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 appal ent homogeneity from an extract of TCP-in duced cells Azotobacter sp, strain GPI, The initial step of TCP degrad ation 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 th e stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol o f O-2 per mol of TCP and the formation of 1 mol of Cl- ions. No eviden ce for membrane association or for a multicomponent system was obtaine d, Molecular masses of 240 kDa for the native enzyme and 60 kDa for th e subunit were determined, indicating a homotetrameric structure, Cros s-linking studies with dimethylsuberimidate were consistent with this finding, TCP was the best substrate for 2,4,6-trichlorophenol-4-monoox ygenase (TCP-4-monooxygenase). The majority of other chlorophenols con verted by the enzyme bear a chloro substituent in the l-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 H2O2. The NH2-terminal amino acid sequence of TCP-4-monooxgenase from Azotobacter sp, strain GP1 reveal ed complete identity with the nucleotide-derived sequence from the ana logous enzyme from Pseudomonas pickettii and a high degree of homology with two nondehalogenating monooxygenases. The similarity in enzyme p roperties and the possible evolutionary relatedness of dehalogenating and nondehalogenating monooxygenases are discussed.