Enhanced biodegradation of polychlorinated biphenyls after site-directed mutagenesis of a biphenyl dioxygenase gene

Abstract

Biphenyl dioxygenase catalyzes the first step in the aerobic degradation of polychlorinated biphenyls (PCBs). The nucleotide and amino acid sequences of the biphenyl dioxygenases from two PCB-degrading strains (Pseudomonas sp. strain LB400 and Pseudomonas pseudoakaligenes KF707) were compared. The sequences were found to be nearly identical, yet these enzymes exhibited dramatically different substrate specificities for PCBs. Site-directed mutagenesis of the LB400 bphA gene resulted in an enzyme combining the broad congener specificity of LB400 with increased activity against several congeners characteristic of KF707. These data strongly suggest that the BphA subunit of biphenyl dioxygenase plays an important role in determining substrate selectivity. Further alteration of this enzyme can be used to develop a greater understanding of the structural basis for congener specificity and to broaden the range of degradable PCB congeners. Polychlorinated biphenyls (PCBs) are a group of synthetic compounds composed of biphenyl molecules containing from 1 to 10 chlorines. The vast majority of PCBs in the environment are derived from commercial mixtures (e.g., Aroclors) which contain 60 to 80 different congeners (20). Bacteria able to aerobically degrade PCBs are relatively common; however, in most cases these organisms have narrow congener specificity and are able to degrade only a small number of lightly chlorinated PCBs

Biodegradation of Tritium Labeled Polychlorinated Biphenyls (PCBS) by Local Salt Tolerant Mesophylic Bacillus Strains

“…Biphenyl is mostly degraded by the initial action of a 2, 3-dioxygenase attack on the 2, 3-carbons and meta-cleavage, producing benzoate. Similarly, there are many bacterial strains from the genera Pseudomonas [3], Arthrobacter [4], Vibrio, Aeromonas, Micrococcus, Acinetobacter, Bacillus, Rhodococcus [5], and Streptomyces that degrade mono-, di-, tri-, and several tetrachlorinated PCBs by meta-cleavage of non-chlorinated 2,3-carbons.…”

Genetic and Biochemical Characterization of the Broad Spectrum Chlorobenzene Dioxygenase from Burkholderia Sp. Strain PS12 — Dechlorination of 1,2,4,5‐Tetrachlorobenzene

“…Two critical features of the degradation of highly substituted organic chemicals by microorganisms are activation of the molecule in aerobic degradation processes by introduction of oxygen atoms and elimination of substituent groups (Harayama and Timmis, 1989). Oxygenases exhibiting relaxed substrate specificities and hence able to attack a range of isomeric chemicals are particularly interesting both for natural degradation processes and for the design of performant biological catalysts for bioremediation purposes (Erickson and Mondello, 1993). Oxygenases that additionally eliminate substituents are of special interest as substituents such as chloro-, nitro-and sulfo groups usually complicate an enzymatic attack.…”

Novel forms of ring‐hydroxylating dioxygenases are widespread in pristine and contaminated soils

“…This level of sequence divergence is equivalent to that observed between classes IIA, IIB and III and considerably less than that between these last three classes and class I RHDs. Regions of peptide conservation are not evenly spread throughout High diversity of dioxygenase genes in soils 647 the a-subunit of known RHDs, but are concentrated in two areas that are predicted by site-directed mutagenesis and structural studies to be of critical importance for enzymatic activity (Erickson and Mondello, 1993;Beil et al, 1998;Kauppi et al, 1998). This same pattern of conservation was observed in the environmental clones.…”

“…The alpha subunit of the terminal oxygenase of RHDs represents a useful molecular target to investigate the significance of uncultured aromatic hydrocarbon degraders. This protein is thought to primarily determine the substrate range of RHDs (Erickson and Mondello, 1993;Furukawa et al, 1993;Tan and Cheong, 1994;Suyama et al, 1996;Beil et al, 1998;Parales et al, 1998). It is relatively conserved among known RHDs and contains a number of diagnostic structural features, including universally conserved residues involved in the co-ordination of iron and electron transfer at the active site (Kauppi et al, 1998).…”