Dihydroxylation and dechlorination of chlorinated biphenyls by purified biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400.

Oxidation of biphenyl and nine chlorinated biphenyls (CBs) by the biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400 was examined. The purified terminal oxygenase required the addition of partially purified electron transport components, NAD(P)H, and ferrous iron to oxidize biphenyl and CBs. cis-Biphenyl 2,3-dihydrodiol was produced with biphenyl as the substrate. Dihydrodiols were produced from all CBs, and more than one compound was produced with most substrates. Catechols were produced when the dioxygenase-catalyzed reaction occurred at the 2,3 position of a 2-chlorophenyl ring, resulting in dechlorination of the substrate. Oxidation at the 3,4 position of a 2,5-dichlorophenyl ring produced a 3,4-dihydrodiol. Compounds resulting from both types of reaction were produced during oxidation of 2,5,2'-trichlorobiphenyl. The broad substrate specificity and the ability to oxidize at different ring positions suggest that the biphenyl 2,3-dioxygenase is responsible for the wide range of CBs oxidized by Pseudomonas sp. strain LB400.     

Biphenyl uptake by psychrotolerant Pseudomonas sp. strain Cam-1 and mesophilic Burkholderia sp. strain LB400

We investigated the uptake of biphenyl by the psychrotolerant, polychlorinated biphenyl (PCB)-degrader, Pseudomonas sp. strain Cam-1 and the mesophilic PCB-degrader, Burkholderia sp. strain LB400. The effects of growth substrates, metabolic inhibitors, and temperature on [14C]biphenyl uptake were studied. Biphenyl uptake by both strains was induced by growth on biphenyl, and was inhibited by dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), which are metabolic uncouplers. The Vmax and Km for biphenyl uptake by Cam-1 at 22 degrees C were 5.4 +/- 1.7 nmol x min(-1) x (mg of cell protein)(-1) and 83.1 +/- 15.9 micromol x L(-1), respectively. The Vmax and Km for biphenyl uptake by LB400 at 22 degrees C were 3.2 +/- 0.3 nmol x min(-1) x (mg of cell protein(-1)) and 51.5 +/- 9.6 micromol x L(-1), respectively. At 15 degrees C, the maximum rate for biphenyl uptake by Cam-1 and LB400 was 3.1 +/- 0.3 nmol x min(-1) x (mg of cell protein)(-1) and 0.89 +/- 0.1 nmol x min(-1) x (mg of cell protein)(-1), respectively. Thus, the maximum rate for biphenyl uptake by Cam-1 at 15 degrees C was more than 3 times higher than that for LB400.     

Family shuffling of soil DNA to change the regiospecificity of Burkholderia xenovorans LB400 biphenyl dioxygenase

Previous work has shown that the C-terminal portion of BphA, especially two amino acid segments designated region III and region IV, influence the regiospecificity of the biphenyl dioxygenase (BPDO) toward 2,2'-dichlorobiphenyl (2,2'-CB). In this work, we evolved BPDO by shuffling bphA genes amplified from polychlorinated biphenyl-contaminated soil DNA. Sets of approximately 1-kb DNA fragments were amplified with degenerate primers designed to amplify the C-terminal portion of bphA. These fragments were shuffled, and the resulting library was used to replace the corresponding fragment of Burkholderia xenovorans LB400 bphA. Variants were screened for their ability to oxygenate 2,2'-CB onto carbons 5 and 6, which are positions that LB400 BPDO is unable to attack. Variants S100, S149, and S151 were obtained and exhibited this feature. Variant S100 BPDO produced exclusively cis-5,6-dihydro-5,6-dihydroxy-2,2'-dichlorobiphenyl from 2,2'-CB. Moreover, unlike LB400 BPDO, S100 BphA catalyzed the oxygenation of 2,2',3,3'-tetrachlorobiphenyl onto carbons 5 and 6 exclusively and it was unable to oxygenate 2,2',5,5'-tetrachlorobiphenyl. Based on oxygen consumption measurements, variant S100 oxygenated 2,2'-CB at a rate of 16 +/- 1 nmol min(-1) per nmol enzyme, which was similar to the value observed for LB400 BPDO. cis-5,6-Dihydro-5,6-dihydroxy-2,2'-dichlorobiphenyl was further oxidized by 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) and 2,3-dihydroxybiphenyl dioxygenase (BphC). Variant S100 was, in addition, able to oxygenate benzene, toluene, and ethyl benzene. Sequence analysis identified amino acid residues M237 S238 and S283 outside regions III and IV that influence the activity toward doubly ortho-substituted chlorobiphenyls.     

Metabolism of dibenzofuran and dibenzo-p-dioxin by the biphenyl dioxygenase of Burkholderia xenovorans LB400 and Comamonas testosteroni B-356

We examined the metabolism of dibenzofuran (DF) and dibenzo-p-dioxin (DD) by the biphenyl dioxygenase (BPDO) of Comamonas testosteroni B-356 and compared it with that of Burkholderia xenovorans LB400. Data showed that both enzymes oxygenated DF at a low rate, but Escherichia coli cells expressing LB400 BPDO degraded DF at higher rate (30 nmol in 18 h) compared with cells expressing B-356 BPDO (2 nmol in 18 h). Furthermore, both BPDOs produced dihydro-dihydroxy-dibenzofuran as a major metabolite, which resulted from the lateral oxygenation of DF. 2,2,3-Trihydroxybiphenyl (resulting from angular oxygenation of DF) was a minor metabolite produced by both enzymes. Deuterated DF was used to demonstrate the production of 2,2,3-dihydroxybiphenyl through angular oxygenation of DF. When tested for their ability to oxygenate DD, both enzymes produced as sole metabolite, 2,2,3-trihydroxybiphenyl ether at about the same rate, indicating similar catalytic properties toward this substrate. Altogether, although LB400 and B-356 BPDOs oxygenate a different range of chlorobiphenyls, their metabolite profiles toward DF and DD are similar. This suggests that co-planarity influences the regiospecificity of BPDO toward DF and DD to a higher extent than the presence of an ortho substituent on the molecule.     

Oxidation of biphenyl by a multicomponent enzyme system from Pseudomonas sp. strain LB400.

Pseudomonas sp. strain LB400 grows on biphenyl as the sole carbon and energy source. This organism also cooxidizes several chlorinated biphenyl congeners. Biphenyl dioxygenase activity in cell extract required addition of NAD(P)H as an electron donor for the conversion of biphenyl to cis-2,3-dihydroxy-2,3-dihydrobiphenyl. Incorporation of both atoms of molecular oxygen into the substrate was shown with 18O2. The nonlinear relationship between enzyme activity and protein concentration suggested that the enzyme is composed of multiple protein components. Ion-exchange chromatography of the cell extract gave three protein fractions that were required together to restore enzymatic activity. Similarities with other multicomponent aromatic hydrocarbon dioxygenases indicated that biphenyl dioxygenase may consist of a flavoprotein and iron-sulfur proteins that constitute a short electron transport chain involved in catalyzing the incorporation of both atoms of molecular oxygen into the aromatic ring.     

Evolution of the biphenyl dioxygenase BphA from Burkholderia xenovorans LB400 by random mutagenesis of multiple sites in region III

It is now established that several amino acids of region III of the biphenyl dioxygenase (BPDO) alpha subunit are involved in substrate recognition and regiospecificity toward chlorobiphenyls. However, the sequence pattern of the amino acids of that segment of seven amino acids located in the C-terminal portion of the alpha subunit is rather limited in BPDOs of natural occurrence. In this work, we have randomly mutated simultaneously four residues (Thr(335)-Phe(336)-Ile(338)-Ile(341)) of region III of Burkholderia xenovorans LB400 BphA. The library was screened for variants able to oxygenate 2,2'-dichlorobiphenyl (2,2'-CB). Replacement of Phe(336) with Met or Ile with a concomitant change of Thr(335) to Ala created new variants that transformed 2,2'-CB into 3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl, which is a dead end metabolite that was not cleaved by BphC. Replacement of Thr(335)-Phe(336) with Ala(335)-Leu(336) did not cause this type of phenotypic change. Regiospecificity toward congeners other than 2,2'-CB that were oxygenated more efficiently by variant Ala(335)-Met(336) than by LB400 BPDO was similar for both enzymes. Thus structural changes that altered the regiospecificity toward 2,2'-CB did not affect the metabolite profile of other congeners, although it affected the rate of conversion of these congeners. It was especially noteworthy that both LB400 BPDO and the Ala(335)-Met(336) variant generated 2,3-dihydroxy-2',4,4'-trichlorobiphenyl as the sole metabolite from 2,4,2',4'-CB and 4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl as the major metabolite from 2,3,2',3'-CB. This shows that 2,4,2',4'-CB is oxygenated principally onto vicinal ortho-meta carbons 2 and 3 and that 2,3,2',3'-CB is oxygenated onto meta-para carbons 4 and 5 by both enzymes. The data suggest that interactions between the chlorine substitutes on the phenyl ring and specific amino acid residues of the protein influence the orientation of the phenyl ring inside the catalytic pocket.     

Mineralization of low-chlorinated biphenyls by Burkholderia sp. strain LB400 and by a two-membered consortium upon directed interspecies transfer of chlorocatechol pathway genes

The biphenyl-mineralizing bacterium Burkholderia sp. strain LB400 also utilized 3-chloro-, 4-chloro-, 2,3′-dichloro- and 2,4′-dichlorobiphenyl for growth. By the attack of the initial enzyme a chlorine was eliminated dioxygenolytically from position 2 of one of the aromatic rings when hydrogens of both were substituted by chlorine. The strain mineralized 3-chloro- and 2,3′-dichlorobiphenyl via the central intermediate 3-chlorobenzoate through its chlorocatechol pathway enzymes, but excreted stoichiometric amounts of 4-chlorobenzoate from 4-chloro- and 2,4^′-dichlorobiphenyl. These two compounds were mineralized by a co-culture of strain LB400 and a derivative of the (methyl-) benzoate-degrading strain Pseudomonas putida mt-2 (TOL). The complete degradation was achieved upon transfer of a cluster of at least five genes, encoding the regulated chlorocatechol pathway operon, from strain LB400 to strain mt-2. This transfer was demonstrated by the polymerase chain reaction. Received: 15 April 1998 / Received revision: 12 June 1998 / Accepted: 19 June 1998     

Characterization of hybrid biphenyl dioxygenases obtained by recombining Burkholderia sp. strain LB400 bphA with the homologous gene of Comamonas testosteroni B-356.

The bacterial degradation of polychlorinated biphenyls depends on the ability of the enzyme biphenyl 2,3-dioxygenase (BPDO) to catalyze their oxygenation. Analysis of hybrid BPDOs obtained using common restriction sites to exchange large DNA fragments between LB400 bphA and B-356 bphA showed that the C-terminal portion of LB400 alpha subunit can withstand extensive structural modifications, and that these modifications can change the catalytic properties of the enzyme. On the other hand, exchanging the C-terminal portion of B-356 BPDO alpha subunit with that of LB400 alpha subunit generated inactive chimeras. Data encourage an enzyme engineering approach, consisting of introducing extensive modifications of the C-terminal portion of LB400 bphA to extend BPDO catalytic properties toward polychlorinated biphenyls.     

Oxidation of polychlorinated biphenyls by Pseudomonas sp. strain LB400 and Pseudomonas pseudoalcaligenes KF707.

Biphenyl-grown cells and cell extracts prepared from biphenyl-grown cells of Pseudomonas sp. strain LB400 oxidize a much wider range of chlorinated biphenyls than do analogous preparations from Pseudomonas pseudoalcaligenes KF707. These results are attributed to differences in the substrate specificity of the biphenyl 2,3-dioxygenases from both organisms.     

Induction of bphA, encoding biphenyl dioxygenase, in two polychlorinated biphenyl-degrading bacteria, psychrotolerant Pseudomonas strain Cam-1 and mesophilic Burkholderia strain LB400

We investigated induction of biphenyl dioxygenase in the psychrotolerant polychlorinated biphenyl (PCB) degrader Pseudomonas strain Cam-1 and in the mesophilic PCB degrader Burkholderia strain LB400. Using a counterselectable gene replacement vector, we inserted a lacZ-Gm(r) fusion cassette between chromosomal genes encoding the large subunit (bphA) and small subunit (bphE) of biphenyl dioxygenase in Cam-1 and LB400, generating Cam-10 and LB400-1, respectively. Potential inducers of bphA were added to cell suspensions of Cam-10 and LB400-1 incubated at 30 degrees C, and then beta-galactosidase activity was measured. Biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately six times greater than the basal level in cells incubated with pyruvate. In contrast, the beta-galactosidase activities in LB400-1 incubated with biphenyl and in LB400-1 incubated with pyruvate were indistinguishable. At a concentration of 1 mM, most of the 40 potential inducers tested were inhibitory to induction by biphenyl of beta-galactosidase activity in Cam-10. The exceptions were naphthalene, salicylate, 2-chlorobiphenyl, and 4-chlorobiphenyl, which induced beta-galactosidase activity in Cam-10, although at levels that were no more than 30% of the levels induced by biphenyl. After incubation for 24 h at 7 degrees C, biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately four times greater than the basal level in cells incubated with pyruvate. The constitutive level of beta-galactosidase activity in LB400-1 grown at 15 degrees C was approximately five times less than the level in LB400-1 grown at 30 degrees C. Thus, there are substantial differences in the effects of physical and chemical environmental conditions on genetic regulation of PCB degradation in different bacteria.     

Revisiting the regiospecificity of Burkholderia xenovorans LB400 biphenyl dioxygenase toward 2,2'-dichlorobiphenyl and 2,3,2',3'-tetrachlorobiphenyl

2,2'-Dichlorobiphenyl (CB) is transformed by the biphenyl dioxygenase of Burkholderia xenovorans LB400 (LB400 BPDO) into two metabolites (1 and 2). The most abundant metabolite, 1, was previously identified as 2,3-dihydroxy-2'-chlorobiphenyl and was presumed to originate from the initial attack by the oxygenase on the chlorine-bearing ortho carbon and on its adjacent meta carbon of one phenyl ring. 2,3,2',3'-Tetrachlorobiphenyl is transformed by LB400 BPDO into two metabolites that had never been fully characterized structurally. We determined the precise identity of the metabolites produced by LB400 BPDO from 2,2'-CB and 2,3,2',3'-CB, thus providing new insights on the mechanism by which 2,2'-CB is dehalogenated to generate 2,3-dihydroxy-2'-chlorobiphenyl. We reacted 2,2'-CB with the BPDO variant p4, which produces a larger proportion of metabolite 2. The structure of this compound was determined as cis-3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl by NMR. Metabolite 1 obtained from 2,2'-CB-d(8) was determined to be a dihydroxychlorobiphenyl-d(7) by gas chromatographic-mass spectrometric analysis, and the observed loss of only one deuterium clearly shows that the oxygenase attack occurs on carbons 2 and 3. An alternative attack at the 5 and 6 carbons followed by a rearrangement leading to the loss of the ortho chlorine would have caused the loss of more than one deuterium. The major metabolite produced from catalytic oxygenation of 2,3,2',3'-CB by LB400 BPDO was identified by NMR as cis-4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl. These findings show that LB400 BPDO oxygenates 2,2'-CB principally on carbons 2 and 3 and that BPDO regiospecificity toward 2,2'-CB and 2,3,2,',3'-CB disfavors the dioxygenation of the chlorine-free ortho-meta carbons 5 and 6 for both congeners.     

Antioxidant compounds improved PCB-degradation by Burkholderia xenovorans strain LB400

Polychlorobiphenyls (PCBs) are toxic and persistent organic pollutants that are widely distributed in the environment. Burkholderia xenovorans LB400 is capable of degrading aerobically an unusually wide range of PCBs. However, during PCB-degradation B. xenovorans LB400 generates reactive oxygen species (ROS) that affect its viability. The aim of this study was to increase the efficiency of PCB-degradation of B. xenovorans LB400 by adding antioxidant compounds that could increase tolerance to oxidative stress. The effect of antioxidant compounds on the growth, morphology and PCB-degradation by B. xenovorans LB400 was evaluated. α-Tocopherol or vitamin E (vitE) and berry extract (BE) increased slightly the growth of strain LB400 on biphenyl, whereas in presence of ascorbic acid or vitamin C (vitC) an inhibition of growth was observed. The growth of B. xenovorans LB400 in glucose was inhibited by the addition of 4-chlorobiphenyl (4-CB). Interestingly, in presence of α-tocopherol the growth of strain LB400 was less affected by 4-CB. By transmission electronic microscopy it was observed that α-tocopherol preserved the cell membranes and improved cell integrity of glucose-grown LB400 cells exposed to 4-CB, suggesting a protective effect of α-tocopherol. Notably, α-tocopherol increased biphenyl and 4-CB degradation by B. xenovorans LB400 in an aqueous solution. The effect of antioxidants compounds on PCB-bioremediation was evaluated in agricultural soil spiked with 2-chlorobiphenyl (2-CB), 4-CB and 2,4'-chlorobiphenyl (2,4'-CB). For bioaugmentation, LB400 cells grown on biphenyl and subsequently incubated with pyruvate were added to the soil. Native soil microbiota was able to remove PCBs. Bioaugmentation with strain LB400 increased strongly the PCB-degradation rate. Bioaugmentation with strain LB400 and biostimulation with α-tocopherol or berry extract increased further the PCB degradation. Half-life of 2,4'-CB decreased by bioaugmentation from 24 days to 4 days and by bioaugmentation in presence of α-tocopherol and berry extract to 2 days. By bioaugmentation with strain LB400, 85% of 2,4'-CB was degraded in 20 days, whereas bioaugmentation with strain LB400 and biostimulation with α-tocopherol or berry extract reduced the time to less than 13 days. This indicates that antioxidant compounds stimulated PCB-degradation in soil. Therefore, the addition of antioxidant compounds constitutes an attractive strategy for the scale-up of aerobic PCB-bioremediation processes.     

Degradation of highly chlorinated PCBs byPseudomonas strain LB400

Summary Congeners of polychlorinated biphenyl (PCB) differ in the number and position of chlorine substituents. Although PCBs are degraded, those congoners with five or more chlorines have been considered resistant to bacterial degradation. Metabolism byPseudomonas strain LB400 of PCBs representing a broad spectrum of chlorination patterns and having from two to six chlorines was investigated. Degradation of pure PCB congeners and synthetic congener mixes was measured in resting cell assays with biphenyl- or Luria broth-grown cells. In addition, the appearance of metabolites was followed using HPLC purification, and GC and GC-MS characterization. 2,4,5,2,4,5-[¹⁴C]hexachlorobiphenyl was also used to follow the accumulation of¹⁴C-labeled metabolites. Evidence indicates that LB400 aerobically metabolizes representatives of all major structural classes of PCB's including several congeners which lack adjacent unchlorinated carbon atoms. The mechanisms by which many of these congeners are degraded are not fully understood, but it is apparent that aerobic bacteria can degrade a broader spectrum of PCB congeners than previously believed and that this broad spectrum of degradative competence can exist in a single strain.     

多氯联苯降解菌Burkholderia xenovorans LB400研究进展

Burkholderia xenovorans LB400是一株多氯联苯(polychlorinated biphenyls,PCBs)降解菌,可以氧化含有1?6个氯取代基的多氯联苯。近年来,由于其广泛的底物谱和优异的降解性能,菌株LB400已成为研究原核生物降解多氯联苯的生物化学和分子生物学方面的模式生物。目前关于PCBs的微生物降解研究已不再局限于对微生物资源的挖掘,而是更多地聚焦在LB400等降解菌的PCBs降解基因、降解酶的酶学特性以及酶的人工分子进化等方面。同时,LB400作为早期发现的降解菌,其对多氯联苯的降解途径、底物范围及相关机制也被广泛探讨;但是对于PCBs降解相关基因的调控研究较少。因此,本文以Burkholderia xenovorans LB400对多氯联苯降解为核心,通过综述其代谢途径、代谢相关基因和酶系以及降解应用等方面的研究进展,以期为深入探讨Burkholderia xenovorans LB400的应用以及进一步在遗传、分子和生化水平研究其他多氯联苯降解菌株提供借鉴。     

Purification and characterization of the oxygenase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400.

The iron-sulfur protein of biphenyl 2,3-dioxygenase (ISPBPH) was purified from Pseudomonas sp. strain LB400. The protein is composed of a 1:1 ratio of a large (alpha) subunit with an estimated molecular weight of 53,300 and a small (beta) subunit with an estimated molecular weight of 27,300. The native molecular weight was 209,000, indicating that the protein adopts an alpha 3 beta 3 native conformation. Measurements of iron and acid-labile sulfide gave 2 mol of each per mol of alpha beta heterodimer. The absorbance spectrum showed peaks at 325 and 450 nm with a broad shoulder at 550 nm. The spectrum was bleached upon reduction of the protein with NADPH in the presence of catalytic amounts of ferredoxinBPH and ferredoxinBPH oxidoreductase. The electron paramagnetic resonance spectrum of the reduced protein showed three signals at gx = 1.74, gy = 1.92, and gz = 2.01. These properties are characteristic of proteins that contain a Rieske-type [2Fe-2S] center. Biphenyl was oxidized to cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene by ISPBPH in the presence of ferredoxinBPH, ferredoxinBPH oxidoreductase, NADPH, and ferrous iron. Naphthalene was also oxidized to a cis-dihydrodiol, but only 3% was converted to product under the same conditions that gave 92% oxidation of biphenyl. Benzene, toluene, 2,5-dichlorotoluene, carbazole, and dibenzothiophene were not oxidized. ISPBPH is proposed to be the terminal oxygenase component of biphenyl 2,3-dioxygenase where substrate binding and oxidation occur via addition of molecular oxygen and two reducing equivalents.     

Burkholderia xenovorans LB400联苯双加氧酶及突变体对DDTs的降解

DDTs（dichlorodiphenyltrichloroethane,1,1,1-三氯-2,2-双氯苯基乙烷）是一种典型的持久性有机污染物,曾在疟疾防治和农业除虫方面被广泛应用。虽然包括我国在内的很多国家已经禁止使用DDTs,但目前对环境中DDTs的检测发现它仍然广泛存在且具有新的输入源。DDTs的持续存在对近海生态系统和人类健康具有一定危害,因此它所造成的环境污染问题仍然值得关注。由于Rieske型芳香羟化双加氧酶能够起始多种持久性污染物的降解,过去的几十年里一直是芳香化合物降解领域的焦点。[目的] 为探讨联苯双加氧酶对DDTs的降解特性及机制,本研究选取了食异生素伯克霍尔德氏菌LB400（Burkholderia xenovorans）联苯双加氧酶及突变体对p,p''-DDT和o,p''-DDT的降解过程进行研究。[方法] 以BphAE_LB400为亲本,通过两步定点突变将283位的丝氨酸突变为蛋氨酸,获得突变体BphAE_S283M。通过比较亲本酶与突变体对DDTs的催化性能,模拟突变蛋白结构和分子对接等方法,探究其降解特性及机制。[结果] BphAE_LB400和突变体BphAE_S283M都无法降解对位的p,p''-DDT,但突变体BphAE_S283M可以代谢o,p''-DDT并产生2个立体异构体。对接p,p''-DDT的BphAE_LB400和BphAE_S283M的结构分析表明,BphAE_LB400和BphAE_S283M中p,p''-DDT的反应环均不与原晶体结构中的联苯反应环重合。而对接o,p''-DDT的BphAE_S283M的结构分析表明o,p''-DDT的反应环与晶体结构中的联苯反应环距离很近,且2、3位的碳原子与单核铁原子催化中心的距离在0.5 nm以内,此外,BphAE_S283M的催化腔表面积和体积比BphAE_LB400更大,这很可能有助于BphAE_S283M与o,p''-DDT的结合。[结论] 283位氨基酸是影响BphAE_LB400对DDTs的催化代谢能力的关键氨基酸残基,它可以通过调节反应碳原子与催化中心的距离以及催化腔的大小来影响底物特异性。本次研究进一步阐明了283位氨基酸残基的影响机理,为更有效修复DDTs污染提供理论依据和技术支持。     

Oxidation of aminonitrotoluenes by 2,4-DNT dioxygenase of Burkholderia sp. strain DNT

Aminonitrotoluenes form rapidly from the reduction of dinitrotoluenes (DNTs) which are priority pollutants and animal carcinogens. For example, 4-amino-2-nitrotoluene (4A2NT) and 2A4NT accumulate from the reduction of 2,4-DNT during its aerobic biodegradation. Here, we show that 2,4-DNT dioxygenase (DDO) from Burkholderia sp. strain DNT oxidizes the aminonitrotoluenes 2A3NT, 2A6NT, 4A3NT, and 5A2NT to 2-amino-3-nitrobenzylalcohol, 2-amino-4-nitro-m-cresol and 3-amino-5-nitro-p-cresol, 4-amino-3-nitrobenzylalcohol and aminonitrocresol, and 2-amino-5-nitro-o-cresol, respectively. 2A5NT and 3A4NT are oxidized to aminonitrocresols and/or aminonitrobenzylalcohols, and 4A2NT is oxidized to aminonitrocresol. Only 2A4NT, a reduced compound derived from 2,4-DNT, was not oxidized by DDO or its three variants. The alpha subunit mutation I204Y resulted in two to fourfold faster oxidization of the aminonitrotoluenes. Though these enzymes are dioxygenases, they acted like monooxygenases by adding a single hydroxyl group, which did not result in the release of nitrite.     

2,4-Dinitrotoluene dioxygenase from Burkholderia sp. strain DNT: similarity to naphthalene dioxygenase.

2,4-Dinitrotoluene (DNT) dioxygenase from Burkholderia sp. strain DNT catalyzes the initial oxidation of DNT to form 4-methyl-5-nitrocatechol (MNC) and nitrite. The displacement of the aromatic nitro group by dioxygenases has only recently been described, and nothing is known about the evolutionary origin of the enzyme systems that catalyze these reactions. We have shown previously that the gene encoding DNT dioxygenase is localized on a degradative plasmid within a 6.8-kb NsiI DNA fragment (W.-C. Suen and J. C. Spain, J. Bacteriol. 175:1831-1837, 1993). We describe here the sequence analysis and the substrate range of the enzyme system encoded by this fragment. Five open reading frames were identified, four of which have a high degree of similarity (59 to 78% identity) to the components of naphthalene dioxygenase (NDO) from Pseudomonas strains. The conserved amino acid residues within NDO that are involved in cofactor binding were also identified in the gene encoding DNT dioxygenase. An Escherichia coli clone that expressed DNT dioxygenase converted DNT to MNC and also converted naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. In contrast, the E. coli clone that expressed NDO did not oxidize DNT. Furthermore, the enzyme systems exhibit similar broad substrate specificities and can oxidize such compounds as indole, indan, indene, phenetole, and acenaphthene. These results suggest that DNT dioxygenase and the NDO enzyme system share a common ancestor.     

Phenotypic and Phylogenetic Characterization of Burkholderia (Pseudomonas) sp. Strain LB400

The relevant phenotypic traits and phylogenetic relationships between Burkholderia (Pseudomonas) sp. strain LB400 and B. cepacia ATCC 25416^T were compared to determine the degree to which these two strains might be related. Strain LB400 degrades chlorinated biphenyls and has been a model system for potential use in the bioremediation of polychlorinated biphenyls, while some strains of B. cepacia are plant and human pathogens. The fatty acid methyl ester profile, sole carbon source utilization, and biochemical tests confirmed that strain LB400 was a member of the genus Burkholderia. The 16S rRNA gene sequence showed that this strain was not as closely related to B. cepacia as previously suspected or to other known pathogens of this genus, but is closely related to B. phenazinium, B. caribensis, B. graminis, and three unnamed Burkholderia spp. not known to be pathogenic. Received: 16 August 2000 / Accepted: 27 September 2000