Biphenyl Dioxygenase Research Articles

Enumeration of aromatic oxygenase genes to evaluate monitored natural attenuation at gasoline-contaminated sites

Monitoring groundwater benzene, toluene, ethylbenzene, and xylene (BTEX) concentrations is the typical method to assess monitored natural attenuation (MNA) and bioremediation as corrective actions at gasoline-contaminated sites. Conclusive demonstration of bioremediation, however, relies on converging lines of chemical and biological evidence to support a decision. In this study, real-time PCR quantification of aromatic oxygenase genes was used to evaluate the feasibility of MNA at two gasoline-impacted sites. Phenol hydroxylase (PHE), ring-hydroxylating toluene monooxygenase (RMO), naphthalene dioxygenase (NAH), toluene monooxygenase (TOL), toluene dioxygenase (TOD), and biphenyl dioxygenase (BPH4) genes were routinely detected in BTEX-impacted wells. Aromatic oxygenase genes were not detected in sentinel wells outside the plume indicating that elevated levels of oxygenase genes corresponded to petroleum hydrocarbon contamination. Total aromatic oxygenase gene copy numbers detected in impacted wells were on the order of 10 6–10 9 copies L −1. PHE, RMO, NAH, TOD, and BPH4 gene copies positively correlated to total BTEX concentration. Mann–Kendall analysis of benzene concentrations was used to evaluate the status of the dissolved BTEX plume. The combination of trend analysis of contaminant concentrations with quantification of aromatic oxygenase genes was used to assess the feasibility of MNA as corrective measures at both sites.

Identification, cloning, and characterization of a multicomponent biphenyl dioxygenase from Sphingobium yanoikuyae B1

Sphingobium yanoikuyae B1 utilizes both polycyclic aromatic hydrocarbons (biphenyl, naphthalene, and phenanthrene) and monocyclic aromatic hydrocarbons (toluene, m- and p-xylene) as its sole source of carbon and energy for growth. The majority of the genes for these intertwined monocyclic and polycyclic aromatic pathways are grouped together on a 39 kb fragment of chromosomal DNA. However, this gene cluster is missing several genes encoding essential enzymatic steps in the aromatic degradation pathway, most notably the genes encoding the oxygenase component of the initial polycyclic aromatic hydrocarbon (PAH) dioxygenase. Transposon mutagenesis of strain B1 yielded a mutant blocked in the initial oxidation of PAHs. The transposon insertion point was sequenced and a partial gene sequence encoding an oxygenase component of a putative PAH dioxygenase identified. A cosmid clone from a genomic library of S. yanoikuyae B1 was identified which contains the complete putative PAH oxygenase gene sequence. Separate clones expressing the genes encoding the electron transport components (ferredoxin and reductase) and the PAH dioxygenase were constructed. Incubation of cells expressing the dioxygenase enzyme system with biphenyl or naphthalene resulted in production of the corresponding cis-dihydrodiol confirming PAH dioxygenase activity. This demonstrates that a single multicomponent dioxygenase enzyme is involved in the initial oxidation of both biphenyl and naphthalene in S. yanoikuyae B1.

Characterization of Biphenyl Dioxygenase of Pandoraea pnomenusa B-356 As a Potent Polychlorinated Biphenyl-Degrading Enzyme

Biphenyl dioxygenase (BPDO) catalyzes the aerobic transformation of biphenyl and various polychlorinated biphenyls (PCBs). In three different assays, BPDO(B356) from Pandoraea pnomenusa B-356 was a more potent PCB-degrading enzyme than BPDO(LB400) from Burkholderia xenovorans LB400 (75% amino acid sequence identity), transforming nine congeners in the following order of preference: 2,3',4-trichloro approximately 2,3,4'-trichloro > 3,3'-dichloro > 2,4,4'-trichloro > 4,4'-dichloro approximately 2,2'-dichloro > 2,6-dichloro > 2,2',3,3'-tetrachloro approximately 2,2',5,5'-tetrachloro. Except for 2,2',5,5'-tetrachlorobiphenyl, BPDO(B356) transformed each congener at a higher rate than BPDO(LB400). The assays used either whole cells or purified enzymes and either individual congeners or mixtures of congeners. Product analyses established previously unrecognized BPDO(B356) activities, including the 3,4-dihydroxylation of 2,6-dichlorobiphenyl. BPDO(LB400) had a greater apparent specificity for biphenyl than BPDO(B356) (k(cat)/K(m) = 2.4 x 10(6) +/- 0.7 x 10(6) M(-1) s(-1) versus k(cat)/K(m) = 0.21 x 10(6) +/- 0.04 x 10(6) M(-1) s(-1)). However, the latter transformed biphenyl at a higher maximal rate (k(cat) = 4.1 +/- 0.2 s(-1) versus k(cat) = 0.4 +/- 0.1 s(-1)). A variant of BPDO(LB400) containing four active site residues of BPDO(B356) transformed para-substituted congeners better than BPDO(LB400). Interestingly, a substitution remote from the active site, A267S, increased the enzyme's preference for meta-substituted congeners. Moreover, this substitution had a greater effect on the kinetics of biphenyl utilization than substitutions in the substrate-binding pocket. In all variants, the degree of coupling between congener depletion and O(2) consumption was approximately proportional to congener depletion. At 2.4-A resolution, the crystal structure of the BPDO(B356)-2,6-dichlorobiphenyl complex, the first crystal structure of a BPDO-PCB complex, provided additional insight into the reactivity of this isozyme with this congener, as well as into the differences in congener preferences of the BPDOs.

Characterization of Two Biphenyl Dioxygenases for Biphenyl/PCB Degradation in A PCB Degrader,Rhodococcussp. Strain RHA1

Rhodococcus sp. RHA1 induces two biphenyl dioxygenases, the BphA and EtbA/EbdA dioxygenases, during growth on biphenyl. Their subunit genes were expressed in R. erythropolis IAM1399 to investigate the involvement of each subunit gene in their activity and their substrate preferences. The recombinant expressing ebdA1A2A3etbA4 and that expressing bphA1A2A3A4 exhibited 4-chlorobiphenyl (4-CB) transformation activity, suggesting that these gene sets are responsible for the EtbA/EbdA and BphA dioxygenases respectively. When bphA4 and etbA4 were swapped to construct the recombinants expressing ebdA1A2A3bphA4 and bphA1A2A3etbA4 respectively, compatibility between BphA4 and EtbA4 was suggested by their 4-CB transformation activities. When bphA3 and ebdA3 were swapped, incompatibility between BphA3 and EbdA3 was suggested. BphA and EtbA/EbdA dioxygenases exhibited the highest transformation activity toward biphenyl and naphthalene respectively, and also attacked dibenzofuran and dibenzo-p-dioxin. The wide substrate preference of EtbA/EbdA dioxygenase suggested that it plays a more important role in polychlorinated biphenyl (PCB) degradation than does BphA dioxygenase.

Synthesis of highly hydroxylated aromatics by evolved biphenyl dioxygenase and subsequent dihydrodiol dehydrogenase

The evolved bphA1 (2049) gene, in which nine amino acids from the Pseudomonas pseudoalcaligenes KF707 BphA1 were changed to those from the Burkholderia xenovorans LB400 BphA1 (M247I, H255Q, V258I, G268A, D303E, -313G, S324T, V325I, and T376N), was expressed in Escherichia coli along with the bphA2A3A4 and bphB genes derived from strain KF707. This recombinant E. coli cells converted biphenyl and several heterocyclic aromatic compounds into the highly hydroxylated products such as biphenyl-2,3,2',3'-tetraol (from biphenyl), 2-(2,3-dihydroxyphenyl)benzoxazole-4,5-diol (from 2-phenylbenzoxazole), and 2-(2,5-dihydroxyphenyl)benzoxazole-4,5-diol [from 2-(2-hydroxyphenyl)benzoxazole]. The antioxidative activity of these generated compounds was markedly higher than that of the original substrate used.

Crystallization and preliminary X-ray analysis of the reduced Rieske-type [2Fe-2S] ferredoxin derived from Pseudomonas sp. strain KKS102.

The reduced form of BphA3, a Rieske-type [2Fe-2S] ferredoxin component of the biphenyl dioxygenase BphA from Pseudomonas sp. strain KKS102, was crystallized by the sitting-drop vapour-diffusion method under anaerobic conditions. The crystal belongs to space group P3(1)21, with unit-cell parameters a = b = 49.6, c = 171.9 A, and diffracts to a resolution of 1.95 A. A molecular-replacement calculation using oxidized BphA3 as a search model yielded a satisfactory solution.

Purification, characterization, and crystallization of the components of a biphenyl dioxygenase system from Sphingobium yanoikuyae B1

Sphingobium yanoikuyae B1 initiates the catabolism of biphenyl by adding dioxygen to the aromatic nucleus to form (+)-cis-(2R, 3S)-dihydroxy-1-phenylcyclohexa-4,6-diene. The present study focuses on the biphenyl 2,3-dioxygenase system, which catalyzes the dioxygenation reaction. This enzyme has been shown to have a broad substrate range, catalyzing the dioxygenation of not only biphenyl, but also three- and four-ring polycyclic aromatic hydrocarbons. Extracts prepared from biphenyl-grown B1 cells contained three protein components that were required for the oxidation of biphenyl. The genes encoding the three components (bphA4, bphA3 and bphA1f,A2f) were expressed in Escherichia coli. Biotransformations of biphenyl, naphthalene, phenanthrene, and benzo[a]pyrene as substrates using the recombinant E. coli strain resulted in the formation of the expected cis-dihydrodiol products previously shown to be produced by biphenyl-induced strain B1. The three protein components were purified to apparent homogeneity and characterized in detail. The reductase component (bphA4), designated reductase(BPH-B1), was a 43 kD monomer containing one mol FAD/mol reductase(BPH-B1). The ferredoxin component (bphA3), designated ferredoxin(BPH-B1), was a 12 kD monomer containing approximately 2 g-atoms each of iron and acid-labile sulfur. The oxygenase component (bphA1f,A2f), designated oxygenase(BPH-B1), was a 217 kD heterotrimer consisting of alpha and beta subunits (approximately 51 and 21 kD, respectively). The iron and acid-labile sulfur contents of oxygenase(BPH-B1) per alphabeta were 2.4 and 1.8 g-atom per mol, respectively. Reduced ferredoxin(BPH-B1) and oxygenase(BPH-B1) each gave EPR signals typical of Rieske [2Fe-2S] proteins. Crystals of reductase(BPH-B1), ferredoxin(BPH-B1) and oxygenase(BPH-B1 )diffracted to 2.5 A, 2.0 A and 1.75 A, respectively. The structures of the three proteins are currently being determined.

Redox and Functional Analysis of the Rieske Ferredoxin Component of the Toluene 4-Monooxygenase

Toluene 4-monooxygenase catalyzes the NADH- and O2-dependent hydroxylation of toluene to form p-cresol. The four-protein complex consists of a diiron hydroxylase, an oxidoreductase, a catalytic effector protein, and a Rieske-type ferredoxin (T4moC). Phylogenetic analysis suggests that T4moC is part of a clade specialized for reaction with diiron hydroxylases, possibly reflected in the conservation of W69, whose indole side chain makes close contacts with a bridging sulfide. In order to further investigate the possible origins of this specialization, T4moC, mutated variants of T4moC, and three other purified ferredoxins (the Thermus Rieske protein, the Burkholderia cepacia Rieske-type biphenyl dioxygenase ferredoxin BphF, and the Ralstonia pickettii PK01 toluene monooxygenase TbuB, the Rieske-type ferredoxin from another diiron monooxygenase complex) were studied by redox potential measurements and their ability to complement the catalytic function of the reconstituted toluene 4-monooxygenase complex. A saturation mutagenesis of T4moC W69 indicates that an aromatic residue may modulate the redox potential and is also necessary for activity and/or stability. The redox potential of T4moC was determined to be -173 mV, W69F T4moC was -139 mV, and TbuB was -150 mV. For comparison, BphF had a redox potential of -157 mV [Couture et al. (2001) Biochemistry 40, 84-92]. Of these ferredoxins, all except BphF were able to provide catalytic activity. Given the range in redox potentials observed in the active ferredoxins, shape and electrostatics are strongly implicated in the catalytic specialization. Mutagenesis of other T4moC surface residues gave further insight into possible origins of catalytic specialization. Thus R65A T4moC gave an alteration in apparent KM only, while D82A/D83A T4moC gave alterations in both apparent kcat and KM. Since the different catalytic results were obtained by mutagenesis of residues lying on different sides of the protein adjacent to the [2Fe-2S] cluster, the results suggest that two different faces of T4moC may be involved in protein-protein interactions during catalysis.

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.

Expression of bacterial biphenyl‐chlorobiphenyl dioxygenase genes in tobacco plants

Optimized plant-microbe bioremediation processes in which the plant initiates the metabolism of xenobiotics and releases the metabolites in the rhizosphere to be further degraded by the rhizobacteria is a promising alternative to restore contaminated sites in situ. However, such processes require that plants produce the metabolites that bacteria can readily oxidize. The biphenyl dioxygenase is the first enzyme of the bacterial catabolic pathway involved in the degradation of polychlorinated biphenyls. This enzyme consists of three components: the two sub-unit oxygenase (BphAE) containing a Rieske-type iron-sulfur cluster and a mononuclear iron center, the Rieske-type ferredoxin (BphF), and the FAD-containing ferredoxin reductase (BphG). In this work, based on analyses with Nicotiana benthamiana plants transiently expressing the biphenyl dioxygenase genes from Burkholderia xenovorans LB400 and transgenic Nicotiana tabacum plants transformed with each of these four genes, we have shown that each of the three biphenyl dioxygenase components can be produced individually as active protein in tobacco plants. Therefore, when BphAE, BphF, and BphG purified from plant were used to catalyze the oxygenation of 4-chlorobiphenyl, detectable amounts of 2,3-dihydro-2, 3-dihydroxy-4'-chlorobiphenyl were produced. This suggests that creating transgenic plants expressing simultaneously all four genes required to produce active biphenyl dioxygenase is feasible.

Naphthalene and biphenyl oxidation by two marine Pseudomonas strains isolated from Venice Lagoon sediment

Abstract A sediment sample from Venice Lagoon was found to be contaminated with 475 mg Kg−1 polycyclic aromatic hydrocarbons (PAHs). Naphthalene was the principal pollutant at 26% of total PAHs. Two strains of Pseudomonas SN1 and SB1 were isolated from sediment amended with 2% naphthalene. 16S rRNA gene sequence analysis indicated that the two strains have about 99% nucleotide identity with strains of the genus Pseudomonas, and are very close to Pseudomonas stutzeri. Their metabolic profiles showed significant nutritional differences, the most significant of which was that SN1 grows in marine mineral medium spiked with naphthalene and SB1 grows with biphenyl as sole carbon and energy sources. Pseudomonas sp. SN1 had a doubling time of 3.1 h with 2% naphthalene and SB1 had a doubling time of 19.5 h with 2% biphenyl. Strain SN1 oxidised naphthalene at 564±32 mg O2 l−1 d−1 and SB1 oxidised biphenyl at 426±25 mg O2 l−1 d−1 in respirometry reaction vessels under controlled conditions. Screening of the two strains for dioxygenase genes involved in the first step of the two hydrocarbon degradation pathways, by polymerase chain reaction, showed naphthalene dioxygenase in SN1 and biphenyl dioxygenase in SB1. The strains each have a different catechol 2,3-dioxygenase responsible for cleavage of the aromatic ring.

Development of an Efficient Microbial Conversion System (CellCombiChem) Through Multiple Use of Biphenyl Catabolic Enzyme Genes

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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.

Crystallization and preliminary X-ray analysis of the Rieske-type [2Fe-2S] ferredoxin component of biphenyl dioxygenase from Pseudomonas sp. strain KKS102.

BphA3, a Rieske-type [2Fe-2S] ferredoxin component of a biphenyl dioxygenase (BphA) from Pseudomonas sp. strain KKS102, was crystallized by the hanging-drop vapour-diffusion method. Two crystal forms were obtained from the same conditions. The form I crystal belongs to space group P2(1)2(1)2, with unit-cell parameters a = 26.3, b = 144.3, c = 61.5 A, and diffracted to 2.45 A resolution. The form II crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 26.2, b = 121.3, c = 142.7 A, and diffracted to 2.8 A resolution. A molecular-replacement calculation using BphF as a search model yielded a satisfactory solution for both forms.

Structure of T4moC, the Rieske-type ferredoxin component of toluene 4-monooxygenase

The structure of the Rieske-type ferredoxin (T4moC) from toluene 4-monooxygenase was determined by X-ray crystallography in the [2Fe-2S](2+) state at a resolution of 1.48 A using single-wavelength anomalous dispersion phasing with the [2Fe-2S] center. The structure consists of ten beta-strands arranged into the three antiparallel beta-sheet topology observed in all Rieske proteins. Trp69 of T4moC is adjacent to the [2Fe-2S] centre, which displaces a loop containing the conserved Pro81 by approximately 8 A away from the [2Fe-2S] cluster compared with the Pro loop in the closest structural and functional homolog, the Rieske-type ferredoxin BphF from biphenyl dioxygenase. In addition, T4moC contains five hydrogen bonds to the [2Fe-2S] cluster compared with three hydrogen bonds in BphF. Moreover, the electrostatic surface of T4moC is distinct from that of BphF. These structural differences are identified as possible contributors to the evolutionary specialization of soluble Rieske-type ferredoxins between the diiron monooxygenases and cis-dihydrodiol-forming dioxygenases.

Generation of Novel-Substrate-Accepting Biphenyl Dioxygenases through Segmental Random Mutagenesis and Identification of Residues Involved in Enzyme Specificity

Aryl-hydroxylating dioxygenases are of interest for the degradation of persistant aromatic pollutants, such as polychlorobiphenyls (PCBs), or as catalysts for the functionalization of aromatic scaffolds. In order to achieve dioxygenation of technical mixtures of PCBs, enzymes with broadened or altered substrate ranges are essential. To alter the substrate specificity of the biphenyl dioxygenase (BphA) of Burkholderia xenovorans LB400, we applied a directed evolution approach that used structure-function relationship data to target random mutageneses to specific segments of the enzyme. The limitation of random amino acid (AA) substitutions to regions that are critical for substrate binding and the exclusion of AA exchanges from positions that are essential for catalytic activity yielded enzyme variants of interest at comparatively high frequencies. After only a single mutagenic cycle, 10 beneficial variants were detected in a library of fewer than 1,000 active enzymes. Compared to the parental BphA, they showed between 5- and 200-fold increased turnover of chlorinated biphenyls, with substituent patterns that rendered them largely recalcitrant to attack by BphA-LB400. Determination of their sequences identified AAs that prevent the acceptance of specific PCBs by the wild-type enzyme, such as Pro334 and Phe384. The results suggest prime targets for subsequent cycles of BphA modification. Correlations with a three-dimensional model of the enzyme indicated that most of the exchanges with major influence on substrate turnover do not involve pocket-lining residues and had not been predictable through structural modeling.

Mutation Mining—A Prospector's Tale

Protein structure visualization tools render images that allow the user to explore structural features of a protein. Context specific information relating to a particular protein or protein family is, however, not easily integrated and must be uploaded from databases or provided through manual curation of input files. Protein Engineers spend considerable time iteratively reviewing both literature and protein structure visualizations manually annotated with mutated residues. Meanwhile, text mining tools are increasingly used to extract specific units of raw text from scientific literature and have demonstrated the potential to support the activities of Protein Engineers. The transfer of mutation specific raw-text annotations to protein structures requires integrated data processing pipelines that can co-ordinate information retrieval, information extraction, protein sequence retrieval, sequence alignment and mutant residue mapping. We describe the Mutation Miner pipeline designed for this purpose and present case study evaluations of the key steps in the process. Starting with literature about mutations made to protein families; haloalkane dehalogenase, bi-phenyl dioxygenase, and xylanase we enumerate relevant documents available for text mining analysis, the available electronic formats, and the number of mutations made to a given protein family. We review the efficiency of NLP driven protein sequence retrieval from databases and report on the effectiveness of Mutation Miner in mapping annotations to protein structure visualizations. We highlight the feasibility and practicability of the approach.

Ｂｉｐｈｅｎｙｌ分解経路系酵素遺伝子群を複合的に利用した効率的微生物変換技術（ＣｅｌｌＣｏｍｂｉＣｈｅｍ）の開発

We have developed a system for the comprehensive bioconversion of a series of organic chemicals through the living cells of recombinant microbes (living cells-based combinatorial chemistry; CellCombiChem). This attempt started with recombinant Escherichia coli expressing bacterial genes involved in biphenyl degradation (metabolism). The biphenyl degradation is carried out through four enzymes : biphenyl dioxygenase (BphA), dihydrodiol dehydrogenase (BphB), 2, 3-dihydrodiol dioxygenase (BphC), and hydrolase (BphD). We succeeded in synthesizing various oxidized compounds from a series of aromatic compounds, e.g. phenyl heterocycles and flavonoids, by using E. coli expressing these enzyme genes systematically (bphA, synthesis of 1, 2-cis-dihydrodiol; bphA+bphB, 1, 2-diol; bphA+bphB+bphC, picolinic acid; bphA+bphB+bphC+bphD, carboxylic acid). In this bphA (bphA1A2A3A4), bphA1 (2072) evolved by family (DNA) shuffling, which has a wide substrate preference, was used. Many of the converted compounds were novel, and some of them showed potent antioxidative activity.

Steady-State Kinetic Characterization of Evolved Biphenyl Dioxygenase, Which Acquired Novel Degradation Ability for Benzene and Toluene

Biphenyl dioxygenase (Bph Dox) catalyzes initial oxygenation in the bacterial biphenyl degradation pathway. Bph Dox in Pseudomonas pseudoalcaligenes KF707 is a Rieske type three-component enzyme in which a large subunit (encoded by the bphA1 gene) plays an important role in the substrate specificity of Bph Dox. Steady-state kinetic assays using purified enzyme components demonstrated that KF707 Bph Dox had a kcat/Km of 33.1 x 10(3) (M(-1) s(-1)) for biphenyl. Evolved 1072 Bph Dox generated by the process of DNA shuffling (Suenaga, H. et al., J. Bacteriol., 184, 3682-3688 (2002)) exhibited enhanced degradation activity not only for biphenyl (kcat/Km of 62.2 x 10(3) [M(-1) s(-1)]) but also for benzene and toluene, compounds that are rarely attacked by KF707 Bph Dox. These results suggest that evolved 1072 Bph Dox acquires higher affinities and catalytic efficiencies for various substrates than the original KF707 enzyme.

Soil Microbial Community Responses to Additions of Organic Carbon Substrates and Heavy Metals (Pb and Cr)

Microcosm experiments were conducted with soils contaminated with heavy metals (Pb and Cr) and aromatic hydrocarbons to determine the effects of each upon microbial community structure and function. Organic substrates were added as a driving force for change in the microbial community. Glucose represented an energy source used by a broad variety of bacteria, whereas fewer soil species were expected to use xylene. The metal amendments were chosen to inhibit the acute rate of organic mineralization by either 50% or 90%, and lower mineralization rates persisted over the entire 31-day incubation period. Significant biomass increases were abolished when metals were added in addition to organic carbon. The addition of organic carbon alone had the most significant impact on community composition and led to the proliferation of a few dominant phylotypes, as detected by PCR-denaturing gradient gel electrophoresis of bacterial 16S rRNA genes. However, the community-wide effects of heavy metal addition differed between the two carbon sources. For glucose, either Pb or Cr produced large changes and replacement with new phylotypes. In contrast, many phylotypes selected by xylene treatment were retained when either metal was added. Members of the Actinomycetales were very prevalent in microcosms with xylene and Cr(VI); gene copy numbers of biphenyl dioxygenase and phenol hydroxylase (but not other oxygenases) were elevated in these microcosms, as determined by real-time PCR. Much lower metal concentrations were needed to inhibit the catabolism of xylene than of glucose. Cr(VI) appeared to be reduced during the 31-day incubations, but in the case of glucose there was substantial microbial activity when much of the Cr(VI) remained. In the case of xylene, this was less clear.