Purification and characterization of 2,4,6-trichlorophenol-4-monooxygenase, a dehalogenating enzyme from Azotobacter sp. strain GP1

Abstract

The enzyme which catalyzes the dehalogenation of 2,4,6-trichlorophenol (TCP) was purified to apparent homogeneity from an extract of TCP-induced cells of Azotobacter sp. strain GP1. The initial step of TCP degradation in this bacterium is inducible by TCP; no activity was found in succinate-grown cells or in phenol-induced cells. NADH, flavin adenine dinucleotide, and O 2 are required as cofactors. As reaction products, 2,6-dichlorohydroquinone and Cl ؊ ions were identified. Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of O 2 per mol of TCP and the formation of 1 mol of Cl ؊ ions. No evidence for membrane association or for a multicomponent system was obtained. Molecular masses of 240 kDa for the native enzyme and 60 kDa for the subunit were determined, indicating a homotetrameric structure. Cross-linking studies with dimethylsuberimidate were consistent with this finding. TCP was the best substrate for 2,4,6-trichlorophenol-4-monooxygenase (TCP-4-monooxygenase). The majority of other chlorophenols converted by the enzyme bear a chloro substituent in the 4-position. 2,6-Dichlorophenol, also accepted as a substrate, was hydroxylated in the 4-position to 2,6-dichlorohydroquinone in a nondehalogenating reaction. NADH and O 2 were consumed by the pure enzyme also in the absence of TCP with simultaneous production of H 2 O 2 . The NH 2 -terminal amino acid sequence of TCP-4-monooxygenase from Azotobacter sp. strain GP1 revealed complete identity with the nucleotide-derived sequence from the analogous enzyme from Pseudomonas pickettii and a high degree of homology with two nondehalogenating monooxygenases. The similarity in enzyme properties and the possible evolutionary relatedness of dehalogenating and nondehalogenating monooxygenases are discussed.Polychlorinated phenols are widespread environmental pollutants, and their degradation has been the subject of numerous investigations (for reviews, see references 5, 9, 10, and 21). While many different microorganisms with the capacity to degrade these compounds have been isolated, relatively few reports have dealt with the initial step of their degradation. Moreover, the majority of these investigations were performed with whole cells, crude extracts, or partially purified enzymes, and so far only two polychlorophenol-dehalogenating enzymes from Burkholderia (formerly Pseudomonas) cepacia AC1100 (33) and from a Flavobacterium sp. (34) have been purified to homogeneity. Other polychlorophenol dehalogenases were partially purified from an Arthrobacter sp. (23) or found to be membrane-associated enzymes in Rhodococcus chlorophenolicus (26,27) and Mycobacterium fortuitum (28).The dehalogenation of 2,4,6-trichlorophenol (TCP) was detected in crude extracts of Streptomyces rochei 303 (7) and Pseudomonas pickettii (12). The conversion of TCP to 2,6-dichlorohydroquinone was also observed with whole cells from Pseudomonas cepacia AC1100 (25).In this paper, we describe the purification and characterization of a TCP-dehalogenating enzyme from t...

Cork Taint of Wines: Role of the Filamentous Fungi Isolated from Cork in the Formation of 2,4,6-Trichloroanisole by O Methylation of 2,4,6-Trichlorophenol

“…In addition, 2,4,6-TCA was not detected after potential bioconversion or catabolism of highly chlorinated precursors, including hexachlorocyclohexane, hexachlorobenzene, PCP, PCA, 2,3,4,6-TeCP, and 2,3,4,6-TeCA. This is not an unexpected result if we bear in mind that the catabolism of some of these compounds, such as PCP, 2,4,5-TCP, and 2,4,6-TCP, proceeds under aerobic conditions mainly through an oxidative dehalogenating mechanism catalyzed by monooxygenase-type enzymes, yielding different quinones as intermediates (3,36,37,38), although reductive dechlorination can also occur in the presence of oxygen (3). Besides, to our knowledge, 2,4,6-TCA has never been detected as an intermediate .…”

Biodegradation of 2,4,6-Tribromophenol byOchrobactrumsp. Strain TB01

“…strain GP1, a primary step in 2,4,6-TCP degradation is NAD(P)Hdependent, and the reaction is catalyzed by homotetrameric TCP-4-monooxygenase, leading to the formation of 2,6-dichlorohydroquinone as a product. 23) To our knowledge, NAD(P)H-dependent reductive dehalogenation of 2,4,6-TBP to 2,4-DBP is a unique reaction. A possible reaction mechanism is proposed in Fig.…”

Two‐Component Flavin‐dependent Pyrrole‐2‐carboxylate Monooxygenase from Rhodococcus Sp.

“…A similar situation exists for trichlorophenol-4-monooxygenases from Azotobacter sp. and R. pickettii, respectively (Takizawa et al, 1995;Wieser et al, 1997). The former organism forms a single-component monooxygenase catalyzing a substrate dehalogenation by hydroxylation at position 4 (Wieser et al, 1997), whereas a two-component monooxygenase catalyzing the same substrate conversion was described for the latter (Takizawa et al, 1995).…”

“…(Hormann and Andreesen, 1994) ; 4, 4-hydroxyphenylacetate-3-hydroxylase from E. coli (Prieto and Garcia, 1994) ; 5, 2,4,6-trichlorophenoI-4-monooxygenase from Azotobacter sp. (Wieser et al, 1997). Amino acids that are identical to the sequence from Rhodococcus sp.…”

“…However, a high identity was observed with the single-component 2,4,6-trichlorophenol monooxygenase from Azotobacter sp. (Wieser et al, 1997) and the twocomponent monooxygenases chlorophenol-4-hydroxylase from Ralstonia pickettii (formerly Pseudomonas) and 4-hydroxyphenylacetate 3-hydroxylase from Escherichia coli (Prieto and Garcia, 1994;Takizawa et al, 1995). Interestingly, the sequences of both subunits of the R. pickettii enzyme contain the typical dinucleotide-binding motif (Takizawa et al, 1995), whereas none is present in the subunits of the Escherichia coli enzyme (Prieto and Garcia, 1994).…”