Abstract
Biphenyl dioxygenase (Bph Dox) is responsible for the initial dioxygenation of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in determination of substrate specificity of biphenyl-related compounds including polychlorinated biphenyls (PCBs). Functional evolution of Bph Dox of Pseudomonas pseudoalcaligenes KF707 was accomplished by random priming recombination of the bphA1 gene, involving two rounds of in vitro recombination and mutation followed by selection for increased activity in vivo. Evolved Bph Dox acquired novel and multifunctional degradation capabilities not only for PCBs but also for dibenzofuran, dibenzo-p-dioxin, dibenzothiophene, and fluorene, the compounds scarcely attacked by the original KF707 Bph Dox. The modes of oxygenation were angular and lateral dioxygenation for dibenzofuran and dibenzo-p-dioxin, sulfoxidation for dibenzothiophene, and mono-oxygenation for fluorene. These enzymes also exhibited enhanced degradation abilities for PCB congeners, retaining 2,3-dioxygenase activity and gaining 3,4-dioxygenase activity, depending on the chlorine substitution of PCB congeners. Further mutation analysis revealed that the amino acid at position 376 in BphA1 is significantly involved in the acquisition of multifunctional oxygenase activities and mode of oxygenation.
Highlights
Bacterial oxygenases are involved in the initial hydroxylation of aromatic hydrocarbons, and they are usually two or three component enzymes
The results demonstrated that a relatively small number of amino acids in the carboxyl-terminal half of BphA1 are involved in the recognition of the chlorinated ring and the site of dioxygenation and are responsible for the degradation of polychlorinated biphenyls (PCBs)
Colonies forming the ring meta-cleavage yellow products from both biphenyl and dibenzofuran were screened, and the extended function of these evolved enzymes was analyzed with PCBs and biphenyl-related compounds
Summary
Bacterial Strains, Plasmid, and Growth Conditions—The biphenylutilizing strain P. pseudoalcaligenes KF707 was grown in basal salt medium as described previously [20]. The pRPF1000 series plasmids contain bphA1 (evolved)-bphA2A3A4BC and were used to assay the production of ring meta-cleavage products from PCBs and biphenyl-related compounds. The EcoRI fragment containing the bphA1 gene was cut from pRPF707, electrophoresed on 0.7% agarose gels, and recovered by a DNA purification kit (TOYOBO). Plasmid pSSF1202, which contains bphA1 from pRPF1202 (described below), was used as the template for mutagenesis. This plasmid was amplified by PCR using two complementary oligonucleotides: 5Ј-CACAACATCCGCACCTTCTCCGCAGGCGGC-3Ј and 5Ј-GCCTGCGGAGAAGGTGCGGATGTTGTGCCG-3Ј for amino acid change V376T, and 5Ј-CACAACATCCGCAACTTCTCCGCAGGCGGC-3Ј and 5Ј-GCCTGCGGAGAAGTTGCGGATGTTGTGCCG-3Ј for V376N (the codon of the amino acid to be changed is underlined). Amounts of substrate depletion were calculated by normalization to the recovery of 2,4,6,2Ј,4Ј-pentachlorobiphenyl, a nondegradable internal standard extracted from heat-treated control cells, and quantified by use of a standard curve [5, 18]
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