Purification and characterization of 2,4,6-trichlorophenol-4-monooxygenase, a dehalogenating enzyme from Azotobacter sp. strain GP1.
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 O2 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 O2 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 O2 were consumed by the pure enzyme also in the absence of TCP with simultaneous production of H2O2. The NH2-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.
Wieser
M
M
Institut für Mikrobiologie, Universität Hohenheim, Stuttgart, Germany.
Wagner
B
B
Eberspächer
J
J
Lingens
F
F
D000595:Amino Acid Sequence; D001395:Azotobacter / Q000201:enzymology*; D001673:Biodegradation, Environmental; D002712:Chlorides / Q000378:metabolism; D002733:Chlorophenols / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010101:Oxygen Consumption; D013379:Substrate Specificity; D013696:Temperature
D002712:Chlorides; D002733:Chlorophenols; D004791:Enzyme Inhibitors; D008670:Metals; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D006899:Mixed Function Oxygenases; C024564:2,4,6-trichlorophenol