Determination of the active site of Sphingobium chlorophenolicum 2,6-dichlorohydroquinone dioxygenase (PcpA)

2,6-Dichlorohydroquinone 1,2-dioxygenase (PcpA) from Sphingobium chlorophenolicum ATCC 39723 is a member of a class of Fe(II)-containing hydroquinone dioxygenases that is involved in the mineralization of the pollutant pentachlorophenol. This enzyme has not been extensively characterized, despite its interesting ring-cleaving activity and use of Fe(II), which are reminiscent of the well-known extradiol catechol dioxygenases. On the basis of limited sequence homology to the extradiol catechol dioxygenases, the residues ligating the Fe(II) center were originally proposed to be H159, H227, and E276 (Xu et al. in Biochemistry 38:7659–7669, 1999). However, PcpA has higher sequence homology to a newly reported, crystallographically characterized zinc metalloenzyme that has a similar predicted fold. We generated a homology model of the structure of PcpA based upon the structure of this zinc metalloenzyme. The homology model predicts that the tertiary structure of PcpA differs significantly from that of the extradiol dioxygenases, and that the residues ligating the Fe(II) are H11, H227, and E276. This structural model was tested by mutating each of H11, H159, H227, and E276 to alanine. An additional residue that is predicted to lie near the active site and is conserved among PcpA, its closest homologues, and the extradiol dioxygenases, Y266, was mutated to phenylalanine. Of these mutants, only H159A retained significant activity, thus confirming the active-site location predicted by the homology-based structural model. The model provides an important basis for understanding the origin of the unique function of PcpA.     

Subcellular localization of pentachlorophenol 4-monooxygenase in Sphingobium chlorophenolicum ATCC 39723

We have studied the subcellular localization of pentachlorophenol 4-monooxygenase (PCP4MO) in Sphingobium chlorophenolicum ATCC 39723 during induction by pentachlorophenol (PCP). Using a monoclonal antibody CL6 specific to the native and recombinant PCP4MO, the enzyme was primarily found soluble as determined by immunoblot and ELISA analyses of cellular fractions. However, the enzyme was observed both in the soluble and membrane-bound forms during induction for 2-4 h, suggesting its translocation out from the cytoplasm. Electron microscopy confirmed that PCP4MO was predominantly present in the cytoplasm at 1 h, whereas at 4 h significant amount was detected also in the membrane and periplasm. After 6 h, the majority of PCP4MO was in the periplasm and only small amount was bound to the inner membrane or present in the cytoplasm. The results indicate that after biosynthesis PCP4MO in S. chlorophenolicum is exported via the inner membrane to the final location in the periplasm.     

Genome shuffling improves degradation of the anthropogenic pesticide pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723

Pentachlorophenol (PCP), a highly toxic anthropogenic pesticide, can be mineralized by Sphingobium chlorophenolicum, a gram-negative bacterium isolated from PCP-contaminated soil. However, degradation of PCP is slow and S. chlorophenolicum cannot tolerate high levels of PCP. We have used genome shuffling to improve the degradation of PCP by S. chlorophenolicum. We have obtained several strains that degrade PCP faster and tolerate higher levels of PCP than the wild-type strain. Several strains obtained after the third round of shuffling can grow on one-quarter-strength tryptic soy broth plates containing 6 to 8 mM PCP, while the original strain cannot grow in the presence of PCP at concentrations higher than 0.6 mM. Some of the mutants are able to completely degrade 3 mM PCP in one-quarter-strength tryptic soy broth, whereas no degradation can be achieved by the wild-type strain. Analysis of several improved strains suggests that the improved phenotypes are due to various combinations of mutations leading to an enhanced growth rate, constitutive expression of the PCP degradation genes, and enhanced resistance to the toxicity of PCP and its metabolites.     

Identification and characterization of hydroxyquinone hydratase activities from Sphingobium chlorophenolicum ATCC 39723

Hydroxyquinol, a common metabolite of aromatic compounds, is readily auto-oxidized to hydroxyquinone. Enzymatic activities that metabolized hydroxyquinone were observed from the cell extracts of Sphingobium chlorophenolicum ATCC 39723. An enzyme capable of transforming hydroxyquinone was partially purified, and its activities were characterized. The end product was confirmed to be 2,5-dihydroxyquinone by comparing UV/Vis absorption spectra, electrospray mass spectra, and gas chromatography-mass spectra of the end product and the authentic compound. We have proposed that the enzyme adds a H₂O molecule to hydroxyquinone to produce 2,5-dihydroxycyclohex-2-ene-1, 4-dione, which spontaneously rearranges to 1, 2,4,5-tetrahydroxybenzene. The latter is auto-oxidized by O₂ to 2,5-dihydroxyquinone. The proposed pathway was supported by the overall reaction stoichiometry. Thus, the transformation of hydroxyquinol to 2,5-dihydroxyquinone involves two auto-oxidation of quinols and one enzymatic reaction catalyzed by a hydratase. The specific enzymatic step did not require O₂, further supporting the assignment as a hydratase. To our knowledge, this is the first identification of a quinone hydratase, enhancing the knowledge on microbial metabolism of hydroxyquinone and possibly leading to the development of enzymatic method for the production of 2,5-dihydroxyquinone, a widely used chemical in various industrial applications.     

Substrate binding site of naphthalene 1,2-dioxygenase: functional implications of indole binding

The three-dimensional structure of the aromatic hydroxylating enzyme naphthalene dioxygenase (NDO) from Pseudomonas sp. NCIB 9816-4 was recently determined. The refinement of the structure together with cyclic averaging showed that in the active site of the enzyme there is electron density for a flat aromatic compound. This compound appears to be an indole adduct, which in Escherichia coli is derived from tryptophan present in the rich culture medium. An indole-dioxygen adduct has been built which fits the electron density convincingly. Support for this interpretation was obtained from crystals of the enzyme purified from cells grown in the absence of tryptophan which had an empty substrate pocket. These types of crystals were soaked in indole solutions and the position of indole in this complex was similar to the corresponding part in the modelled indole-oxygen adduct. This suggests that a peroxide bound to iron end-on attacks the substrate and forms this intermediate. The substrate position has implications for the substrate specificity of the enzyme. Docking studies with indole, naphthalene and biphenyl inside the substrate pocket of NDO suggest the presence of subpockets where the one close to the active site iron is reserved for the binding of the aromatic ring which is hydroxylated upon catalysis. The plausible location for the binding of dioxygen is between this pocket and the catalytic iron. This is in accordance with the enantiospecificity of the products.     

Conversion of Sphingobium chlorophenolicum ATCC 39723 to a Hexachlorobenzene Degrader by Metabolic Engineering

The gene cassette (camA⁺ camB⁺ camC) encoding a cytochrome P-450_cam variant was integrated into the nonessential gene pcpM of the pentachlorophenol degrader Sphingobium chlorophenolicum ATCC 39723 by homologous recombination. The recombinant strain could degrade hexachlorobenzene at a rate of 0.67 nmol · mg (dry weight)⁻¹ · h⁻¹, and intermediate pentachlorophenol was also identified.     

Conversion of Sphingobium chlorophenolicum ATCC 39723 to a hexachlorobenzene degrader by metabolic engineering

The gene cassette (camA+ camB+ camC) encoding a cytochrome P-450cam variant was integrated into the nonessential gene pcpM of the pentachlorophenol degrader Sphingobium chlorophenolicum ATCC 39723 by homologous recombination. The recombinant strain could degrade hexachlorobenzene at a rate of 0.67 nmol.mg (dry weight)-1.h-1, and intermediate pentachlorophenol was also identified.     

Sphingobium chlorophenolicum dichlorohydroquinone dioxygenase (PcpA) is alkaline resistant and thermally stable

Dichlorohydroquinone dioxygenase (PcpA) is the ring-cleavage enzyme in the PCP biodegradation pathway in Sphingobium chlorophenolicum strain ATCC 39723. PcpA dehalogenates and oxidizes 2,6-dichlorohydroquinone to form 2-chloromaleylacetate, which is subsequently converted to succinyl coenzyme A and acetyl coenzyme A via 3-oxoadipate. Previous studies have shown that PcpA is highly substrate-specific and only uses 2,6-dichlorohydroquinone as its substrate. In the current study, we overexpressed and purified recombinant PcpA and showed that PcpA was highly alkaline resistant and thermally stable. PcpA exhibited two activity peaks at pH 7.0 and 10.0, respectively. The apparent k(cat) and K(m) were measured as 0.19 ± 0.01 s(-1) and 0.24 ± 0.08 mM, respectively at pH 7.0, and 0.17 ± 0.01 s(-1) and 0.77 ± 0.29 mM, respectively at pH 10.0. Electron paramagnetic resonance studies showed rapid oxidation of Fe(II) to Fe(III) in PcpA and the formation of a stable radical intermediate during the enzyme catalysis. The stable radical was predicted to be an epoxide type dichloro radical with the unpaired electron density localized on C3.     

Genome Shuffling Improves Degradation of the Anthropogenic Pesticide Pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723

Pentachlorophenol (PCP), a highly toxic anthropogenic pesticide, can be mineralized by Sphingobium chlorophenolicum, a gram-negative bacterium isolated from PCP-contaminated soil. However, degradation of PCP is slow and S. chlorophenolicum cannot tolerate high levels of PCP. We have used genome shuffling to improve the degradation of PCP by S. chlorophenolicum. We have obtained several strains that degrade PCP faster and tolerate higher levels of PCP than the wild-type strain. Several strains obtained after the third round of shuffling can grow on one-quarter-strength tryptic soy broth plates containing 6 to 8 mM PCP, while the original strain cannot grow in the presence of PCP at concentrations higher than 0.6 mM. Some of the mutants are able to completely degrade 3 mM PCP in one-quarter-strength tryptic soy broth, whereas no degradation can be achieved by the wild-type strain. Analysis of several improved strains suggests that the improved phenotypes are due to various combinations of mutations leading to an enhanced growth rate, constitutive expression of the PCP degradation genes, and enhanced resistance to the toxicity of PCP and its metabolites.     

Pentachlorophenol hydroxylase, a poorly functioning enzyme required for degradation of pentachlorophenol by Sphingobium chlorophenolicum

Several strains of Sphingobium chlorophenolicum have been isolated from soil that was heavily contaminated with pentachlorophenol (PCP), a toxic pesticide introduced in the 1930s. S. chlorophenolicum appears to have assembled a poorly functioning pathway for degradation of PCP by patching enzymes recruited via two independent horizontal gene transfer events into an existing metabolic pathway. Flux through the pathway is limited by PCP hydroxylase. PCP hydroxylase is a dimeric protein that belongs to the family of flavin-dependent phenol hydroxylases. In the presence of NADPH, PCP hydroxylase converts PCP to tetrachlorobenzoquinone (TCBQ). The k(cat) for PCP (0.024 s(-1)) is very low, suggesting that the enzyme is not well evolved for turnover of this substrate. Structure-activity studies reveal that substrate binding and activity are enhanced by a low pK(a) for the phenolic proton, increased hydrophobicity, and the presence of a substituent ortho to the hydroxyl group of the phenol. PCP hydroxylase exhibits substantial uncoupling; the C4a-hydroxyflavin intermediate, instead of hydroxylating the substrate, can decompose to produce H(2)O(2) in a futile cycle that consumes NADPH. The extent of uncoupling varies from 0 to 100% with different substrates. The extent of uncoupling is increased by the presence of bulky substituents at position 3, 4, or 5 and decreased by the presence of a chlorine in the ortho position. The effectiveness of PCP hydroxylase is additionally hindered by its promiscuous activity with tetrachlorohydroquinone (TCHQ), a downstream metabolite in the degradation pathway. The conversion of TCHQ to TCBQ reverses flux through the pathway. Substantial uncoupling also occurs during the reaction with TCHQ.     

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     

Maintenance role of a glutathionyl-hydroquinone lyase (PcpF) in pentachlorophenol degradation by Sphingobium chlorophenolicum ATCC 39723

Pentachlorophenol (PCP) is a toxic pollutant. Its biodegradation has been extensively studied in Sphingobium chlorophenolicum ATCC 39723. All enzymes required to convert PCP to a common metabolic intermediate before entering the tricarboxylic acid cycle have been characterized. One of the enzymes is tetrachloro-p-hydroquinone (TeCH) reductive dehalogenase (PcpC), which is a glutathione (GSH) S-transferase (GST). PcpC catalyzes the GSH-dependent conversion of TeCH to trichloro-p-hydroquinone (TriCH) and then to dichloro-p-hydroquinone (DiCH) in the PCP degradation pathway. PcpC is susceptible to oxidative damage, and the damaged PcpC produces glutathionyl (GS) conjugates, GS-TriCH and GS-DiCH, which cannot be further metabolized by PcpC. The fate and effect of GS-hydroquinone conjugates were unknown. A putative GST gene (pcpF) is located next to pcpC on the bacterial chromosome. The pcpF gene was cloned, and the recombinant PcpF was purified. The purified PcpF was able to convert GS-TriCH and GS-DiCH conjugates to TriCH and DiCH, respectively. The GS-hydroquinone lyase reactions catalyzed by PcpF are rather unusual for a GST. The disruption of pcpF in S. chlorophenolicum made the mutant lose the GS-hydroquinone lyase activities in the cell extracts. The mutant became more sensitive to PCP toxicity and had a significantly decreased PCP degradation rate, likely due to the accumulation of the GS-hydroquinone conjugates inside the cell. Thus, PcpF played a maintenance role in PCP degradation and converted the GS-hydroquinone conjugates back to the intermediates of the PCP degradation pathway.     

Dioxygenative cleavage of C-methylated hydroquinones and 2,6-dichlorohydroquinone by Pseudomonas sp. HH35.

The dioxygenolytic catabolism of five C-methylated hydroquinones and 2,6-dichlorohydroquinone in Pseudomonas sp. strain HH35 was elucidated. This organism, which is known to catabolise 2,6-dimethylhydroquinone by 1,2-cleavage, accumulated metabolites from 2-methyl-, 2,3-dimethyl-, 2,5-dimethyl-, 2,3,5-trimethyl- and 2,3,5,6-tetramethylhydroquinone which we isolated and characterised by mass spectrometry and (1)H NMR and UV spectroscopy. The identification of these metabolites defined the impact of methyl groups present in the hydroquinone and showed how each substitution pattern determined the site of the initial enzymic attack. With the exception of the 2,3,5,6-tetramethylhydroquinone, all C-methylated hydroquinones were catabolised by an initial dioxygenolytic cleavage occurring adjacent (1,2- or 3,4-cleavage) to a hydroxy group. In addition, our results indicated that the 2,6-dichlorohydroquinone is catabolised in a similar way by this strain.     

Gentisate 1,2-dioxygenase from Pseudomonas. Substrate coordination to active site Fe2+ and mechanism of turnover

Gentisate 1,2-dioxygenase catalyzes the oxygenolytic ring cleavage of gentisate (2,5-dihydroxybenzoate) between carbons 1 and 2 to form maleylpyruvate. The essential active site Fe2+ of the enzyme binds NO to yield an EPR-active (S = 3/2) complex. Hyperfine broadening from 17O (I = 5/2) is observed in the spectrum of the enzyme-nitrosyl complex prepared in 17O-enriched water, demonstrating that water is an iron ligand. Association of gentisate with the enzyme-nitrosyl complex causes the broadening due to [17O]water to disappear, suggesting that water is displaced. Hyperfine broadening of the EPR spectrum for the gentisate-bound complex is observed when 17O is incorporated into either the carbon 1 carboxylate or carbon 2 hydroxyl substituents of gentisate, but not when it is placed in the carbon 5 hydroxyl substituent. Thus, substrate apparently binds directly to the iron through the carbon 1 carboxylate and carbon 2 hydroxyl substituents, thereby bringing the site of ring cleavage close to the active site iron. Since NO must bind to the iron to elicit an EPR signal, a total of three sites in the iron coordination appear to be available for exogenous ligands. The role of the substrate functional groups in catalysis is investigated through comparison of the reaction kinetics of gentisate analogs using the gentisate 1,2-dioxygenases isolated from Pseudomonas acidovorans and Pseudomonas testosteroni. Turnover is either eliminated or substantially reduced on replacement of any of the functional groups of gentisate. Furthermore, an electron-donating group that can tautomerize (hydroxyl or amine) is required in a ring position either ortho or para to the carbon 2 substituent for turnover. The best alternate substrate of this group is 5-aminosalicylate, which is turned over at approximately 7% of the rate of gentisate by the enzyme from P. testosteroni. Both atoms from O2 are shown to be incorporated into the product of 5-aminosalicylate turnover. This is the first direct demonstration of dioxygenase stoichiometry in the reaction of any ferrous, non-heme, aromatic ring-cleaving dioxygenase. It is proposed that the enzyme-catalyzed O2 attack on the aromatic ring of gentisate is initiated from a complex in which O2 and substrate are simultaneously coordinated to the active site iron. Subsequent dioxygen bond cleavage and insertion are proposed to be promoted by a resonance shift involving ketonization of the carbon 5 hydroxyl group.     

Substrate specificity of chlorophyllase

Apparent Km and V_max values were obtained for hydrolysis of methyl and ethyl chlorophyllides a, methyl and ethyl pheophorbide a, and 9-hydroxymethyl pheophorbide a by chlorophyllase from Ailanthus altissima. Analysis of substrate specificity data for chlorophyllase indicates that the presence of a 9-keto group and a methyl alcohol group esterified at the 7-position in chlorophyll derivatives results in maximum binding affinity for substrates. Data on maximum reaction rates indicate that the rate-controlling step of hydrolysis occurs after release of the alcohol from the ester. Probable high affinity chlorophyllase inhibitors can be predicted on the basis of these specificity studies.     

Aromatic metabolism in Rhizobium trifolii-catechol 1,2-dioxygenase

Catechol 1,2-dioxygenase has been purified 46-fold from cells of Rhizobium trifolii TA1 grown on benzoate plus glucose. The dioxygenase had a molecular weight of 107,000 and a sub-unit molecular weight of 59,000. The enzyme had a K _m of 2 M for catechol and also cleaved 4-methylcatechol. The dioxygenase contained 2 g atoms of Fe³⁺ per mole of enzyme which could be removed by treatment with 1,10-phenanthroline, resulting in a complete loss of activity; reactivation of the enzyme occurred specifically with Fe³⁺.