Catechol Dioxygenase Research Articles

Biomimetic iron(iii) complexes of N3O and N3O2 donor ligands: protonation of coordinated ethanolate donor enhances dioxygenase activity

A series of iron(III) complexes 1-4 of the tripodal tetradentate ligands N,N-bis(pyrid-2-ylmethyl)-N-(2-hydroxyethyl)amine H(L1), N,N-bis(pyrid-2-ylmethyl)-N-(2-hydroxy- propyl)amine H(L2), N,N-bis(pyrid-2-ylmethyl)-N-ethoxyethanolamine H(L3), and N-((pyrid-2-ylmethyl)(1-methylimidazol-2-ylmethyl))-N-(2-hydroxyethyl)amine H(L4), have been isolated, characterized and studied as functional models for intradiol-cleaving catechol dioxygenases. In the X-ray crystal structure of [Fe(L1)Cl(2)] 1, the tertiary amine nitrogen and two pyridine nitrogen atoms of H(L1) are coordinated meridionally to iron(III) and the deprotonated ethanolate oxygen is coordinated axially. In contrast, [Fe(HL3)Cl(3)] 3 contains the tertiary amine nitrogen and two pyridine nitrogen atoms coordinated facially to iron(III) with the ligand ethoxyethanol moiety remaining uncoordinated. The X-ray structure of the bis(μ-alkoxo) dimer [{Fe(L5)Cl}(2)](ClO(4))(2)5, where HL is the tetradentate N(3)O donor ligand N,N-bis(1-methylimidazol-2-ylmethyl)-N-(2-hydroxyethyl)amine H(L5), contains the ethanolate oxygen donors coordinated to iron(III). Interestingly, the [Fe(HL)(DBC)](+) and [Fe(HL3)(HDBC)X] adducts, generated by adding ∼1 equivalent of piperidine to solutions containing equimolar quantities of iron(III) complexes 1-5 and H(2)DBC (3,5-di-tert-butylcatechol), display two DBC(2-)→ iron(III) LMCT bands (λ(max): 1, 577, 905; 2, 575,915; 3, 586, 920; 4, 563, 870; 5, 557, 856 nm; Δλ(max), 299-340 nm); however, the bands are blue-shifted (λ(max): 1, 443, 700; 2, 425, 702; 3, 424, 684; 4, 431, 687; 5, 434, 685 nm; Δλ(max), 251-277 nm) on adding 1 more equivalent of piperidine to form the adducts [Fe(L)(DBC)] and [Fe(HL3)(HDBC)X]. Electronic spectral and pH-metric titration studies in methanol disclose that the ligand in [Fe(HL)(DBC)](+) is protonated. The [Fe(L)(DBC)] adducts of iron(III) complexes of bis(pyridyl)-based ligands (1,2) afford higher amounts of intradiol-cleavage products, whereas those of mono/bis(imidazole)-based ligands (4,5) yield mainly the auto-oxidation product benzoquinone. It is remarkable that the adducts [Fe(HL)(DBC)](+)/[Fe(HL3)(DBC)X] exhibit higher rates of oxygenation affording larger amounts of intradiol-cleavage products and lower amounts of benzoquinone.

Mononuclear iron(iii) complexes of 3N ligands in organized assemblies: spectral and redox properties and attainment of regioselective extradiol dioxygenase activity

Four octahedral iron(III) complexes of the type [Fe(L)Cl(3)], where L is a tridentate 3N ligand like N,N-bis(pyrid-2-ylmethyl)amine (bpa, L1), N,N-bis(benzimidazol-2-ylmethyl)amine (bba, L2), 1,4,7-triazacyclononane (tacn, L3) and 2,2';6',2''-terpyridine (terpy, L4), have been isolated and their catechol dioxygenase activity investigated in dichloromethane, water and different aqueous micellar media. The positions of both the catecholato-to-iron(III) LMCT bands observed for the DBC(2-) (H(2)DBC = 3,5-di-tert-butylcatechol) adducts reveal that the adducts are present as cationic [Fe(L)(DBC)(H(2)O)](+) species, which interact strongly with anionic SDS micelles and dock themselves on the anionic micellar surface, and that they exist in the aqueous phase in CTAB and TX 100 micelles. The Fe(III)/Fe(II) redox potentials of the complexes throw light on the Lewis acidity of the iron(III) center as modified by the ligand donor atoms and hence the interaction of the complexes with different micelles. The DBSQ/DBC(2-) redox potentials in SDS micellar media are more positive than those in aqueous solution confirming the presence of the aqua species [Fe(L)(DBC)(H(2)O)](+). The DBC(2-) adducts of the iron(III) complexes of bpa, bba and tacn ligands, all with facial coordination, elicit extradiol (E) cleavage to different extents while the adduct of the terpy complex with meridional coordination of the ligand shows always intradiol (I) cleavage. It is remarkable that the bpa complex shows the highest yield of extradiol product and high product selectivity in aqueous SDS solution (E, 84.0%; E/I, 61.0 : 1) and in SDS : n-hexane reverse micellar medium (E, 93.7%) illustrating that a vacant or solvent coordinated site is essential for observing extradiol cleavage. Interestingly, the rates of dioxygenase reactions in aqueous and aqueous micellar solutions are significantly higher than those in non-aqueous solvents. Also, they diminish in the order, SDS > TX-100 > CTAB, illustrating the facile substitution of coordinated water molecule by molecular oxygen in [Fe(L)(DBC)(H(2)O)](+) bound to anionic SDS micelles.

Iron(III) complexes of N2O and N3O donor ligands as functional models for catechol dioxygenase enzymes: ether oxygen coordination tunes the regioselectivity and reactivity

A series of mononuclear iron(III) complexes of the type [Fe(L)Cl(3)], where L is a systematically modified N(2)O or N(3)O ligand with a methoxyethyl/tetrahydrofuryl ether oxygen donor atom, have been isolated and studied as models for catechol dioxygenases. The X-ray crystal structures of [Fe(L2)Cl(3)] 2, [Fe(L6)Cl(3)] 6, [Fe(L5)(TCC)Cl] 5a, where H(2)TCC = tetrachlorocatechol, [Fe(L6)(TCC)Br] 6a, and the μ-oxo dimer [{Fe(L6)Cl}(2)O](ClO(4))(2) 6b have been successfully determined. In [Fe(L2)Cl(3)] 2 the N(2)O ligand is facially coordinated to iron(III) through the pyridine and secondary amine nitrogen atoms and the tetrahydrofuryl oxygen atom. In [Fe(L6)Cl(3)] 6, [Fe(L5)(TCC)Cl] 5a and [Fe(L6)(TCC)Br] 6a the N(3)O donor ligands L5 and L6 act as a tridentate N3 donor ligand coordinated through two pyridine and one secondary amine nitrogen atoms, whereas the ether oxygen is not coordinated. The spectral and electrochemical properties of the adducts [Fe(L)(DBC)Cl] of 1-8, where H(2)DBC = 3,5-di-tert-butylcatechol, in DMF and their solvated adduct species [Fe(L)(DBC)(Sol)](+), where Sol = DMF/H(2)O, generated in situ in dichloromethane, respectively, have been investigated. The product analysis demonstrates that the adducts [Fe(L)(DBC)Cl] effect cleavage of catechol in the presence of O(2) in DMF to give mainly the intradiol (I) product with a small amount of the extradiol (E) product (E/I, 0.2:1-0.7:1). Interestingly, the solvated species [Fe(L)(DBC)(Sol)](+) derived from 1-4 cleave H(2)DBC to provide mainly the extradiol cleavage products with lower amounts of intradiol products (E/I, 2.3:1-4.3:1) in dichloromethane. In contrast, the solvated species [Fe(L)(DBC)(Sol)](+) derived from 5-8 cleave H(2)DBC to provide both extradiol and intradiol products (E/I, 0.6:1-2.3:1) due to the involvement of the ether oxygen donor of the methoxyethyl/tetrahydrofuryl arm in the coordination to iron(III) upon removal of a chloride ion.

Analysis of bacterial degradation pathways for long-chain alkylphenols involving phenol hydroxylase, alkylphenol monooxygenase and catechol dioxygenase genes

Eighteen 4-t-octylphenol-degrading bacteria were isolated and screened for the presence of degradative genes by polymerase chain reaction method using four designed primer sets. The primer sets were designed to amplify specific fragments from multicomponent phenol hydroxylase, single component monooxygenase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase genes. Seventeen of the 18 isolates exhibited the presence of a 232bp amplicon that shared 61–92% identity to known multicomponent phenol hydroxylase gene sequences from short and/or medium-chain alkylphenol-degrading strains. Twelve of the 18 isolates were positive for a 324bp region that exhibited 78–95% identity to the closest published catechol 1,2-dioxygenase gene sequences. The two strains, Pseudomonas putida TX2 and Pseudomonas sp. TX1, contained catechol 1,2-dioxygenase genes also have catechol 2,3-dioxygenase genes. Our result revealed that most of the isolated bacteria are able to degrade long-chain alkylphenols via multicomponent phenol hydroxylase and the ortho-cleavage pathway.

A hyperactive cobalt-substituted extradiol-cleaving catechol dioxygenase.

Homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum (HPCD) has an Fe(II) center in its active site that can be replaced with Mn(II) or Co(II). Whereas Mn-HPCD exhibits steady-state kinetic parameters comparable to those of Fe-HPCD, Co-HPCD behaves somewhat differently, exhibiting significantly higher [Formula: see text] and k (cat). The high activity of Co-HPCD is surprising, given that cobalt has the highest standard M(III/II) redox potential of the three metals. Comparison of the X-ray crystal structures of the resting and substrate-bound forms of Fe-HPCD, Mn-HPCD, and Co-HPCD shows that metal substitution has no effect on the local ligand environment, the conformational integrity of the active site, or the overall protein structure, suggesting that the protein structure does not differentially tune the potential of the metal center. Analysis of the steady-state kinetics of Co-HPCD suggests that the Co(II) center alters the relative rate constants for the interconversion of intermediates in the catalytic cycle but still allows the dioxygenase reaction to proceed efficiently. When compared with the kinetic data for Fe-HPCD and Mn-HPCD, these results show that dioxygenase catalysis can proceed at high rates over a wide range of metal redox potentials. This is consistent with the proposed mechanism in which the metal mediates electron transfer between the catechol substrate and O(2) to form the postulated [M(II)(semiquinone)superoxo] reactive species. These kinetic differences and the spectroscopic properties of Co-HPCD provide new tools with which to explore the unique O(2) activation mechanism associated with the extradiol dioxygenase family.

Growth of Pseudomonas putida F1 on styrene requires increased catechol-2,3-dioxygenase activity, not a new hydrolase

Pseudomonas putida F1 cannot grow on styrene despite being able to degrade it through the toluene degradation (tod) pathway. Previous work had suggested that this was because TodF, the meta-fission product (MFP) hydrolase, was unable to metabolize the styrene MFP 2-hydroxy-6-vinylhexa-2,4-dienoate. Here we demonstrate via kinetic and growth analyses that the substrate specificity of TodF is not the limiting factor preventing F1 from growing on styrene. Rather, we found that the metabolite 3-vinylcatechol accumulated during styrene metabolism and that micromolar concentrations of this intermediate inactivated TodE, the catechol-2,3-dioxygenase (C23O) responsible for its cleavage. Analysis of cells growing on styrene suggested that inactivation of TodE and the subsequent accumulation of 3-vinylcatechol resulted in toxicity and cell death. We found that simply overexpressing TodE on a plasmid (pTodE) was all that was necessary to allow F1 to grow on styrene. Similar results were also obtained by expressing a related C23O, DmpB from Pseudomonas sp. CF600, in tandem with its plant-like ferredoxin, DmpQ (pDmpQB). Further analysis revealed that the ability of F1 (pDmpQB) and F1 (pTodE) to grow on styrene correlated with increased C23O activity as well as resistance of the enzyme to 3-vinylcatechol-mediated inactivation. Although TodE inactivation by 3-halocatechols has been studied before, to our knowledge, this is the first published report demonstrating inactivation by a 3-vinylcatechol. Given the ubiquity of catechol intermediates in aromatic hydrocarbon metabolism, our results further demonstrate the importance of C23O inactivation as a determinant of growth substrate specificity.

Functional consortia for cresol-degrading activated sludges: Toxicity-to-extinction approach

The conventional roll tube and plating techniques are typically time consuming and can culture in vitro only a small fraction of microbes in natural microflora. This study utilizes a novel, simple, and rapid method, the toxicity-to-extinction approach, to obtain the minimal functional consortium that can effectively degrade meta- (m-), para- (p-), and ortho- (o-) cresols. The original sludge had 16 major bands by denaturing gradient gel electrophoresis (DGGE). Microbial diversity decreased as the cresol concentration increased. The functional strains acquired under toxic stress by dosed cresols that individually degraded m-, p-, and o-cresols were identified. Catechol 1,2-dioxygenase (C12D) and catechol 2,3-dioxygenase (C23D) activities in cell-free extracts were determined spectrophotometrically and were correlated with noted changes in microbial communities under cresol stress. The proposed toxicity-to-extinction approach is feasible for isolating a functional consortium from sludge for cresol degradation.

Quantification of catechol dioxygenase gene expression in soil during degradation of 2,4-dichlorophenol

The tfdC and C23O genes encode two catechol dioxygenases that catalyse ortho and meta cleavage of a key metabolite (chlorocatechol) of 2,4-dichlorophenol (2,4-DCP) metabolism, respectively. Primers were designed and a real-time PCR assay was developed to assess the abundance and expression of both tfdC and C23O genes in a soil amended with 2,4-DCP over a 21-day period. tfdC, the gene encoding the ortho cleaving dioxygenase, was significantly more abundant than the meta cleaving dioxygenase gene (C23O) throughout the experiment. The highest levels of tfdC were observed 2 days after amendment of soil with 2,4-DCP, at which stage the rate of 2,4-DCP degradation was at its maximum. In contrast, C230 copy numbers declined initially and peaked when degradation had slowed considerably. mRNA of the two chlorocatechol dioxygenase genes was not detected on day 0, but both genes were expressed after this time point. tfdC was expressed at a significantly higher level than C23O in 2,4-DCP-amended soil throughout the course of the microcosm, indicating the dominance of the ortho metabolic pathway. Phylogenetic analysis revealed a wide diversity of chlorocatechol dioxygenase genes in the 2,4-DCP-exposed soil examined.

The fungal pathogen Cochliobolus heterostrophus responds to maize phenolics: novel small molecule signals in a plant-fungal interaction

The transcription factor ChAP1 of the fungal pathogen of maize, Cochliobolus heterostrophus, responds to oxidative stress by migration to the nucleus and activation of antioxidant genes. Phenolic and related compounds found naturally in the host also trigger nuclear localization of ChAP1, but only slight upregulation of some antioxidant genes. ChAP1 thus senses phenolic compounds without triggering a strong antioxidant response. We therefore searched for genes whose expression is regulated by phenolic compounds and/or ChAP1. The C. heterostrophus genome contains a cluster of genes for metabolism of phenolics. One such gene, catechol dioxygenase CCHD1, was induced at least 10-fold by caffeic and coumaric acids. At high phenolic concentrations (≥ 1.6 mM), ChAP1 is needed for maximum CCHD1 expression. At micromolar levels of phenolics CCHD1 is as strongly induced in chap1 mutants as in the wild type. The pathogen thus detects phenolics by at least two signalling pathways: one causing nuclear retention of ChAP1, and another triggering induction of CCHD1 expression. The low concentrations required for induction of CCHD1 indicate fungal receptors for plant phenolics. Symbiotic and pathogenic bacteria are known to detect phenolics, and our findings generalize this to a eukaryotic pathogen. Phenolics and related compounds thus provide a ubiquitous plant-derived signal.

Iron(III) complexes of tridentate N 3 ligands as models for catechol dioxygenases: Stereoelectronic effects of pyrazole coordination

The iron(III) complexes of the tridentate N 3 ligands pyrazol-1-ylmethyl(pyrid-2-ylmethyl)amine (L1), 3,5-dimethylpyrazol-1-ylmethyl(pyrid-2-ylmethyl)amine (L2), 3- iso-propylpyrazol-1-ylmethyl(pyrid-2-ylmethyl)amine (L3) and (1-methyl-1 H-imidazol-2-ylmethyl)pyrid-2-ylmethylamine (L4) have been isolated and studied as functional models for catechol dioxygenases. They have been characterized by elemental analysis and spectral and electrochemical methods. The X-ray crystal structure of the complex [Fe(L1)Cl 3] 1 has been successfully determined. The complex possesses a distorted octahedral coordination geometry in which the tridentate ligand facially engages iron(III) and the Cl − ions occupy the remaining coordination sites. The Fe–N pz bond distance (2.126(5) Å) is shorter than the Fe–N py bond (2.199(5) Å). The systematic variation in the ligand donor substituent significantly influences the Lewis acidity of the iron(III) center and hence the interaction of the present complexes with a series of catechols. The catecholate adducts [Fe(L)(DBC)Cl], where H 2DBC = 3,5-di- tert-butylcatechol, have been generated in situ and their spectral and redox properties and dioxygenase activities have been studied in N, N-dimethylformamide solution. The adducts [Fe(L)(DBC)Cl] undergo cleavage of DBC 2− in the presence of dioxygen to afford major amounts of intradiol and smaller amounts extradiol cleavage products. In dichloromethane solution the [Fe(L)(DBC)Cl] adducts afford higher amounts of extradiol products (64.1–22.2%; extradiol-to-intradiol product selectivity E/ I, 2.6:1–4.5:1) than in DMF (2.5–6.6%; E/ I, 0.1:1–0.4:1). The results are in line with the recent understanding of the function of intra- and extradiol-cleaving catechol dioxygenases.

A Functional Model of Extradiol-Cleaving Catechol Dioxygenases: Mimicking the 2-His-1-Carboxylate Facial Triad

The synthesis and characterization of an iron-catecholate model complex of a tridentate 2-N-1-carboxylate ligand derived from L-proline are reported. The X-ray crystal structure of the complex [(L)(3)Fe(3)(DBC)(3)] (1) (where L is 1-(2-pyridylmethyl)pyrrolidine-2-carboxylate and DBC is the dianion of 3,5-di-tert-butyl catechol) reveals that the tridentate ligand binds to the iron center in a facial manner and mimics the 2-his-1-carboxylate facial triad motif observed in extradiol-cleaving catechol dioxygenases. The iron(III)-catecholate complex (1) reacts with dioxygen in acetonitrile in ambient conditions to cleave the C-C bond of catecholate. In the reaction, an equal amount of extra- and intradiol cleavage products are formed without any auto-oxidation product. The iron-catecholate complex is a potential functional model of extradiol-cleaving catechol dioxygenases.

Biomimetic Formation of 2‐Tropolones by Dioxygenase‐Catalysed Ring Expansion of Substituted 2,4‐Cyclohexadienones

Substituted 2-tropolone natural products are found in plants and fungi. Their biosynthesis is thought to occur by ring expansion from a cyclohexadienone precursor, but this reaction has not previously been demonstrated experimentally. Treatment of 6-hydroxy-6-hydroxymethylcyclohexa-2,4-dienone with the non-haem iron(II)-dependent extradiol catechol dioxygenase MhpB from Escherichia coli results in the formation of the 2-tropolone ring-expansion product through a pinacol-type rearrangement. Three further substituted cyclohexa-2,4-dienone analogues were prepared, and treatment of each analogue was found to give the substituted 2-tropolone ring-expansion product. This ring expansion could also be effected nonenzymatically by treatment with 1,4,7-triazacyclononane and FeCl(2). This is a novel transformation for non-haem iron-dependent enzymes, and this is the first experimental demonstration of the proposed ring-expansion reaction in tropolone biosynthesis.

Long-term Performance and Community Analysis of Spirodela Polyrrhiza-bacteria Association Treating Phenol-contaminated Water

『大阪大学大学院工学研究科環境・エネルギー工学専攻生物圏環境工学領域 研究活動報告』, (2010.4.1～2011.3.31), pp.83-94, 大阪大学大学院工学研究科環境・エネルギー工学専攻環境資源・材料学講座生物圏環境工学領域, 2011.5 に掲載

Identification of Phenol-Degrading Nocardia Sp. Strain C-14-1 and Characterization of Its Ring-Cleavage 2,3-Dioxygenase

An aerobic bacterial strain C-14-1 was isolated from an acrylic fiber wastewater. The strain was found to belong toNocardia sp. according to morphological, physiological and its 16S rRNA gene sequence. This strain was able todegrade both alkanes and succinonitrile such as phenol. Catechol 2,3-dioxygenase (C23O) gene was found andamplified with the designed primers from the total DNA of C-14-1. The result of Southern blot indicated that there isonly one C23O gene in the genome of C-14-1.

Detection, expression and quantitation of the biodegradative genes in Antarctic microorganisms using PCR

In this study, 28 hydrocarbon-degrading bacterial isolates from oil-contaminated Antarctic soils were screened for the presence of biodegradative genes such as alkane hydroxylase (alks), the ISPalpha subunit of naphthalene dioxygenase (ndoB), catechol 2,3-dioxygenase (C23DO) and toluene/biphenyl dioxygenase (todC1/bphA1) by using polymerase chain reaction (PCR) methods. All naphthalene degrading bacterial isolates exhibited the presence of a 648 bp amplicon that shared 97% identity to a known ndoB sequence from Pseudomonas putida. Twenty-two out of the twenty-eight isolates screened were positive for one, two or all three different regions of the C23DO gene. For alkane hydroxylase, all 6 Rhodococcus isolates were PCR-positive for a 194 bp and a 552 bp segment of the alkB gene, but exhibited variable results with primers located at different segments of this gene. Three Pseudomonas spp. 4/101, 19/1, 30/3 amplified 552 bp segment of alkB. Only two Pseudomonas sp. 7/156 and 4/101 amplified a segment of alkB exhibiting 89-94% nucleotide sequence identity with the existing sequence of alkB in the GenBank sequence database. Transcripts of three genes, alkB2, C23DO and ndoB, that were amplified by DNA-PCR in three different bacterial isolates also exhibited positive amplification by reverse transcriptase PCR (RT-PCR) method confirming that these genes are functional. A competitive PCR (cPCR) method was developed for a quantitative estimation of ndoB from pure cultures of the naphthalene-degrading Pseudomonas sp. 30/2. A minimum of 1 x 10(7) copies of the ndoB gene was detected based on the comparison of the intensities of the competitor and target DNA bands. It is expected that the identification and characterization of the biodegradative genes will provide a better understanding of the catabolic pathways in Antarctic psychrotolerant bacteria, and thereby help support bioremediation strategies for oil-contaminated Antarctic soils.

Thermal Effects on the Activity and Structural Conformation of Catechol 2,3-Dioxygenase from Pseudomonas putida SH1

A bacterium, Pseudomonas putida SH1, which can catabolize phenol, naphthalene, or cresol as the sole carbon and energy source, was isolated from a petroleum-contaminated site in Taiwan. The catechol 2,3-dioxygenase (C23O) was purified from this bacterial strain when grown on naphthalene as the sole carbon and energy source. The enzyme is composed of four identical subunits with a native molecular weight of 128 +/- 5 kD. Small-angle neutron scattering (SANS) techniques were employed to study the thermal effects on the structural conformation of this enzyme in solution. The SANS measurements revealed distinct changes in the size of the enzyme between 50 and 80 degrees C, and the size was not restored during the subsequent cooling. The enzyme started to denature at 55 degrees C, and the structure was destroyed by the time the temperature reached 80 degrees C, at which the enzyme had become more than twice the original size. The optimal catalytic temperature of the enzyme was at 50 degrees C. The half-life of the activity at this temperature was 45 min. The enzyme activity increases starting from 25 degrees C and reaches its maximum at 50 degrees C, below which no obvious change in the size of the enzyme is found. Noticeable enlargement of the enzyme is revealed when the enzymatic activity starts to fall. By combination of SANS measurement and biochemical properties of the enzyme, this study demonstrates the correlation of enzyme size in solution and catalytic activity upon a heat treatment. In addition, for a protein composed of multiple subunits, the shape of the enzyme and the dissociation of the enzyme subunits in a thermal cycle were also demonstrated by SANS methodology.

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.

Iron(III) Complexes of Tripodal Monophenolate Ligands as Models for Non-Heme Catechol Dioxygenase Enzymes: Correlation of Dioxygenase Activity with Ligand Stereoelectronic Properties

The iron(III) complexes [Fe(L)Cl(2)] 1-6 of the tripodal monophenolate ligands N,N-bis(2-pyridylmethyl)-N'-(2-hydroxybenzyl)amine H(L1), [(1-methylimidazol-2-ylmethyl)-(pyrid-2-ylmethyl)aminomethyl]-phenol H(L2), 2,4-dimethyl-6-[(1-methylimidazol-2-ylmethyl)(pyrid-2-ylmethyl)aminomethyl]phenol H(L3), N,N-dimethyl-N'-(pyrid-2-ylmethyl)-N'-(2-hydroxybenzyl)ethylenediamine H(L4), N,N-dimethyl-N'-(1-methylimidazol-2-ylmethyl)-N'-(2-hydroxybenzyl)ethylenediamine H(L5), and N,N-dimethyl-N'-(1-methylimidazol-2-ylmethyl)-N'-(2-hydroxy-3,5-dimethylbenzyl)ethylenediamine H(L6) have been isolated and studied as structural and functional models for the intradiol-cleaving catechol dioxygenase enzymes. The complexes have been characterized using elemental analysis, electrospray ionization mass spectrometry, and absorption spectral and electrochemical methods. The single crystal X-ray structures of [Fe(L3)Cl(2)] 3 and [Fe(L6)Cl(2)] 6 have been successfully determined, and the rhombically distorted octahedral coordination geometry around iron(III) in them are constituted by the phenolate oxygen and pyridyl/-NMe(2), and N-methylimidazolyl and tertiary amine nitrogens of the tripodal tetradentate ligands H(L3)/H(L6) and two cis-coordinated chloride ions. The sterically demanding -NMe(2) group as in 6 imposes an Fe-O-C bond angle (139.8 degrees) and Fe-O bond length (1.852 A), which are very close to those (Fe-O-C, 133, 148 degrees; Fe-O(tyrosinate), 1.81, 1.91 A) of 3,4-PCD enzymes. The Fe-O-C bond angle observed for 6 is higher than that for 3 (125.1 degrees), and the Fe-O(phenolate) bond distance in 6 is shorter than that in 3 (1.905 A). In methanol solution all the complexes exhibit two phenolate-to-Fe(III) ligand-to-metal charge transfer (LMCT) bands in the ranges 536-622 and 329-339 nm. Further, when 3,5-di-tert-butylcatechol (H(2)DBC) pretreated with two moles of Et(3)N is added to 1-6, two new intense DBC(2-)-to-iron(III) LMCT bands (466-489, 676-758 nm) are observed, which are similar to those observed for 3,4-PCD enzyme-substrate complex. All the complexes elicit oxidative intradiol cleavage of H(2)DBC in the presence of O(2). Interestingly, among the present complexes, 3 containing coordinated N-methylimidazolyl nitrogen shows the highest rate of intradiol cleavage, which correlates with the highest energy of DBC(2-)-to-iron(III) LMCT band and the most negative DBSQ/DBC(2-) redox potential. Also, the catecholate adducts of complexes 4 and 5, both containing a -NMe(2) donor group, react faster and produce higher amounts of intradiol cleavage products (4: 55.3; 5, 50.6%) than the analogous complexes 1 (43.2%) and 2 (32.7%), both containing a pyridyl nitrogen donor, which is consistent with the more negative DBSQ/DBC(2-) redox potentials for 4 and 5. The increase in rate of catechol dioxygenation with increase in the DBC(2-)-to-iron(III) LMCT band energy and decrease in DBSQ/DBC(2-) redox potential is illustrated by invoking a facile alpha-electron transfer from iron(III) to catecholate-bound molecular oxygen in the substrate activation mechanism proposed for the intradiol-cleaving catechol dioxygenases. Also, when the substituents on the phenolate arm are varied to tune the Lewis acidity of iron(III) center, the reaction rate decreases with decrease in Lewis acidity and, interestingly, extradiol cleavage is also observed when the Lewis acidity is decreased further by incorporating a 3,5-dimethylphenolate arm as in 6.

Rational Design of Catechol-2, 3-dioxygenase for Improving the Enzyme Characteristics

Catechol-2, 3-dioxygenase (C23O) from Pseudomonas sp. CGMCC2953 identified in our laboratory, which is one of the key enzymes responsible for phenanthrene biodegradation, was expected to get better characteristics tolerant to environment for its further application. With the aim of improving the enzyme properties by introducing intermolecular disulfide bonds, X-ray structure of a C23O from Pseudomonas putida MT-2, a highly conserved homologous with the C23O from Pseudomonas sp. CGMCC2953, was directly used to find the potential sites for forming disulfide bonds between two monomers of the target C23O. Two sites, Ala229 and His294, were identified and mutated to cysteine, respectively, by using site mutagenesis. The expected disulfide bond between these two CYS residues was confirmed with both molecular modeling and experimental results. The optimum temperature of the mutated enzyme was widened from 40 to 40 approximately 50 degrees C. The mutated C23O became more alkalescency stable compared with the wild-type enzyme, e.g., 75% of the maximal enzyme activity retained even under pH 9.5 while 50% residue for the wild-type one. Improvement of thermostability of the mutated C230 with the redesigned disulfide was also confirmed.

Enrichment of bacteria possessing catechol dioxygenase genes in the rhizosphere of Spirodela polyrrhiza: A mechanism of accelerated biodegradation of phenol

The bacterial community structure in bulk water and in rhizosphere fractions of giant duckweed, Spirodela polyrrhiza, was quantitatively and qualitatively investigated by PCR-based methods using 6 environmental water samples to elucidate the mechanisms underlying selective accumulation of aromatic compound-degrading bacteria in the rhizosphere of S. polyrrhiza. S. polyrrhiza selectively accumulated a diverse range of aromatic compound-degrading bacteria in its rhizosphere, regardless of the origin of water samples, despite no exposure to phenol. The relative abundances of the catechol 1,2-dioxygenase (C12O) gene (C12O DNA) and catechol 2,3-dioxygenase (C23O) gene (C23O DNA) were calculated as the ratios of the copy numbers of these genes to the copy number of 16S rDNA and are referred to as the rhizosphere effect (RE) value. The RE values for C12O DNA and C23O DNA were 1.0 × 10 1–9.3 × 10 3 and 1.7 × 10 2–1.5 × 10 4 times as high, respectively, in rhizosphere fractions as in bulk water fractions, and these higher values were associated with a notably higher sequence diversity of C12O DNA and C23O DNA. The RE values during phenol degradation were 3.6 × 10 0–4.3 × 10 2 and 2.2 × 10 0–1.7 × 10 2, respectively, indicating the ability of S. polyrrhiza to selectively accumulate aromatic compound-degrading bacteria in its rhizosphere during phenol degradation. The bacterial communities in the rhizosphere fractions differed from those in the bulk water fractions, and those in the bulk water fractions were notably affected by the rhizosphere bacterial communities. S. polyrrhiza released more than 100 types of phenolic compound into its rhizosphere as root exudates at the considerably high specific release rate of 1520 mg TOC and 214 mg phenolic compounds/d/g root (wet weight). This ability of S. polyrrhiza might result in the selective recruitment and accumulation of a diverse range of bacteria harboring genes encoding C12O and C23O, and the subsequent accelerated degradation of phenol in the rhizosphere.