Discovery of a eugenol oxidase from Rhodococcus sp. strain RHA1

A gene encoding a eugenol oxidase was identified in the genome from Rhodococcus sp. strain RHA1. The bacterial FAD-containing oxidase shares 45% amino acid sequence identity with vanillyl alcohol oxidase from the fungus Penicillium simplicissimum. Eugenol oxidase could be expressed at high levels in Escherichia coli, which allowed purification of 160 mg of eugenol oxidase from 1 L of culture. Gel permeation experiments and macromolecular MS revealed that the enzyme forms homodimers. Eugenol oxidase is partly expressed in the apo form, but can be fully flavinylated by the addition of FAD. Cofactor incorporation involves the formation of a covalent protein-FAD linkage, which is formed autocatalytically. Modeling using the vanillyl alcohol oxidase structure indicates that the FAD cofactor is tethered to His390 in eugenol oxidase. The model also provides a structural explanation for the observation that eugenol oxidase is dimeric whereas vanillyl alcohol oxidase is octameric. The bacterial oxidase efficiently oxidizes eugenol into coniferyl alcohol (KM=1.0 microM, kcat=3.1 s-1). Vanillyl alcohol and 5-indanol are also readily accepted as substrates, whereas other phenolic compounds (vanillylamine, 4-ethylguaiacol) are converted with relatively poor catalytic efficiencies. The catalytic efficiencies with the identified substrates are strikingly different when compared with vanillyl alcohol oxidase. The ability to efficiently convert eugenol may facilitate biotechnological valorization of this natural aromatic compound.     

Catabolic potential of multiple PCB transformation systems in Rhodococcus sp. strain RHA1

Summary Polychlorinated biphenyl (PCB) transformation activity of a strong PCB degrader, Rhodococcus sp. strain RHA1, was examined in different concentrations of PCBs. A extremely strong PCB transformation activity was observed on 30 g PCB/ml. At 50 and 100 g/ml, transformation activities were diminished. In the case of bphA insertion mutant, RDA1, transformation activity in the presence of ethylbezene was poor even at 30 g/ml. This indicated that the bphA dependent system would play a major role in PCB transformation by RHA1. Greater transformation activity of RHA1 was observed in the presence of ethylbenzene than in the presence of biphenyl.     

Growth inhibition of Rhodococcus sp. strain RHA1 in the course of PCB transformation

Summary Growth of a PCB degrader Rhodococcus sp. RHA1 on biphenyl and ethylbenzene was inhibited by 100 g/ml PCB 48. A PCB tolerant derivative of RHA1 designated RCD1 was deficient in growth on biphenyl. Southern hybridization experiments suggested that RCD1 has the bphDE gene deletion in a 390-kb linear plasmid of RHA1. The bphD gene complementation restored growth deficiency on biphenyl and growth inhibition on ethylbenzene by PCB 48, indicating that PCB metabolites are the cause of growth inhibition.     

Functional characterization of a catabolic plasmid from polychlorinated- biphenyl-degrading Rhodococcus sp. strain RHA1

Rhodococcus sp. strain RHA1, a potent polychlorinated-biphenyl (PCB)-degrading strain, contains three linear plasmids ranging in size from 330 to 1,100 kb. As part of a genome sequencing project, we report here the complete sequence and characterization of the smallest and least-well-characterized of the RHA1 plasmids, pRHL3. The plasmid is an actinomycete invertron, containing large terminal inverted repeats with a tightly associated protein and a predicted open reading frame (ORF) that is similar to that of a mycobacterial rep gene. The pRHL3 plasmid has 300 putative genes, almost 21% of which are predicted to have a catabolic function. Most of these are organized into three clusters. One of the catabolic clusters was predicted to include limonene degradation genes. Consistent with this prediction, RHA1 grew on limonene, carveol, or carvone as the sole carbon source. The plasmid carries three cytochrome P450-encoding (CYP) genes, a finding consistent with the high number of CYP genes found in other actinomycetes. Two of the CYP genes appear to belong to novel families; the third belongs to CYP family 116 but appears to belong to a novel class based on the predicted domain structure of its reductase. Analyses indicate that pRHL3 also contains four putative "genomic islands" (likely to have been acquired by horizontal transfer), insertion sequence elements, 19 transposase genes, and a duplication that spans two ORFs. One of the genomic islands appears to encode resistance to heavy metals. The plasmid does not appear to contain any housekeeping genes. However, each of the three catabolic clusters contains related genes that appear to be involved in glucose metabolism.     

Catabolism of 1,3-dinitrobenzene by Rhodococcus sp. QT-1

The 1,3-dinitrobenzene-degrading Rhodococcus strain QT-1 was isolated under nitrogen limiting conditions from contaminated soil samples. Experimental data indicate that 1,3-dinitrobenzene is metabolized via 4-nitrocatechol. Both compounds were oxidized by resting cells and nitro groups were completely eliminated as nitrite. Strain QT-1 utilizes both 1,3-dinitrobenzene and 4-nitrocatechol as source of nitrogen in the absence as well as in the presence of high amounts of ammonia. Growth on 4-nitrocatechol does not induce the enzyme(s) for the initial oxidation of 1,3-dinitrobenzene.Abbreviations TNT 2,4,6-trinitrotoluene - 1,3DNB 1,3-dinitrobenzene - 4NC 4-nitrocatechol - 3NA 3-nitroaniline - NB nutrient broth; t_d doubling time - OD₅₄₆ optical density at 546 nm     

Crystallization of the terminal oxygenase component of biphenyl dioxygenase derived from Rhodococcus sp. strain RHA1

The terminal oxygenase component of the biphenyl dioxygenase (BphA1A2 complex) was over-expressed with a novel over expression system in recombinant Rhodococcus strain and purified. The purified enzyme has been crystallized by the hanging drop vapor diffusion method and subjected to X-ray diffraction analysis. The crystals belong to the tetragonal system in the space group P4(1)2(1)2 or P4(3)2(1)2 and diffract to better than 2.2A resolution.     

Crystal structure of the terminal oxygenase component of biphenyl dioxygenase derived from Rhodococcus sp. strain RHA1

Biphenyl dioxygenase is the enzyme that catalyzes the stereospecific dioxygenation of the aromatic ring. This enzyme has attracted the attention of researchers due to its ability to oxidize polychlorinated biphenyls, which is one of the serious environmental contaminants. We determined the crystal structure of the terminal oxygenase component of the biphenyl dioxygenase (BphA1A2) derived from Rhodococcus strain sp. RHA1 in substrate-free and complex forms. These crystal structures revealed that the substrate-binding pocket makes significant conformational changes upon substrate binding to accommodate the substrate into the pocket. Our analysis of the crystal structures suggested that the residues in the substrate-binding pocket can be classified into three groups, which, respectively, seem to be responsible for the catalytic reaction, the orientation/conformation of the substrate, and the conformational changes of the substrate-binding pocket. The cooperative actions of residues in the three groups seem to determine the substrate specificity of the enzyme.     

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.     

Cloning and expression of the benzoate dioxygenase genes from Rhodococcus sp. strain 19070

The bopXYZ genes from the gram-positive bacterium Rhodococcus sp. strain 19070 encode a broad-substrate-specific benzoate dioxygenase. Expression of the BopXY terminal oxygenase enabled Escherichia coli to convert benzoate or anthranilate (2-aminobenzoate) to a nonaromatic cis-diol or catechol, respectively. This expression system also rapidly transformed m-toluate (3-methylbenzoate) to an unidentified product. In contrast, 2-chlorobenzoate was not a good substrate. The BopXYZ dioxygenase was homologous to the chromosomally encoded benzoate dioxygenase (BenABC) and the plasmid-encoded toluate dioxygenase (XylXYZ) of gram-negative acinetobacters and pseudomonads. Pulsed-field gel electrophoresis failed to identify any plasmid in Rhodococcus sp. strain 19070. Catechol 1,2- and 2,3-dioxygenase activity indicated that strain 19070 possesses both meta- and ortho-cleavage degradative pathways, which are associated in pseudomonads with the xyl and ben genes, respectively. Open reading frames downstream of bopXYZ, designated bopL and bopK, resembled genes encoding cis-diol dehydrogenases and benzoate transporters, respectively. The bop genes were in the same order as the chromosomal ben genes of P. putida PRS2000. The deduced sequences of BopXY were 50 to 60% identical to the corresponding proteins of benzoate and toluate dioxygenases. The reductase components of these latter dioxygenases, BenC and XylZ, are 201 residues shorter than the deduced BopZ sequence. As predicted from the sequence, expression of BopZ in E. coli yielded an approximately 60-kDa protein whose presence corresponded to increased cytochrome c reductase activity. While the N-terminal region of BopZ was approximately 50% identical in sequence to the entire BenC or XylZ reductases, the C terminus was unlike other known protein sequences.     

Characterization of the 450-kb linear plasmid in a polychlorinated biphenyl degrader, Rhodococcus sp. strain RHA1

A strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, has diverse biphenyl/PCB degradative genes and harbors huge linear plasmids, including pRHL1 (1,100 kb), pRHL2 (450 kb), and pRHL3 (330 kb). The diverse degradative genes are distributed mainly on the pRHL1 and pRHL2 plasmids. In this study, the structural and functional characteristics of pRHL2 were determined. We constructed a physical map of pRHL2, and the degradative enzyme genes, including bphB2, etbD2, etbC, bphDEF, bphC2, and bphC4, were localized in three regions. Conjugal transfer of pRHL2 between RHA1 mutant derivatives was observed at a frequency of 7.5 x 10(-5) transconjugant per recipient. These results suggested that the linear plasmid is a possible determinant of propagation of the diverse degradative genes in rhodococci. The termini of pRHL2 were cloned and sequenced. The left and right termini of pRHL2 had 3-bp perfect terminal inverted repeats and were not as similar to each other (64% identity) as the known actinomycete linear replicons are. Southern hybridization analysis with pRHL2 terminal probes suggested that the right terminus of pRHL2 is similar to pRHL1 and pRHL3 termini. Retardation of both terminal fragments in the gel shift assay indicated that each terminus of pRHL2 is linked to a protein. We suggest that pRHL2 has invertron termini, as has been reported previously for Streptomyces linear replicons.     

A Novel Transformation of Polychlorinated Biphenyls by Rhodococcus sp. Strain RHA1

We have characterized a biphenyl degrader, Rhodococcus sp. strain RHA1. Biphenyl-grown cells of strain RHA1 efficiently transformed 45 components in the 62 major peaks of a polychlorinated biphenyl (PCB) mixture of Kanechlors 200, 300, 400, and 500 within 3 days, which includes mono- to octachlorobiphenyls. Among the intermediate metabolites of PCB transformation, di- and trichlorobenzoic acids were identified. The gradual decrease of these chlorobenzoic acids during incubation indicated that these chlorobenzoic acids would also be degraded by this strain. The effect of the position of chlorine substitution was determined by using PCB mixtures that have chlorine substitutions mainly at either the ortho or the meta position. This strain transformed both types of congeners, and strong PCB transformation activity of RHA1 was indicated. RHA1 accumulated 4-chlorobenzoic acid temporally during the transformation of 4-chlorobiphenyl. The release of most chloride in the course of 2,2(prm1)-dichlorobiphenyl degradation was observed. These results suggested that RHA1 would break down at least some PCB congeners into smaller molecules to a considerable extent.