Optimization of 2,3-dihydroxybiphenyl 1,2-dioxygenase expression and its application for biosensor

In this study, two statistical experimental designs, Plackett-Burman design (PBD) and response surface methodology (RSM), were employed to enhance the expression of 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC_LA-4), which was subsequently used for the construction of catechol biosensor. Ten important factors were evaluated by PBD, and four significant parameters were then optimized by RSM. Under the favorable fermentation conditions, the maximal specific activity of BphC_LA-4 was about 0.58U/mg with catechol as substrate. Meanwhile, homology modeling and molecular docking were utilized to help understand the interaction between BphC_LA-4 and catecholic substrates, which illustrated that BphC_LA-4 presented lower binding affinity towards 4-methylcatechol in comparison with 3-methylcatechol and catechol. Interestingly, the BphC_LA-4 enzyme electrode prepared by SiO2 sol-gel showed good response to all these three catecholic compounds. The differences of selectivity to 4-methylcatechol between free and immobilized enzyme implied that the introduction of electro-catalysis might have an effect on the enzyme-catalysis process.     

The mechanism-based inactivation of 2,3-dihydroxybiphenyl 1,2-dioxygenase by catecholic substrates.

2,3-Dihydroxybiphenyl 1,2-dioxygenase (EC ), the extradiol dioxygenase of the biphenyl biodegradation pathway, is subject to inactivation during the steady-state cleavage of catechols. Detailed analysis revealed that this inactivation was similar to the O(2)-dependent inactivation of the enzyme in the absence of catecholic substrate, resulting in oxidation of the active site Fe(II) to Fe(III). Interestingly, the catecholic substrate not only increased the reactivity of the enzyme with O(2) to promote ring cleavage but also increased the rate of O(2)-dependent inactivation. Thus, in air-saturated buffer, the apparent rate constant of inactivation of the free enzyme was (0.7 +/- 0.1) x 10(-3) s(-1) versus (3.7 +/- 0.4) x 10(-3) s(-1) for 2,3-dihydroxybiphenyl, the preferred catecholic substrate of the enzyme, and (501 +/- 19) x 10(-3) s(-1) for 3-chlorocatechol, a potent inactivator of 2,3-dihydroxybiphenyl 1,2-dioxygenase (partition coefficient = 8 +/- 2, K(m)(app) = 4.8 +/- 0.7 microm). The 2,3-dihydroxybiphenyl 1,2-dioxygenase-catalyzed cleavage of 3-chlorocatechol yielded predominantly 2-pyrone-6-carboxylic acid and 2-hydroxymuconic acid, consistent with the transient formation of an acyl chloride. However, the enzyme was not covalently modified by this acyl chloride in vitro or in vivo. The study suggests a general mechanism for the inactivation of extradiol dioxygenases during catalytic turnover involving the dissociation of superoxide from the enzyme-catecholic-dioxygen ternary complex and is consistent with the catalytic mechanism.     

Inactivation of 2,3-dihydroxybiphenyl 1,2-dioxygenase fromPseudomonas sp. strain CB406 by 3,4-dihydroxybiphenyl (4-phenylcatechol)

Summary 3,4-dihydroxybiphenyl is not a substrate for the 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) from biphenyldegradingPseudomonas sp. strain CB406. It acts as both a reversible inhibitor and a potent inactivator of the enzyme. The inactivation process requires the presence of O₂ and can be reversed by the removal of the 3,4-dihydroxybiphenyl followed by incubation of the enzyme in the presence of dithioerythritol and Fe²⁺ under anaerobic conditions. Two other extradiol dioxygenases behave similarly, the catechol 2,3-dioxygenase (BphE) from strain CB406 and the BphC fromPseudomonas sp. strain LB400. The BphC fromP. testosteroni B-356 also did not cleave 3,4-dihydroxybiphenyl but it was not inactivated.Abbreviations C23o Catechol 2,3-dioxygenase - 34DHBP 3,4-dihydroxybiphenyl     

Inhibition of catechol 2,3-dioxygenase from Pseudomonas putida by 3-chlorocatechol.

Partially purified preparations of catechol 2,3-dioxygenase from toluene-grown cells of Pseudomonas putida catalyzed the stoichiometric oxidation of 3-methylcatechol to 2-hydroxy-6-oxohepta-2,4-dienoate. Other substrates oxidized by the enzyme preparation were catechol, 4-methylcatechol, and 4-fluorocatechol. The apparent Michaelis constants for 3-methylcatechol and catechol were 10.6 and 22.0 muM, respectively. Substitution at the 4-position decreases the affinity and activity of the enzyme for the substrate. Catechol 2,3-dioxygenase preparations did not oxidize 3-chlorocatechol. In addition, incubation of the enzyme with 3-chlorocatechol led to inactivation of the enzyme. Kinetic analyses revealed that both 3-chlorocatechol and 4-chlorocatechol were noncompetitive or mixed-type inhibitors of the enzyme. 3-Chlorocatechol (Ki = 0.14 muM) was a more potent inhibitor than 4-chlorocatechol (Ki = 50 muM). The effect of the ion-chelating agents Tiron and o-phenanthrolene were compared with that of 3-chlorocatechol on the inactivation of the enzyme. Each inhibitor appeared to remove iron from the enzyme, since inactive enzyme preparations could be fully reactivated by treatment with ferrous iron and a reducing agent.     

Rapid purification of an active recombinant His-tagged 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83

2,3-Dihydroxybiphenyl 1,2-dioxygenase (2,3-DBPD) is an extradiol-type dioxygenase that catalyzes the aromatic ring fission of 2,3-dihydroxybiphenyl, the third step in the biphenyl degradation pathway. The nucleotide sequence of the Pseudomonas putida OU83 gene bphC, which encodes 2,3-DBPD, was cloned into a plasmid pQE31. The His-tagged 2,3-DBPD produced by a recombinant Escherichia coli strain, SG13009(pREP4)(pAKC1), and purified with a Ni-nitrilotriacetic acid resin affinity column using the His-bind Qiagen system. The His-tagged 2,3-DBPD construction, carrying a single 6×His tail on the N-terminal of the polypeptide, was active. SDS-PAGE analysis of the purified active 2,3-DBPD gave a single band of 34 kDa; this is in agreement with the size of the bphC coding region. The K_m for 2,3-dihydroxybiphenyl was 14.5±2 μM. The enzyme activity was enhanced by ferrous ion but inhibited by ferric ion. The enzyme activity was inhibited by thiol-blocking reagents and heavy metals HgCl₂, CuSO₄, NiSO₄, and CdCl₂. The yield was much higher and the time required to purify recombinant 2,3-DBPD from clone pAKC1 was faster than by the conventional chromatography procedures.     

Oxidation of carbazole to 3-hydroxycarbazole by naphthalene 1,2-dioxygenase and biphenyl 2,3-dioxygenase

Abstract Naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4 and biphenyl dioxygenase from Beijerinckia sp. B8/36 oxidized the aromatic N-heterocycle carbazole to 3-hydroxycarbazole. Toluene dioxygenase from Pseudomonas putida F39/D did not oxidize carbazole. Transformations were carried out by mutant strains which oxidize naphthalene and biphenyl to cis -dihydrodiols, and with a recombinant E. coli strain expressing the structural genes of naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4. 3-Hydroxycarbazole is presumed to result from the dehydration of an unstable cis -dihydrodiol.     

Isolation, characterization and docking studies of 2,3-dihydroxybiphenyl 1,2-dioxygenase from an activated sludge metagenome

A 2,3-dihydroxybiphenyl-1,2-dioxygenase gene (designated as bphC_meta) was identified in activated sludge metagenome by PCR. This gene shared 99% sequence identity with BphC from Burkholderia xenovorans LB400. The enzyme was purified from recombinant Escherichia coli with a subunit molecular mass of 32 ± 1 kDa. It was optimally active at pH 9.0 and 40°C, using 2,3-dihydroxybiphenyl as a substrate. Activity toward substituted catechols was: 2,3-dihydroxybiphenyl > 3-methylcatechol > catechol > 4-chlorocatechol (4-methylcatechol). The prediction made by molecular docking was consistent with the kinetic experimental data, and further explained the substrate preference of BphC_meta. The present study could pave the way for the improved understanding and application of BphCs derived from metagenomes.     

Enzyme–substrate interaction and characterization of a 2,3-dihydroxybiphenyl 1,2-dioxygenase from Dyella ginsengisoli LA-4

A bphC gene (915 bp) encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) was amplified by PCR from Dyella ginsengisoli LA-4, which was heterologously expressed in Escherichia coli . The purified His-Tag BphC was able to catalyze the meta -cleavage reaction of the dihydroxylated aromatic rings. According to the specificity constant ( K _cat/ K _m) of BphC_LA-4, the specificity of BphC_LA-4 was determined in the following order: 2,3-dihydroxybiphenyl>3-methylcatechol>catechol>4-chlorocatechol>4-methylcatechol. The experimental data were consistent with the prediction of enzyme–substrate complexes. The highest specific activity of BphC_LA-4 was 118.3 U mg⁻¹ for 2,3-dihydroxybiphenyl.     

Purification of 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83 and characterization of the gene (bphC).

The 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DBPD) of Pseudomonas putida OU83 was constitutively expressed and purified to apparent homogeneity. The apparent molecular mass of the native enzyme was 256 kDa, and the subunit molecular mass was 32 kDa. The data suggested that 2,3-DBPD was an octamer of identical subunits. The nucleotide sequence of a DNA fragment containing the bphC region was determined. The deduced protein sequence for 2,3-DBPD consisted of 292 amino acid residues, with a calculated molecular mass of 31.9 kDa, which was in agreement with data for the purified 2,3-DBPD. Nucleotide and amino acid sequence analyses of the bphC gene and its product, respectively, revealed that there was a high degree of homology between the OU83 bphC gene and the bphC genes of Pseudomonas cepacia LB400 and Pseudomonas pseudoalcaligenes KF707.     

Functionality of biphenyl 2,3-dioxygenase components in naphthalene 1,2-dioxygenase

Naphthalene 1,2-dioxygenase (Nap dox) and biphenyl 2,3-dioxygenase (Bph dox) are related enzymes that have differentiated during evolution as their specificity has changed. Although their component arrangement is similar, the structure of each component has been modified quite extensively. The purpose of this work was to determine the catalytic capacity of purified Nap dox toward chlorobiphenyls and to investigate the functionality of Bph dox components in the Nap dox system. Both enzyme systems were purified by affinity chromatography as histidine-tagged fused proteins. Data show for the first time that Nap dox can catalyze the oxygenation of all three monochlorobiphenyl isomers, but it is unable to hydroxylate 2,5-, 2,2′-, 3,3′-, 4,4′-di- and 2,2′,5,5′-tetrachlorobiphenyl. The rates of cytochrome c reduction by the ferredoxin components of the two enzymes were identical when the Bph dox reductase component was used in the assay, showing an efficient electron transfer between the Bph dox reductase component and the Nap dox ferredoxin. However, when the Bph dox ferredoxin was used to reconstitute a hybrid Nap dox, the enzyme was only 22% as active as the parental enzyme. These data are discussed in terms of the potential use of Nap dox for the development of enhanced chlorobiphenyl-degrading dioxygenases. Received: 15 October 1998 / Received revision: 21 January 1999 / Accepted: 31 January 1999     

Identification of an alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase in Rhodococcus sp. strain RHA1 and cloning of the gene.

Gram-positive Rhodococcus sp. strain RHA1 possesses strong polychlorinated biphenyl-degrading capabilities. An RHA1 bphC gene mutant, strain RDC1, had been previously constructed (E. Masai, A. Yamada, J. M. Healy, T. Hatta, K. Kimbara, M. Fukuda, and K. Yano, Appl. Environ. Microbiol. 61:2079-2085, 1995). An alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD), designated EtbC, was identified in RDC1 cells grown on ethylbenzene. EtbC contained the broadest substrate specificity of any meta cleavage dioxygenase identified in a Rhodococcus strain to date, including RHA1 BphC. EtbC was purified to near homogeneity from RDC1 cells grown on ethylbenzene, and a 58-amino-acid NH2-terminal sequence was determined. The NH2-terminal amino acid sequence was used for the identification of the etbC gene from an RDC1 chromosomal DNA 2,3-DHBD expression library. The etbC gene was successfully cloned, and we report here the determination of its nucleotide sequence. The substrate specificity patterns of cell extract and native nondenaturing polyacrylamide gel electrophoresis analysis identified the coexpression of two 2,3-DHBDs (BphC and EtbC) in RHA1 cells grown on either biphenyl or ethylbenzene. The possible implication of coexpressed BphC extradiol dioxygenases in the strong polychlorinated-biphenyl degradation activity of RHA1 was suggested.     

Molecular evolution of bacterial indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that catalyze the first step in L-Trp catabolism via the kynurenine pathway. In mammals, TDO is mainly expressed in the liver and primarily supplies nicotinamide adenine dinucleotide (NAD⁺). TDO is widely distributed from mammals to bacteria. Active IDO enzymes have been reported only in vertebrates and fungi. In mammals, IDO activity plays a significant role in the immune system while in fungal species, IDO is constitutively expressed and supplies NAD⁺, like mammalian TDO. A search of genomic databases reveals that some bacterial species also have a putative IDO gene. A phylogenetic analysis clustered bacterial IDOs into two groups, group I or group II bacterial IDOs. The catalytic efficiencies of group I bacterial IDOs were very low and they are suspected not to contribute significantly to L-Trp metabolism. The bacterial species bearing the group I bacterial IDO are scattered across a few phyla and no phylogenetically close relationship is observed between them. This suggests that the group I bacterial IDOs might be acquired by horizontal gene transmission that occurred in each lineage independently. In contrast, group II bacterial IDOs showed rather high catalytic efficiency. Particularly, the enzymatic characteristics (K_m, V_max and inhibitor selectivity) of the Gemmatimonas aurantiaca IDO are comparable to those of mammalian IDO1, although comparison of the IDO sequences does not suggest a close evolutionary relationship. In several bacteria, TDO and the kynureninase gene (kynU) are clustered on their chromosome suggesting that these genes could be transcribed in an operon. Interestingly, G. aurantiaca has no TDO, and the IDO is clustered with kynU on its chromosome. Although the G. aurantiaca also has NadA and NadB to synthesize a quinolinic acid (a precursor of NAD⁺) via the aspartate pathway, the high activity of the G. aurantiaca IDO flanking the kynU gene suggests its IDO has a function similar to eukaryotic enzymes.     

Crystal structure of 4-chlorocatechol 1,2-dioxygenase from the chlorophenol-utilizing gram-positive Rhodococcus opacus 1CP

The crystal structure of the 4-chlorocatechol 1,2-dioxygenase from the Gram-positive bacterium Rhodococcus opacus (erythropolis) 1CP, a Fe(III) ion-containing enzyme involved in the aerobic biodegradation of chloroaromatic compounds, has been solved by multiple wavelength anomalous dispersion using the weak anomalous signal of the two catalytic irons (1 Fe/257 amino acids) and refined at a 2.5 A resolution (R(free) 28.7%; R factor 21.4%). The analysis of the structure and its comparison with the structure of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus ADP1 (Ac 1,2-CTD) highlight significant differences between these enzymes. The general topology of the present enzyme comprises two catalytic domains (one for each subunit) related by a noncrystallographic 2-fold axis and separated by a common alpha-helical zipper motif consisting of five N-terminal helices from each subunit; furthermore the C-terminal tail is shortened significantly with respect to the known Ac 1,2-CTD. The presence of two phospholipids binding in a hydrophobic tunnel along the dimer axis is shown here to be a common feature for this class of enzyme. The active site cavity presents several dissimilarities with respect to the known catechol-cleaving enzyme. The catalytic nonheme iron(III) ion is bound to the side chains of Tyr-134, Tyr-169, His-194, and His-196, and a cocrystallized benzoate ion, bound to the metal center, reveals details on a novel mode of binding of bidentate inhibitors and a distinctive hydrogen bond network with the surrounding ligands. Among the amino acid residues expected to interact with substrates, several are different from the corresponding analogs of Ac 1,2-CTD: Asp-52, Ala-53, Gly-76, Phe-78, and Cys-224; in addition, regions of largely conserved amino acid residues in the catalytic cleft show different shapes resulting from several substantial backbone and side chain shifts. The present structure is the first of intradiol dioxygenases that specifically catalyze the cleavage of chlorocatechols, key intermediates in the aerobic catabolism of toxic chloroaromatics.     

Multiple genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase in the gram-positive polychlorinated biphenyl-degrading bacterium Rhodococcus erythropolis TA421, isolated from a termite ecosystem.

Rhodococcus erythropolis TA421 was isolated from a termite ecosystem and is able to degrade a wide range of polychlorinated biphenyl (PCB) congeners. Genetic and biochemical analyses of the PCB catabolic pathway of this organism revealed that there are four different bphC genes (bphC1, bphC2, bphC3, and bphC4) which encode 2,3-dihydroxybiphenyl dioxygenases. As determined by Southern hybridization, none of the bphC genes exhibits homology to any other bphC gene. bphC1, bphC2, and bphC4 encode enzymes that have narrow substrate specificities and cleave the first aromatic ring in the meta position. In contrast, bphC3 encodes a meta cleavage dioxygenase with broad substrate specificity. Asturias et al. have shown that the closely related organism Rhodococcus globerulus P6 contains three different bphC genes (bphC1, bphC2, and bpHC3) which encode meta cleavage dioxygenases. The data suggest that there is a diverse family of bphC genes which encode PCB meta cleavage dioxygenases in members of the genus Rhodococcus.     

Characterization of a novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl 1,2-dioxygenase from a polychlorinated biphenyl- and naphthalene-degrading Bacillus sp. JF8

A novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC_JF8) catalyzing the meta-cleavage of the hydroxylated biphenyl ring was purified from the thermophilic biphenyl and naphthalene degrader, Bacillus sp. JF8, and the gene was cloned. The native and recombinant BphC enzyme was purified to homogeneity. The enzyme has a molecular mass of 125 +/- 10 kDa and was composed of four identical subunits (35 kDa). BphC_JF8 has a temperature optimum of 85 degrees C and a pH optimum of 7.5. It exhibited a half-life of 30 min at 80 degrees C and 81 min at 75 degrees C, making it the most thermostable extradiol dioxygenase studied. Inductively coupled plasma mass spectrometry analysis confirmed the presence of 4.0-4.8 manganese atoms per enzyme molecule. The EPR spectrum of BphC_JF8 exhibited g = 2.02 and g = 4.06 signals having the 6-fold hyperfine splitting characteristic of Mn(II). The enzyme can oxidize a wide range of substrates, and the substrate preference was in the order 2,3-dihydroxybiphenyl > 3-methylcatechol > catechol > 4-methylcatechol > 4-chlorocatechol. The enzyme is resistant to denaturation by various chelators and inhibitors (EDTA, 1,10-phenanthroline, H2O2, 3-chlorocatechol) and did not exhibit substrate inhibition even at 3 mm 2,3-dihydroxybiphenyl. A decrease in Km accompanied an increase in temperature, and the Km value of 0.095 microm for 2,3-dihydroxybiphenyl (at 60 degrees C) is among the lowest reported. The kinetic properties and thermal stability of the native and recombinant enzyme were identical. The primary structure of BphC_JF8 exhibits less than 25% sequence identity to other 2,3-dihydroxybiphenyl 1,2-dioxygenases. The metal ligands and active site residues of extradiol dioxygenases are conserved, although several amino acid residues found exclusively in enzymes that preferentially cleave bicyclic substrates are missing in BphC_JF8. A three-dimensional homology model of BphC_JF8 provided a basis for understanding the substrate specificity, quaternary structure, and stability of the enzyme.     

Molecular breeding of polymerases for resistance to environmental inhibitors

Potent inhibitors limit the use of PCR assays in a wide spectrum of specimens. Here, we describe the engineering of polymerases with a broad resistance to complex environmental inhibitors using molecular breeding of eight different polymerase orthologues from the genus Thermus and directed evolution by CSR in the presence of inhibitors. Selecting for resistance to the inhibitory effects of Neomylodon bone powder, we isolated 2D9, a chimeric polymerase comprising sequence elements derived from DNA polymerases from Thermus aquaticus, Thermus oshimai, Thermus thermophilus and Thermus brockianus. 2D9 displayed a striking resistance to a broad spectrum of complex inhibitors of highly divergent composition including humic acid, bone dust, coprolite, peat extract, clay-rich soil, cave sediment and tar. The selected polymerase promises to have utility in PCR-based applications in a wide range of fields including palaeobiology, archaeology, conservation biology, forensic and historic medicine.     

Purification, characterization, and substrate specificity of two 2,3-dihydroxybiphenyl 1,2-dioxygenase from Rhodococcus sp. R04, showing their distinct stability at various temperature

The genes of two 2,3-dihydroxybiphenyl 1,2-dioxygenases (BphC1 and BphC2) were obtained from the gene library of Rhodococcus sp. R04. The enzymes have been purified to apparent electrophoretic homogeneity from the cell extracts of the recombinant harboring bphC1 and bphC2. Both BphC1 and BphC2 were hexamers, consisting of six subunits of 35 and 33kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The enzymes had similar optimal pH (pH 9.0), but different temperatures for their maximum activity (30 degrees C for BphC1, 80 degrees C for BphC2). In addition, they exhibited distinct stability at various temperatures. The enzymes could cleave a wide range of catechols, with 2,3-dihydroxybiphenyl being the optimum substrate for BphC1 and BphC2. BphC1 was inhibited by 2,3-dihydroxybiphenyl, catechol and 3-chlorocatechol, whereas BphC2 showed strong substrate inhibition for all the given substrates. BphC2 exhibited a half-life of 15min at 80 degrees C and 50min at 70 degrees C, making it the most thermostable extradiol dioxygenase studied in mesophilic bacteria. After disruption of bphC1 and bphC2 genes, R04DeltaC1 (bphC1 mutant) delayed the time of their completely eliminating biphenyl another 15h compared with its parent strain R04, but R04DeltaC2 (bphC2 mutant) lost the ability to grow on biphenyl, suggesting that BphC1 plays an assistant role in the degrading of biphenyl by strain R04, while BphC2 is essential for the growth of strain R04 on biphenyl.     

Crystal structure of 3-chlorocatechol 1,2-dioxygenase key enzyme of a new modified ortho-pathway from the Gram-positive Rhodococcus opacus 1CP grown on 2-chlorophenol

The crystal structure of the 3-chlorocatechol 1,2-dioxygenase from the Gram-positive bacterium Rhodococcus opacus (erythropolis) 1CP, a Fe(III) ion-containing enzyme specialized in the aerobic biodegradation of 3-chloro- and methyl-substituted catechols, has been solved by molecular replacement techniques using the coordinates of 4-chlorocatechol 1,2-dioxygenase from the same organism (PDB code 1S9A) as a starting model and refined at 1.9 A resolution (R(free) 21.9%; R-factor 17.4%). The analysis of the structure and of the kinetic parameters for a series of different substrates, and the comparison with the corresponding data for the 4-chlorocatechol 1,2-dioxygenase isolated from the same bacterial strain, provides evidence of which active site residues are responsible for the observed differences in substrate specificity. Among the amino acid residues expected to interact with substrates, only three are altered Val53(Ala53), Tyr78(Phe78) and Ala221(Cys224) (3-chlorocatechol 1,2-dioxygenase(4-chlorocatechol 1,2-dioxygenase)), clearly identifying the substitutions influencing substrate selectivity in these enzymes. The crystallographic asymmetric unit contains eight subunits (corresponding to four dimers) that show heterogeneity in the conformation of a co-crystallized molecule bound to the catalytic non-heme iron(III) ion resembling a benzohydroxamate moiety, probably a result of the breakdown of recently discovered siderophores synthesized by Gram-positive bacteria. Several different modes of binding benzohydroxamate into the active site induce distinct conformations of the interacting protein ligands Tyr167 and Arg188, illustrating the plasticity of the active site origin of the more promiscuous substrate preferences of the present enzyme.     

Substrate specificity and expression of three 2,3-dihydroxybiphenyl 1,2-dioxygenases from Rhodococcus globerulus strain P6

Rhodococcus globerulus strain P6 contains at least three genes, bphC1, bphC2, and bphC3, coding for 2,3-dihydroxybiphenyl 1,2-dioxygenases; the latter two specify enzymes of the family of one-domain extradiol dioxygenases. In order to assess the importance of these different isoenzymes for the broad catabolic activity of this organism towards the degradation of polychlorinated biphenyls (PCBs), the capacities of recombinant enzymes expressed in Escherichia coli to transform different chlorosubstituted dihydroxybiphenyls formed by the action of R. globerulus P6 biphenyl dioxygenase and biphenyl 2,3-dihydrodiol dehydrogenase were determined. Whereas both BphC2 and BphC3 showed similar activities for 2,3-dihydroxybiphenyl and all monochlorinated 2,3-dihydroxybiphenyls, BphC1 exhibited only weak activity for 2'-chloro-2,3-dihydroxybiphenyl. More highly chlorinated 2'-chlorosubstituted 2,3-dihydroxybiphenyls were also transformed at high rates by BphC2 and BphC3 but not BphC1. In R. globerulus P6, BphC2 was constitutively expressed, BphC1 expression was induced during growth on biphenyl, and BphC3 was not expressed at significant levels under the experimental conditions. Although we cannot rule out the expression of BphC3 under certain environmental conditions, it seems that the contrasting substrate specificities of BphC1 and BphC2 contribute significantly to the versatile PCB-degrading phenotype of R. globerulus P6.