation via hydroxyquinol appears to be encoded by a specialized gene cluster comprising most of the genes for the pathway including a maleylacetate reductase gene (Armengaud et al, 1999).…”

Purification and characterization of 2,6-dichloro-p-hydroquinone chlorohydrolase from Flavobacterium sp. strain ATCC 39723

“…The breakdown of mono-or dichlorophenols is usually through a catechol intermediate before ring cleavage (6). On the other hand, most polychlorinated phenols are degraded through a chlorohydroxyquinol (CHQ) intermediate before ring cleavage (1,3,24).The reaction mechanisms and enzymes responsible for the degradation of polychlorinated phenols have been studied in several microorganisms (11,20,(22)(23)(24). Flavobacterium sp.…”

“…The breakdown of mono-or dichlorophenols is usually through a catechol intermediate before ring cleavage (6). On the other hand, most polychlorinated phenols are degraded through a chlorohydroxyquinol (CHQ) intermediate before ring cleavage (1,3,24).…”

Genomic Analysis and Identification of Catabolic Pathways for Aromatic Compounds in Corynebacterium glutamicum

“…Second, the degradation of the dihydroxylated benzene derivatives involves ring fission and subsequent reactions linked to central metabolism in the cell. Ring fission is catalyzed by ring-cleavage dioxygenases that use molecular oxygen to open the aromatic ring between the two hydroxyl groups (ortho-cleavage, catalyzed by intradiol dioxygenases) 39,40) or proximal to one of the two hydroxyl groups (meta-cleavage, catalyzed by extradiol dioxygenases) 11,17,35) . For many years, investigations of the degradation of aromatic compounds have focused almost exclusively on Gram-negative bacteria such as species of Pseudomonas and a considerable body of knowledge has emerged 23) .…”