Identification and characterization of genes involved in the downstream degradation pathway of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26

Sphingomonas paucimobilis UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH) as a sole source of carbon and energy. In our previous study, we cloned and characterized genes that are involved in the conversion of gamma-HCH to maleylacetate (MA) via chlorohydroquinone (CHQ) in UT26. In this study, we identified and characterized an MA reductase gene, designated linF, that is essential for the utilization of gamma-HCH in UT26. A gene named linEb, whose deduced product showed significant identity to LinE (53%), was located close to linF. LinE is a novel type of ring cleavage dioxygenase that catalyzes the conversion of CHQ to MA. LinEb expressed in Escherichia coli transformed CHQ and 2,6-dichlorohydroquinone to MA and 2-chloromaleylacetate, respectively. Our previous and present results indicate that UT26 (i) has two gene clusters for degradation of chlorinated aromatic compounds via hydroquinone-type intermediates and (ii) uses at least parts of both clusters for gamma-HCH utilization.     

Metabolism of 2-chloro-4-nitrophenol in a gram negative bacterium, Burkholderia sp. RKJ 800

A 2-chloro-4-nitrophenol (2C4NP) degrading bacterial strain designated as RKJ 800 was isolated from a pesticide contaminated site of India by enrichment method and utilized 2C4NP as sole source of carbon and energy. The stoichiometric amounts of nitrite and chloride ions were detected during the degradation of 2C4NP. On the basis of thin layer chromatography, high performance liquid chromatography and gas chromatography-mass spectrometry, chlorohydroquinone (CHQ) and hydroquinone (HQ) were identified as major metabolites of the degradation pathway of 2C4NP. Manganese dependent HQ dioxygenase activity was observed in the crude extract of 2C4NP induced cells of the strain RKJ 800 that suggested the cleavage of the HQ to γ-hydroxymuconic semialdehyde. On the basis of the 16S rRNA gene sequencing, strain RKJ 800 was identified as a member of genus Burkholderia. Our studies clearly showed that Burkholderia sp. RKJ 800 degraded 2-chloro-4-nitrophenol via hydroquinone pathway. The pathway identified in a gram negative bacterium, Burkholderia sp. strain RKJ 800 was differed from previously reported 2C4NP degradation pathway in another gram-negative Burkholderia sp. SJ98. This is the first report of the formation of CHQ and HQ in the degradation of 2C4NP by any gram-negative bacteria. Laboratory-scale soil microcosm studies showed that strain RKJ 800 is a suitable candidate for bioremediation of 2C4NP contaminated sites.     

Elucidation of the 4-hydroxyacetophenone catabolic pathway in Pseudomonas fluorescens ACB

The catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB is known to proceed through the intermediate formation of hydroquinone. Here, we provide evidence that hydroquinone is further degraded through 4-hydroxymuconic semialdehyde and maleylacetate to beta-ketoadipate. The P. fluorescens ACB genes involved in 4-hydroxyacetophenone utilization were cloned and characterized. Sequence analysis of a 15-kb DNA fragment showed the presence of 14 open reading frames containing a gene cluster (hapCDEFGHIBA) of which at least four encoded enzymes are involved in 4-hydroxyacetophenone degradation: 4-hydroxyacetophenone monooxygenase (hapA), 4-hydroxyphenyl acetate hydrolase (hapB), 4-hydroxymuconic semialdehyde dehydrogenase (hapE), and maleylacetate reductase (hapF). In between hapF and hapB, three genes encoding a putative intradiol dioxygenase (hapG), a protein of the Yci1 family (hapH), and a [2Fe-2S] ferredoxin (hapI) were found. Downstream of the hap genes, five open reading frames are situated encoding three putative regulatory proteins (orf10, orf12, and orf13) and two proteins possibly involved in a membrane efflux pump (orf11 and orf14). Upstream of hapE, two genes (hapC and hapD) were present that showed weak similarity with several iron(II)-dependent extradiol dioxygenases. Based on these findings and additional biochemical evidence, it is proposed that the hapC and hapD gene products are involved in the ring cleavage of hydroquinone.     

Identification of a serine hydrolase which cleaves the alicyclic ring of tetralin

A gene designated thnD, which is required for biodegradation of the organic solvent tetralin by Sphingomonas macrogoltabidus strain TFA, has been identified. Sequence comparison analysis indicated that thnD codes for a carbon-carbon bond serine hydrolase showing highest similarity to hydrolases involved in biodegradation of biphenyl. An insertion mutant defective in ThnD accumulates the ring fission product which results from the extradiol cleavage of the aromatic ring of dihydroxytetralin. The gene product has been purified and characterized. ThnD is an octameric thermostable enzyme with an optimum reaction temperature at 65 degrees C. ThnD efficiently hydrolyzes the ring fission intermediate of the tetralin pathway and also 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the ring fission product of the biphenyl meta-cleavage pathway. However, it is not active towards the equivalent intermediates of meta-cleavage pathways of monoaromatic compounds which have small substituents in C-6. When ThnD hydrolyzes the intermediate in the tetralin pathway, it cleaves a C-C bond comprised within the alicyclic ring of tetralin instead of cleaving a linear C-C bond, as all other known hydrolases of meta-cleavage pathways do. The significance of this activity of ThnD for the requirement of other activities to mineralize tetralin is discussed.     

Pathway for Degradation of 2-Chloro-4-Nitrophenol in <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. SJCon

Degradation of 2-Chloro-4-nitrophenol (2C4NP) was studied by Arthrobacter sp. SJCon, isolated from the soil of a pesticide contaminated site. This strain utilized 2C4NP as sole source of carbon and energy and degraded 2C4NP with stoichiometric release of nitrite and chloride ions. A metabolite was detected during the study of 2C4NP degradation and identified as chlorohydroquinone (CHQ) by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC–MS). Inhibition study using 2,2′-dipyridyl showed that CHQ is a terminal aromatic compound in degradation pathway of 2C4NP. CHQ dioxygenase activity was observed in the crude extract of 2C4NP induced cells of the strain SJCon that suggested the cleavage of the CHQ to maleylacetate (MA). Our study clearly showed that Arthrobacter sp. SJCon degraded 2C4NP via formation of CHQ that further cleaved to MA by CHQ dioxygenase. This mechanism of degradation of 2C4NP differs from previously reported degradation pathways of 2C4NP.     

Ring-cleaving dioxygenases with a cupin fold

Ring-cleaving dioxygenases catalyze key reactions in the aerobic microbial degradation of aromatic compounds. Many pathways converge to catecholic intermediates, which are subject to ortho or meta cleavage by intradiol or extradiol dioxygenases, respectively. However, a number of degradation pathways proceed via noncatecholic hydroxy-substituted aromatic carboxylic acids like gentisate, salicylate, 1-hydroxy-2-naphthoate, or aminohydroxybenzoates. The ring-cleaving dioxygenases active toward these compounds belong to the cupin superfamily, which is characterized by a six-stranded β-barrel fold and conserved amino acid motifs that provide the 3His or 2- or 3His-1Glu ligand environment of a divalent metal ion. Most cupin-type ring cleavage dioxygenases use an Fe(II) center for catalysis, and the proposed mechanism is very similar to that of the canonical (type I) extradiol dioxygenases. The metal ion is presumed to act as an electron conduit for single electron transfer from the metal-bound substrate anion to O(2), resulting in activation of both substrates to radical species. The family of cupin-type dioxygenases also involves quercetinase (flavonol 2,4-dioxygenase), which opens up two C-C bonds of the heterocyclic ring of quercetin, a wide-spread plant flavonol. Remarkably, bacterial quercetinases are capable of using different divalent metal ions for catalysis, suggesting that the redox properties of the metal are relatively unimportant for the catalytic reaction. The major role of the active-site metal ion could be to correctly position the substrate and to stabilize transition states and intermediates rather than to mediate electron transfer. The tentative hypothesis that quercetinase catalysis involves direct electron transfer from metal-bound flavonolate to O(2) is supported by model chemistry.     

Substrate specificity of Sphingobium chlorophenolicum 2,6-dichlorohydroquinone 1,2-dioxygenase

PcpA is an aromatic ring-cleaving dioxygenase that is homologous to the well-characterized Fe(II)-dependent catechol extradiol dioxygenases. This enzyme catalyzes the oxidative cleavage of 2,6-dichlorohydroquinone in the catabolism of pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723. (1)H NMR and steady-state kinetics were used to determine the regiospecificity of ring cleavage and the substrate specificity of the enzyme. PcpA exhibits a high degree of substrate specificity for 2,6-disubstituted hydroquinones, with halogens greatly preferred at those positions. Notably, the k(cat)(app)/K(mA)(app) of 2,6-dichlorohydroquinone is ~40-fold higher than that of 2,6-dimethylhydroquinone. The asymmetric substrate 2-chloro-6-methylhydroquinone yields a mixture of 1,2- and 1,6-cleavage products. These two modes of cleavage have different K(mO(2))(app) values (21 and 260 μM, respectively), consistent with a mechanism in which the substrate binds in two catalytically productive orientations. In contrast, monosubstituted hydroquinones show a limited amount of ring cleavage but rapidly inactivate the enzyme in an O(2)-dependent fashion, suggesting that oxidation of the Fe(II) may be the cause. Potent inhibitors of PcpA include ortho-disubstituted phenols and 3-bromocatechol. 2,6-Dibromophenol is the strongest competitive inhibitor, consistent with PcpA's substrate specificity. Several factors that could yield this specificity for halogen substituents are discussed. Interestingly, 3-bromocatechol also inactivates the enzyme, while 2,6-dihalophenols do not, indicating a requirement for two hydroxyl groups for ring cleavage and for enzyme inactivation. These results provide mechanistic insights into the hydroquinone dioxygenases.     

Cloning and characterization of a chromosome-encoded catechol 2,3-dioxygenase gene from Pseudomonas aeruginosa ZD 4-3

Catechol 2,3-dioxygenase (C23O), one of extradiol-type dioxygenases cleaving the aromatic C-C bond at the meta-position of dihydroxylated aromatic substrates, catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde. Based on curing experiment, PCR identification, and Southern hybridization, the gene responsible for C23O was localized on a 3.5-kb EcoRI/BamHI fragment and cloned from P. aeruginosa ZD 4-3 able to degrade both single and bicyclic compounds via the meta-cleavage pathway. A complete nucleotide sequence analysis of the C23O revealed that it had one ORF, which showed a strong amino acid sequence similarity to the known C23Os of mesophilic gram-negative bacteria. The alignment analysis indicated that distinct difference existed between the C23O in this study and the 2,3-dihydroxybiphenyl dioxygenases cleaving bicyclic aromatic compounds. The heterogenous expression of the pheB gene in Escherichia coli BL21(DE3) demonstrated that this C23O possessed a meta-cleavage activity.     

A Two-Component para-Nitrophenol Monooxygenase Initiates a Novel 2-Chloro-4-Nitrophenol Catabolism Pathway in Rhodococcus imtechensis RKJ300

Rhodococcus imtechensis RKJ300 (DSM 45091) grows on 2-chloro-4-nitrophenol (2C4NP) and para-nitrophenol (PNP) as the sole carbon and nitrogen sources. In this study, by genetic and biochemical analyses, a novel 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with hydroxyquinol (hydroxy-1,4-hydroquinone or 1,2,4-benzenetriol [BT]) as the ring cleavage substrate. Real-time quantitative PCR analysis indicated that the pnp cluster located in three operons is likely involved in the catabolism of both 2C4NP and PNP. The oxygenase component (PnpA1) and reductase component (PnpA2) of the two-component PNP monooxygenase were expressed and purified to homogeneity, respectively. The identification of chlorohydroquinone (CHQ) and BT during 2C4NP degradation catalyzed by PnpA1A2 indicated that PnpA1A2 catalyzes the sequential denitration and dechlorination of 2C4NP to BT and catalyzes the conversion of PNP to BT. Genetic analyses revealed that pnpA1 plays an essential role in both 2C4NP and PNP degradations by gene knockout and complementation. In addition to catalyzing the oxidation of CHQ to BT, PnpA1A2 was also found to be able to catalyze the hydroxylation of hydroquinone (HQ) to BT, revealing the probable fate of HQ that remains unclear in PNP catabolism by Gram-positive bacteria. This study fills a gap in our knowledge of the 2C4NP degradation mechanism in Gram-positive bacteria and also enhances our understanding of the genetic and biochemical diversity of 2C4NP catabolism.     

Novel Pathway for Conversion of Chlorohydroxyquinol to Maleylacetate in Burkholderia cepacia AC1100

Burkholderia cepacia AC1100 metabolizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) via formation of 5-chlorohydroxyquinol (5-CHQ), hydroxyquinol (HQ), maleylacetate, and β-oxoadipate. The step(s) leading to the dechlorination of 5-CHQ to HQ has remained unidentified. We demonstrate that a dechlorinating enzyme, TftG, catalyzes the conversion of 5-CHQ to hydroxybenzoquinone, which is then reduced to HQ by a hydroxybenzoquinone reductase (HBQ reductase). HQ is subsequently converted to maleylacetate by hydroxyquinol 1,2-dioxygenase (HQDO). All three enzymes were purified. We demonstrate specific product formation by colorimetric assay and mass spectrometry when 5-CHQ is treated successively with the three enzymes: TftG, TftG plus HBQ reductase, and TftG plus HBQ reductase plus HQDO. This study delineates the complete enzymatic pathway for the degradation of 5-CHQ to maleylacetate.     

Purification and characterization of a 1,2,4-trihydroxybenzene 1,2-dioxygenase from the basidiomycete Phanerochaete chrysosporium.

1,2,4-Trihydroxybenzene (THB) is an intermediate in the Phanerochaete chrysosporium degradation of vanillate and aromatic pollutants. A P. chrysosporium intracellular enzyme able to oxidatively cleave the aromatic ring of THB was purified by ammonium sulfate precipitation, hydrophobic and ion-exchange chromatographies, and native gel electrophoresis. The native protein has a molecular mass of 90 kDa and a subunit mass of 45 kDa. The enzyme catalyzes an intradiol cleavage of the substrate aromatic ring to produce maleylacetate. 18O2 incorporation studies demonstrate that molecular oxygen is a cosubstrate in the reaction. The enzyme exhibits high substrate specificity for THB; however, catechol cleavage occurs at approximately 20% of the optimal rate. THB dioxygenase catalyzes a key step in the degradation pathway of vanillate, an intermediate in lignin degradation. Maleylacetate, the product of THB cleavage, is reduced to beta-ketoadipate by an NADPH-requiring enzyme present in partially purified extracts.     

The ins and outs of ring-cleaving dioxygenases

Ring-cleaving dioxygenases catalyze the oxygenolytic fission of catecholic compounds, a critical step in the aerobic degradation of aromatic compounds by bacteria. Two classes of these enzymes have been identified, based on the mode of ring cleavage: intradiol dioxygenases utilize non-heme Fe(III) to cleave the aromatic nucleus ortho to the hydroxyl substituents; and extradiol dioxygenases utilize non-heme Fe(II) or other divalent metal ions to cleave the aromatic nucleus meta to the hydroxyl substituents. Recent genomic, structural, spectroscopic, and kinetic studies have increased our understanding of the distribution, evolution, and mechanisms of these enzymes. Overall, extradiol dioxygenases appear to be more versatile than their intradiol counterparts. Thus, the former cleave a wider variety of substrates, have evolved on a larger number of structural scaffolds, and occur in a wider variety of pathways, including biosynthetic pathways and pathways that degrade non-aromatic compounds. The catalytic mechanisms of the two enzymes proceed via similar iron-alkylperoxo intermediates. The ability of extradiol enzymes to act on a variety of non-catecholic compounds is consistent with proposed differences in the breakdown of this iron-alkylperoxo intermediate in the two enzymes, involving alkenyl migration in extradiol enzymes and acyl migration in intradiol enzymes. Nevertheless, despite recent advances in our understanding of these fascinating enzymes, the major determinant of the mode of ring cleavage remains unknown.     

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.     

Evidence of two pathways for the metabolism of phenol by Aspergillus fumigatus

Aspergillus fumigatus (ATCC 28282), a thermotolerant fungus, has been shown to be capable of growth on phenol as the sole carbon and energy source. During growth of the organism on phenol, catechol and hydroquinone accumulated transiently in the medium; cells grown on phenol oxidised these compounds without a lag period. Two different routes operating simultaneously, leading to different ring-fission substrates, are proposed for the metabolism of phenol. In one route, phenol undergoes ortho-hydroxylation to give catechol, which is then cleaved by an intradiol mechanism leading to 3-oxoadipate. In the other route, phenol is hydroxylated in the para-position to produce hydroquinone, which is then converted into 1,2,4-trihydroxybenzene for ring fission by ortho-cleavage to give maleylacetate. Cell-free extracts of phenol-grown mycelia were found to contain enzymic activities for the proposed steps. Two ring-fission dioxygenases, one active towards 1,2,4-trihydroxybenzene, but not catechol, and one active towards both ring-fission substrates, were separated by FPLC. Succinate-grown mycelia did not oxidise any of the intermediates until a clear lag period had elapsed and did not contain any of the enzymic activities for phenol metabolism.     

Flavin-containing monooxygenases from <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> responsible for fungal metabolism of phenolic compounds

We investigated the cellular responses of the white-rot basidiomycete Phanerochaete chrysosporium against vanillin. Based upon a proteomic survey, it was demonstrated that two flavin-containing monooxygenases (PcFMO1 and PcFMO2) are translationally up-regulated in response to exogenous addition of vanillin. To elucidate their catalytic functions, we cloned cDNAs and heterologously expressed them in Escherichia coli. The recombinant PcFMO1 showed catalytic activities against monocyclic phenols such as phenol, hydroquinone, and 4-chlorophenol. In addition, the product from hydroquinone was identified as 1,2,4-trihydroxybenzene, an important intermediate in a metabolic pathway of aromatic compounds in which the aromatic ring of 1,2,4-trihydroxybenzene can be further cleaved by fungal dioxygenases for mineralization. Thus, the ortho-cleavage pathway of phenolic compounds would presumably be associated with PcFMO1.     

The Ins and Outs of Ring-Cleaving Dioxygenases

ABSTRACT

Ring-cleaving dioxygenases catalyze the oxygenolytic fission of catecholic compounds, a critical step in the aerobic degradation of aromatic compounds by bacteria. Two classes of these enzymes have been identified, based on the mode of ring cleavage: intradiol dioxygenases utilize non-heme Fe(III) to cleave the aromatic nucleus ortho to the hydroxyl substituents; and extradiol dioxygenases utilize non-heme Fe(II) or other divalent metal ions to cleave the aromatic nucleus meta to the hydroxyl substituents. Recent genomic, structural, spectroscopic, and kinetic studies have increased our understanding of the distribution, evolution, and mechanisms of these enzymes. Overall, extradiol dioxygenases appear to be more versatile than their intradiol counterparts. Thus, the former cleave a wider variety of substrates, have evolved on a larger number of structural scaffolds, and occur in a wider variety of pathways, including biosynthetic pathways and pathways that degrade non-aromatic compounds. The catalytic mechanisms of the two enzymes proceed via similar iron-alkylperoxo intermediates. The ability of extradiol enzymes to act on a variety of non-catecholic compounds is consistent with proposed differences in the breakdown of this iron-alkylperoxo intermediate in the two enzymes, involving alkenyl migration in extradiol enzymes and acyl migration in intradiol enzymes. Nevertheless, despite recent advances in our understanding of these fascinating enzymes, the major determinant of the mode of ring cleavage remains unknown.     

Cloning and expression of genes encoding meta-cleavage enzymes from 4,6-dimethyldibenzothiophene-degrading Sphingomonas strain TZS-7

Sphingomonas strain TZS-7 was reported as the first strain to have the ability to degrade 4,6-dimethyldibenzothiophene (4,6-dmDBT) by the ring-destructive pathway. Two genes for meta-cleavage dioxygenases were cloned from strain TZS-7. Expression of each gene showed that one enzyme was specific for 2,3-dihydroxybiphenyl while another was more specific for catechol. The genes for the two enzymes were named dmdC and catA. The analysis of deduced amino acid sequences indicates that CatA falls into the class of meta-cleavage dioxygenases acting on dihydroxylated monocyclic compounds and DmdC falls into the class of meta-cleavage dioxygenases acting on dihydroxylated polycyclic compounds.     

Structural characterization of 2,6‐dichloro‐p‐hydroquinone 1,2‐dioxygenase (PcpA) from Sphingobium chlorophenolicum,a new type of aromatic ring‐cleavage enzyme

PcpA (2,6‐dichloro‐p‐hydroquinone 1,2‐dioxygenase) from Sphingobium chlorophenolicum, a non‐haem Fe(II) dioxygenase capable of cleaving the aromatic ring of p‐hydroquinone and its substituted variants, is a member of the recently discovered p‐hydroquinone 1,2‐dioxygenases. Here we report the 2.6 Å structure of PcpA, which consists of four βαβββ motifs, a hallmark of the vicinal oxygen chelate superfamily. The secondary co‐ordination sphere of the Fe(II) centre forms an extensive hydrogen‐bonding network with three solvent exposed residues, linking the catalytic Fe(II) to solvent. A tight hydrophobic pocket provides p‐hydroquinones access to the Fe(II) centre. The p‐hydroxyl group is essential for the substrate‐binding, thus phenols and catechols, lacking a p‐hydroxyl group, do not bind to PcpA. Site‐directed mutagenesis and kinetic analysis confirm the critical catalytic role played by the highly conserved His10, Thr13, His226 and Arg259. Based on these results, we propose a general reaction mechanism for p‐hydroquinone 1,2‐dioxygenases.     

Three different 2,3-dihydroxybiphenyl-1,2-dioxygenase genes in the gram-positive polychlorobiphenyl-degrading bacterium Rhodococcus globerulus P6.

Rhodococcus globerulus P6 (previously designated Acinetobacter sp. strain P6, Arthrobacter sp. strain M5, and Corynebacterium sp. strain MB1) is able to degrade a wide range of polychlorinated biphenyl (PCB) congeners. The genetic and biochemical analyses of the PCB catabolic pathway reported here have revealed the existence of a PCB gene cluster--bphBC1D--and two further bphC genes--bphC2 and bphC3--that encode three narrow-substrate-specificity enzymes (2,3-dihydroxybiphenyl dioxygenases) that meta cleave the first aromatic ring. None of the bphC genes show by hybridization homology to each other or to bphC genes in other bacteria, and the three bphC gene products have different kinetic parameters and sensitivities to inactivation by 3-chlorocatechol. This suggests that there exists a wide diversity in PCB meta cleavage enzymes.