Metabolism Of Biphenyl Research Articles

A poplar short-chain dehydrogenase reductase plays a potential key role in biphenyl detoxification

Polychlorinated biphenyls (PCBs) are persistent organic pollutants with severe effects on human health and the biosphere. Plant-based remediation offers many benefits over conventional PCB remediation, but its development has been hampered by our poor understanding of biphenyl metabolism in eukaryotes, among other factors. We report here a major PCB-responsive protein in poplar, a plant model system capable of PCB uptake and translocation. We provide structural and functional evidence that this uncharacterized protein, termed SDR57C, belongs to the heterogeneous short-chain dehydrogenase reductase (SDR) superfamily. Despite sequence divergence, structural modeling hinted at structural and functional similarities between SDR57C and BphB, a central component of the Bph pathway for biphenyl/PCB degradation in aerobic bacteria. By combining gas chromatography/mass spectrometry (GC/MS) profiling with a functional complementation scheme, we found that poplar SDR57C can replace BphB activity in the upper Bph pathway of Pseudomonas furukawaii KF707 and therefore catalyze the oxidation of 2,3-dihydro-2,3-dihydroxybiphenyl (2,3-DHDB) to 2,3-dihydroxybiphenyl (2,3-DHB). Consistent with this biochemical activity, we propose a mechanism of action based on prior quantum studies, general properties of SDR enzymes, and the modeled docking of 2,3-DHDB to the SDR57C-NAD+ complex. The putative detoxifying capacity of SDR57C was substantiated through reverse genetics in Arabidopsis thaliana Phenotypic characterization of the SDR lines underscored an inducible plant pathway with the potential to catabolize toxic biphenyl derivatives. Partial similarities with aerobic bacterial degradation notwithstanding, real-time messenger RNA quantification indicates the occurrence of plant-specific enzymes and features. Our results may help explain differences in degradative abilities among plant genotypes and also provide elements to improve them.

Nontarget analysis reveals gut microbiome-dependent differences in the fecal PCB metabolite profiles of germ-free and conventional mice.

Mammalian polychlorinated biphenyl (PCB) metabolism has not been systematically explored with nontarget high-resolution mass spectrometry (Nt-HRMS). Here we investigated the importance of the gut microbiome in PCB biotransformation by Nt-HRMS analysis of feces from conventional (CV) and germ-free (GF) adult female mice exposed to a single oral dose of an environmental PCB mixture (6mg/kg or 30mg/kg in corn oil). Feces were collected for 24h after PCB administration, PCB metabolites were extracted from pooled samples, and the extracts were analyzed by Nt-HRMS. Twelve classes of PCB metabolites were detected in the feces from CV mice, including PCB sulfates, hydroxylated PCB sulfates (OH-PCB sulfates), PCB sulfonates, and hydroxylated methyl sulfone PCBs (OH-MeSO2-PCBs) reported previously. We also observed eight additional PCB metabolite classes that were tentatively identified as hydroxylated PCBs (OH-PCBs), dihydroxylated PCBs (DiOH-PCBs), monomethoxylated dihydroxylated PCBs (MeO-OH-PCBs), methoxylated PCB sulfates (MeO-PCB sulfates), mono-to tetra-hydroxylated PCB quinones ((OH)x-quinones, x=1-4), and hydroxylated polychlorinated benzofurans (OH-PCDF). Most metabolite classes were also detected in the feces from GF mice, except for MeO-OH-PCBs, OH-MeSO2-PCBs, and OH-PCDFs. Semi-quantitative analyses demonstrate that relative PCB metabolite levels increased with increasing dose and were higher in CV than GF mice, except for PCB sulfates and MeO-PCB sulfates, which were higher in GF mice. These findings demonstrate that the gut microbiome plays a direct or indirect role in the absorption, distribution, metabolism, or excretion of PCB metabolites, which in turn may affect toxic outcomes following PCB exposure.

Autochthonous bioaugmentation with non-direct degraders: A new strategy to enhance wastewater bioremediation performance

Autochthonous bioaugmentation (ABA) strategies are primarily carried out using a single, highly efficient type of bacteria that is capable of directly degrading the target compound. However, no studies have examined the use of non-direct degraders (NDDs), which are involved in the metabolic pathway of target compounds instead of direct degradation. Here, to evaluate the bioremediation efficiency and mechanism of ABA by NDDs, we demonstrated the use of an NDD on the biodegradation of biphenyl, a model compound used to study polychlorinated biphenyl (PCB) degradation. The NDD examined in this study, Marmoricola LJ-33, was isolated from activated sludge. Although Marmoricola LJ-33 alone did not directly degrade biphenyl under laboratory conditions, it did contribute to in situ biphenyl biodegradation in the activated sludge, as evidenced by DNA-stable-isotope-probing (DNA-SIP). Implementation of ABA with strain LJ-33 was shown to significantly accelerate biphenyl degradation efficiency, demonstrating the potential of NDD strains for degradation in ABA. More importantly, LJ-33 amendment altered the diversity of the microbial communities involved in biphenyl metabolism. Our findings suggest that a combination of pre-screening followed by DNA-SIP analysis is a practical strategy to precisely separate NDDs. Additionally, our work indicates a new mechanism of ABA strategy with NDDs as a promising in situ bioremediation strategy, broadening our concept in constructing functional consortia to enhance the biodegradation performance of activated sludge in wastewater treatment plants.

Degradation Mechanism of 4-Chlorobiphenyl by Consortium ofPseudomonas sp. Strain CB-3 and Comamonas sp. Strain CD-2.

Polychlorinated biphenyls (PCBs) are types of lasting environmental pollutants which are widely used in various industries. 4-chlorobiphenyl (4CBP) is a PCB which is harmful to the environment as well as humans. Two strains, CB-3 and CD-2, were isolated from the polluted soil of a chemical factory and could completely degrade 50mg/L 4CBP within 12h by co-culture. The consortium comprising strains CB-3 and CD-2 was effective in the degradation of 4CBP. 4CBP was degraded initially by strain CB-3 to accumulate 4-chlorobenzoate (4CBA) and further oxidised by strain CD-2. Based on 16S rRNA gene sequence analysis and phenotypic typing, strain CB-3 and strain CD-2 were identified as Pseudomonas sp. and Comamonas sp., respectively. The substrate spectra experiment showed that strain CB-3 could degrade PCBs with no more than three chlorine atoms. A gene cluster of biphenyl metabolism was found in the genome of strain CB-3. Besides, a dechlorination gene cluster and a gene cluster of protocatechuate (PCA) metabolic were found in the genome of strain CD-2. These gene clusters are supposed to be involved in 4CBP degradation. The ability of strains CB-3 and CD-2 to degrade 4CBP in soil was assessed by soil experiment, and 4CBP at the initial concentration of 10mg/kg was 80.5% removed within 15days.

Biotransformation of the Fluorinated Nonsteroidal Anti-Inflammatory Pharmaceutical Flurbiprofen in Activated Sludge Results in Accumulation of a Recalcitrant Fluorinated Aromatic Metabolite.

Flurbiprofen is a fluorinated, nonsteroidal, anti‐inflammatory pharmaceutical with potential application in a wide range of maladies. Currently, there is no information regarding its environmental fate. To address this, flurbiprofen is spiked at 500 and 50 ppm into activated sewage sludge taken from the municipal treatment plant of Ankara, Turkey. Flurbiprofen is partially degraded after 80 days, with removal proportion varying from 33% to 48%. Isolation of organisms able to use flurbiprofen as a sole carbon and energy source is unsuccessful. A transient, acid‐labile yellow coloration appears in supernatants after addition of flurbiprofen. During disappearance, a novel potential metabolite is detected by high‐performance liquid chromatography (HPLC) analyses, a chemical that does not appear in killed controls or in nonflurbiprofen‐amended controls. Mass spectra of the novel chemical obtained at low and high collision energies are consistent with 4‐(1‐carboxyethyl)‐2‐fluorobenzoic acid, suggesting the application of a canonical metabolic paradigm for halogenated biphenyl metabolism by bacteria in which the nonhalogenated ring is metabolized by dioxygenation and metacleavage, leaving the halogenated aromatic ring behind. This metabolite shows no signs of disappearance after the 80‐day monitoring period, implying that the environmental release of flurbiprofen might be of concern.

Identification of biphenyl-metabolising microbes in activated biosludge using cultivation-independent and -dependent approaches

Microbes have important roles in removing organic pollutants in wastewater treatment plants (WWTPs), especially in mineralising recalcitrant persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs). However, the majority of the microorganisms that metabolise these pollutants in situ remain elusive owing to barriers of traditional techniques in unravel yet-to-be-cultivated microbes. In this study, DNA stable-isotope probing (SIP) coupled with high-throughput sequencing was applied to identify the microbes responsible for PCB degradation in the activated biosludge of a WWTP using 13C-labelled biphenyl (BP). Results of time-course SIP revealed different bacteria and archaea involved in BP metabolism, which dominated the BP-degrading community at different time points. BP degradation by the genera Spartobacteria, Alicyclobacillus, Flavobacterium and the order Cenarchaeales has not been reported previously. The abundance of biphenyl dioxygenase (bphA) genes increased over time and a novel bphA gene was identified from the 13C-heavy DNA fraction. In addition, three cultivable BP degraders were isolated, but did not participate in BP degradation in situ or contain the identified bphA genes. Taken together, these data reveal the huge potential and important roles of yet-to-be-cultivated microbes responsible for PCB degradation in activated biosludge, providing fundamental knowledge on WWTP management to remove POPs.

Benzaldehyde dehydrogenase-driven phytoalexin biosynthesis in elicitor-treated Pyrus pyrifolia cell cultures

Pyrus pyrifolia (Asian pear) cell cultures respond to yeast extract (YE) treatment by accumulating benzoate-derived biphenyl phytoalexins, namely, noraucuparin and aucuparin. Biphenyl phytoalexins are defense-marker metabolites of the sub-tribe Malinae of the family Rosaceae. The substrates for biphenyl biosynthesis are benzoyl-CoA and malonyl-CoA, which combine in the presence of biphenyl synthase (BIS) to produce 3,5-dihydroxybiphneyl. In the non-β-oxidative pathway, benzoyl-CoA is directly derived from benzoic acid in a reaction catalyzed by benzoate-CoA ligase (BZL). Although the core β-oxidative pathway of benzoic acid biosynthesis is well-understood, the complete cascade of enzymes and genes involved in the non-β-oxidative pathway at the molecular level is poorly understood. In this study, we report the detection of benzaldehyde dehydrogenase (BD) activity in YE-treated cell cultures of P. pyrifolia. BD catalyzes the conversion of benzaldehyde to benzoic acid. BD and BIS activities were coordinately induced by elicitor treatment, suggesting their involvement in biphenyl metabolism. Changes in phenylalanine ammonia-lyase (PAL) activity preceded the increases in BD and BIS activities. Benzaldehyde was the preferred substrate for BD (Km=52.0μM), with NAD+ being the preferred co-factor (Km=64μM). Our observations indicate the contribution of BD towards biphenyl phytoalexin biosynthesis in the Asian pear.

Bioluminescence Inhibition Assay for the Detection of Hydroxylated Polychlorinated Biphenyls

Hydroxylated polychlorinated biphenyls (OH-PCBs) are an important class of contaminants that mainly originate from polychlorinated biphenyl metabolism. They may conceivably be as dangerous and persistent as the parent compounds; most prominently, OH-PCBs are endocrine disruptors. Due to increasing evidence of the presence of OH-PCBs in the environment and in living organisms, including humans, and of their toxicity, methods of detection for OH-PCBs are needed in the environmental and medical fields. Herein, we describe the development and optimization of a protein-based inhibition assay for the quantification of OH-PCBs. Specifically, the photoprotein aequorin was utilized for the detection of OH-PCBs. We hypothesized that OH-PCBs interact with aequorin, and we established that OH-PCBs actually inhibit the bioluminescence of aequorin in a dose-dependent manner. We took advantage of this phenomenon to develop an assay that is capable of detecting a wide variety of OH-PCBs with a range of detection limits, the best detection limit being 11 nM for the compound 2-hydroxy-2',3,4',5',6-pentachorobiphenyl. The viability of this system for the screening of OH-PCBs in spiked biological and environmental samples was also established. We envision the implementation of this novel bioluminescence inhibition assay as a rapid, sensitive, and cost-effective method for monitoring OH-PCBs. Furthermore, to the best of our knowledge, this is the first time aequorin has been employed to detect an analyte by the inhibition of its bioluminescence reaction. Hence, this strategy may prove to be a general approach for the development of a new generation of protein-based inhibition assays.

Characterization of the 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) degradation system in Janibacter sp. TYM3221

Bacterial degradation of 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) has been previously reported, however, its degradation enzyme system has not been characterized. In this study, a DDE-degrading bacterium, Janibacter sp. TYM3221, was isolated and characterized. Transformation of DDE was demonstrated by TYM3211 resting cells grown in LB in the presence and absence of biphenyl. Gas chromatography–mass spectrometry analysis revealed five metabolites of DDE containing a meta-ring cleavage product and 4-chlorobenzoic acid, suggesting that TYM3221 degrades DDE to 4-chlorobenzoic acid via a meta-ring cleavage product. A gene cluster, bphAaAbAcAd, which codes for biphenyl dioxygenase subunits, was cloned from TYM3221. A mutant strain with a bphAa-gene inactivation did not grow on biphenyl, and showed no DDE degradation activity. These results indicate that in strain TYM3221, the bphAa-coded biphenyl dioxygenase is involved not only in the metabolism of biphenyl but also in the degradation of DDE.

Preliminary analysis of glutathione S-transferase homolog fromLactobacillus casei Zhang

Glutathione S-transfera ses (GSTs) constitute a large protein family. Some of them are proved to be associated with biphenyl metabolism. In this study, we described a homolog of GST gene inLactobacillus casei Zhang that revealed by genome analysis. The GST gene itself as well as the adjacent gene context was highly conserved among differentL. casei isolates. Phylogenetic investigation suggested that the GST gene was acquired via horizontal gene transfer event, possibly from gamma subdivision. Additional survival experiment indicated that it may not benefit theL. casei host for growing in the presence of biphenyl. It was for the first time discovered that GST was encoded byLactobacillaceae species.

Biphenyl-Metabolizing Bacteria in the Rhizosphere of Horseradish and Bulk Soil Contaminated by Polychlorinated Biphenyls as Revealed by Stable Isotope Probing

DNA-based stable isotope probing in combination with terminal restriction fragment length polymorphism was used in order to identify members of the microbial community that metabolize biphenyl in the rhizosphere of horseradish (Armoracia rusticana) cultivated in soil contaminated with polychlorinated biphenyls (PCBs) compared to members of the microbial community in initial, uncultivated bulk soil. On the basis of early and recurrent detection of their 16S rRNA genes in clone libraries constructed from [(13)C]DNA, Hydrogenophaga spp. appeared to dominate biphenyl catabolism in the horseradish rhizosphere soil, whereas Paenibacillus spp. were the predominant biphenyl-utilizing bacteria in the initial bulk soil. Other bacteria found to derive carbon from biphenyl in this nutrient-amended microcosm-based study belonged mostly to the class Betaproteobacteria and were identified as Achromobacter spp., Variovorax spp., Methylovorus spp., or Methylophilus spp. Some bacteria that were unclassified at the genus level were also detected, and these bacteria may be members of undescribed genera. The deduced amino acid sequences of the biphenyl dioxygenase alpha subunits (BphA) from bacteria that incorporated [(13)C]into DNA in 3-day incubations of the soils with [(13)C]biphenyl are almost identical to that of Pseudomonas alcaligenes B-357. This suggests that the spectrum of the PCB congeners that can be degraded by these enzymes may be similar to that of strain B-357. These results demonstrate that altering the soil environment can result in the participation of different bacteria in the metabolism of biphenyl.

Polychlorinated Biphenyl/Biphenyl Degrading Gene Clusters inRhodococcussp. K37, HA99, and TA431 Are Different from Well-KnownbphGene Clusters of Rhodococci

Four kinds of polychlorinated biphenyl (PCB)-degrading Rhodococcus sp. (TA421, TA431, HA99, and K37) have been isolated from termite ecosystem and under alkaline condition. The bph gene cluster involved in the degradation of PCB/biphenyl has been analyzed in strain TA421. This gene cluster was highly homologous to bph gene clusters in R. globerulus P6 and Rhodococcus sp. RHA1. In this study, we cloned and analyzed the bph gene cluster essential to PCB/biphenyl degradation from R. rhodochrous K37. The order of the genes and the sequence were different in K37 than in P6, RHA1, and TA421. The bphC8(K37) gene was more homologous to the meta-cleavage enzyme involved in phenanthrene metabolism than bphC genes involved in biphenyl metabolism. Two other Rhodococcus strains (HA99 and TA431) had PCB/biphenyl degradation gene clusters similar to that in K37. These findings suggest that these bph gene clusters evolved separately from the well-known bph gene clusters of PCB/biphenyl degraders.

Metamorphosis increases biotransformation of polychlorinated biphenyls: A comparative study of polychlorinated biphenyl metabolism in green frogs (Rana clamitans) and leopard frogs (Rana pipiens) at various life stages

Polychlorinated biphenyl (PCB) elimination rates were used to determine whether green frogs (Rana clamitans) and leopard frogs (Rana pipiens) at three life stages (tadpole, metamorph, and adult) were capable of metabolic biotransformation of PCBs. Polychlorinated biphenyls were grouped according to their structural properties to determine whether congeners with specific structures were eliminated more rapidly than others, which would suggest the occurrence of metabolic biotransformation. For both species and at all life stages studied, PCBs with adjacent unsubstituted meta-para sites (group-2 PCBs) were eliminated significantly faster than PCBs with adjacent unsubstituted ortho-meta sites (group-3 PCBs). Because these groups of PCBs span similar hydrophobicity ranges, it was concluded that green and leopard frogs can selectively metabolize group-2 PCBs. Metabolic activity was more pronounced in the metamorph stage than in any other life stage. In green frog metamorphs, the average elimination rate of the group-2 PCBs was 4.9-fold greater than that of the group-3 PCBs. By contrast, in green frog tadpoles, group-2 PCBs were eliminated 1.9-fold faster than group-3 PCBs, and in green frog adults, group-2 PCBs were eliminated 1.5-fold faster than group-3 PDBs.

Active-site engineering of biphenyl dioxygenase: effect of substituted amino acids on substrate specificity and regiospecificity

Biphenyl dioxygenase (Bph Dox) catalyzes the initial dioxygenation step in the metabolism of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in the determination of the substrate specificity of biphenyl-related compounds including polychlorinated biphenyls (PCBs). Previously, the substitution of Asn at Thr-376 near the active-site iron in the BphA1 of Pseudomonas pseudoalcaligenes KF707 expanded the oxidation range and altered the regiospecificity of Bph Dox for PCBs. In this study, we replaced Thr-376 with Gly, Ser, Gln, Tyr, Val, Phe, Asp, and Lys and expressed these enzymes in Escherichia coli. Bph Dox mutants of Thr376Asn, Thr376Val, Thr376Phe, and Thr376Lys showed novel degradation activity for dibenzofuran, which is a poor substrate for KF707 Bph Dox. All active Bph Dox mutants showed altered regiospecificity with 2,2'-dichlorobiphenyl and 2,5,4'-trichlorobiphenyl. The Thr376Gly, Thr376Val, Thr376Phe, and Thr376Asp Bph Dox mutants introduced molecular oxygen at the 2,3 position of 2,2'-dichlorobiphenyl, forming 2-chloro-2',3'-dihydroxybiphenyl with concomitant dechlorination. The Bph Dox mutants of Thr376Gly, Thr376Ser, Thr376Asp, and Thr376Lys attacked 2,5,4'-trichlorobiphenyl via both 2',3'- and 3,4-dioxygenation activities. In particular, the Thr376Phe Bph Dox mutant exhibited enhanced and expanded degradation activities toward all of the compounds tested. Further site-directed mutation was induced to change the oxidizing character of KF707 Bph Dox to that of the Bph Dox of Burkholderia xenovorans LB400 by the substitution of two amino acids, Ile335Phe and Thr376Asn, near the active-site.

Oxidative stress by biphenyl metabolites induces inhibition of bacterial cell separation

Cell-cell separation of a polychlorinated biphenyl (PCB)-degrading bacterium Comamonas testosteroni TK102 was monitored by flow cytometry. When monohydroxy metabolites of biphenyl (BP) (2-hydroxybiphenyl and 3-hydroxybiphenyl) were added to the culture, cell-cell separation of strain TK102 was inhibited at stationary phase. This inhibition was reproduced on non-PCB degrading bacteria such as Pseudomonas putida PpY101 and Escherichia coli MV1184, but was not observed on Pseudomonas aeruginosa PAO1. An opportunistic pathogen, P. aeruginosa PAO1, produces exopolysaccharide, which is known to scavenge damaging chemicals such as reactive oxygen species (ROS). The higher level of ROS and lipid peroxidants were detected in the cells treated by monohydroxybiphenyls. Fat-soluble vitamin E, which is a lipid radical scavenger, maintained bacterial cell separation during monohydroxybiphenyls treatment. Our results demonstrated that intracellular oxidative stress played an important role in the inhibition of bacterial cell separation during BP metabolism. This study shows that metabolites of environmental pollutants, such as monohydroxylated BP, inhibit bacterial cell separation by oxidative stress.

Metabolism of aromatic hydrocarbons by the filamentous fungus Cyclothyrium sp.

The metabolism of biphenyl, naphthalene, anthracene, phenanthrene, pyrene and benzo[ a]pyrene by Cyclothyrium sp. CBS 109850, a coelomycete isolated for the first time in Brazil from industrially polluted estuarine sediment, was studied. The metabolites were extracted and separated by high performance liquid chromatography (HPLC) and characterized by UV spectral analyses and mass, and proton nuclear magnetic resonance ( 1H NMR) spectrometry. Cyclothyrium sp. transformed biphenyl to 4-hydroxybiphenyl and anthracene to anthracene trans-1,2-dihydrodiol. This isolate metabolized 90% of [9- 14C]phenanthrene, producing phenanthrene trans-9,10-dihydrodiol as a major metabolite, phenanthrene trans-3,4-dihydrodiol, 1-hydroxyphenanthrene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene, and a novel metabolite, 2-hydroxy-7-methoxyphenanthrene. Circular dichroism spectra analyses indicated that the major enantiomers of phenanthrene trans-9, 10-dihydrodiol, phenanthrene trans-3,4-dihydrodiol and pyrene trans-4,5-dihydrodiol, a pyrene metabolite produced previously by Cyclothyrium sp. CBS 109850, were predominantly in the ( R, R) configuration, revealing a high stereoselectivity for initial monooxygenation and enzymatic hydration of phenanthrene and pyrene by Cyclothyrium sp. CBS109850. The results also show a high regioselectivity since the K-regions of phenanthrene and pyrene were the major sites of metabolism.

BphK shows dechlorination activity against 4-chlorobenzoate, an end product of bph-promoted degradation of PCBs

A bphK gene encoding glutathione S-transferase (GST) activity is located in the bph operon in Burkholderia sp. strain LB400 but its role in polychlorinated biphenyl (PCB) metabolism is unknown. This gene was over-expressed in Escherichia coli and an in vivo assay based on growth of E. coli containing GST activity was used to identify potential novel substrates for this enzyme. Using this assay, 4-chlorobenzoate (4-CBA) was identified as a substrate for the BphK enzyme. High pressure liquid chromatography analysis and chloride ion detection showed removal of 4-CBA and an equivalent increase of chloride in cell extracts when incubated with this enzyme. These results would indicate that this BphK enzyme has dechlorination activity in relation to 4-CBA and may have a role in protection of other Bph enzymes against certain chlorinated metabolites of PCB degradation.

Alteration of regiospecificity in biphenyl dioxygenase by active-site engineering.

Biphenyl dioxygenase (Bph Dox) is responsible for the initial dioxygenation step during the metabolism of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in the determination of the substrate specificity of biphenyl-related compounds, including polychlorinated biphenyls (PCBs). Based on crystallographic analyses of naphthalene dioxygenase (B. Kauppi, K. Lee, E. Carredano, R. E. Parales, D. T. Gibson, H. Eklund, and S. Ramaswamy, Structure 6:571-586, 1998), we developed a three-dimensional model of KF707 BphA1 of Pseudomonas pseudoalcaligenes KF707. Based on structural information about the amino acids which coordinate the catalytic nonheme iron center, we constructed 12 site-directed BphA1 mutants with changes at positions 227, 332, 335, 376, 377, and 383 and expressed these enzymes in Escherichia coli. The Ile335Phe, Thr376Asn, and Phe377Leu Bph Dox mutants exhibited altered regiospecificities for various PCBs compared with wild-type Bph Dox. In particular, the Ile335Phe mutant acquired the ability to degrade 2,5,2',5'-tetrachlorobiphenyl by 3,4-dioxygenation and showed bifunctional 2,3-dioxygenase and 3,4-dioxygenase activities for 2,5,2'-trichlorobiphenyl and 2,5,4'-trichlorobiphenyl. Furthermore, two mutants, the Phe227Val and Phe377Ala mutants, introduced molecular oxygen at the 2,3 position, forming 3-chloro-2',3'-dihydroxy biphenyl with concomitant dechlorination.

Two‐year Study of Carcinogenicity and Chronic Toxicity of Biphenyl in Rats

Two‐year Study of Carcinogenicity and Chronic Toxicity of Biphenyl in Rats: Yumi Umeda, et al. Japan Bioassay Research Center, Japan Industrial Safety and Health Association— Carcinogenicity and chronic toxicity of biphenyl were examined in 50 male and 50 female F344 rats exposed to 0, 500, 1,500 or 4,500 ppm biphenyl in the diet for 105 weeks. Bladder tumors were found in the 4,500 ppm males, as evidenced by significantly increased incidence of carcinoma (24/50) and papilloma (10/50) of the transitional cells as well as one rarely observed case both of carcinoma and papilloma of the squamous cells. The survival rate of the 4500 ppm males significantly decreased, due to the bladder tumors and the hematuria accompanied by bladder calculi. The bladder calculi were found in 43 males in the 4,500 ppm group, but in only 8 females. Urinary pH significantly increased in the males, and occult blood was observed both in males and females in the 4,500 ppm group. The pre‐neoplastic lesions were hyperplasia of transitional epithelium (simple, nodular and papillary hyperplasia) in the bladder of the 4,500 ppm males. Incidences of calculus formation and transitional cell hyperplasia in the renal pelvis also significantly increased in the 4,500 ppm males and females. On the other hand, the incidences of the transitional cell hyperplasia and the calculus formation in the bladder and the renal pelvis were far lower in females than in males, and no bladder tumors were observed in the females. Causative factors of the bladder tumors and their male predominance were discussed with reference to the findings reported in the literature and the previous study of biphenyl metabolism.

Flux control analysis for biphenyl metabolism by Rhodococcus pyridinovorans R04

The flux control coefficients of the four enzymes involved in the upper pathway of biphenyl degradation were determined from transient metabolite concentrations. The first enzyme was indicated as the major rate-limiting step of the pathway with a control coefficient of 0.48. The flux control coefficients of the other three enzymes were 0.03, 0.23 and 0.27, respectively. This is the first experimental evidence of the control step in the pathway of biphenyl degradation using metabolic control analysis.