Biochemical and Genetic Characterization of a Gentisate 1,2-Dioxygenase from Sphingomonas sp. Strain RW5

A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC 1.13.11.4), gtdA, from Sphingomonas sp. strain RW5 was cloned and sequenced. The gtdA gene encodes a 350-amino-acid polypeptide with a predicted size of 38.85 kDa. Comparison of the gtdA gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no significant homology except for a low similarity (27%) to the 1-hydroxy-2-naphthoate dioxygenase (phdI) of the phenanthrene degradation in Nocardioides sp. strain KP7 (T. Iwabuchi and S. Harayama, J. Bacteriol. 179:6488–6494, 1997). This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases. The gene was subcloned and hyperexpressed in E. coli. The resulting product was purified to homogeneity and partially characterized. Under denaturing conditions, the polypeptide exhibited an approximate size of 38.5 kDa and migrated on gel filtration as a species with a molecular mass of 177 kDa. The enzyme thus appears to be a homotetrameric protein. The purified enzyme stoichiometrically converted gentisate to maleylpyruvate, which was identified by gas chromatography-mass spectrometry analysis as its methyl ester. Values of affinity constants (K_m) and specificity constants (K_cat/K_m) of the enzyme were determined to be 15 μM and 511 s⁻¹ M⁻¹ × 10⁴ for gentisate and 754 μM and 20 s⁻¹ M⁻¹ × 10⁴ for 3,6-dichlorogentisate. Three further open reading frames (ORFs) were found downstream of gtdA. The deduced amino acid sequence of ORF 2 showed homology to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomerase superfamily and glutathione reductase superfamily, respectively.Large amounts of aromatic compounds have been released into the environment during the last decades as a result of extensive production of industrial chemicals and agricultural applications of pesticides. Many of these compounds, particularly the chlorinated derivatives, are toxic, even at low concentrations, and persist in the environment (14, 39). Numerous microorganisms have been isolated which degrade xenobiotic aromatic compounds through aerobic or anaerobic degradative reactions (16, 17, 34, 46, 47). A wide variety of polycyclic and homocyclic aromatic compounds are aerobically transformed to a limited number of central dihydroxylated intermediates like catechol, protocatechuate, or gentisate. Whereas catabolic pathways for catechol and protocatechuate have been extensively characterized (22, 35), little is known about gentisate degradation.Gentisic acid (2,5-dihydroxybenzoic acid) is a key intermediate in the aerobic degradation of such aromatic compounds as dibenzofuran (15), naphthalene (18, 48), salicylate (40, 55), anthranilate (32), and 3-hydroxybenzoate (26). Degradation of gentisate is initiated by gentisate 1,2-dioxygenase (GDO; EC 1.13.11.4, gentisate:oxygen oxidoreductase), which cleaves the aromatic ring between the carboxyl and the vicinal hydroxyl group to form maleylpyruvate (30). Maleylpyruvate can be converted to central metabolites of the Krebs cycle either by cleavage to pyruvate and maleate (5, 24) or by isomerization to fumarylpyruvate and subsequent cleavage to fumarate and pyruvate (10, 31, 51).Of the two well-studied classes of ring cleavage dioxygenases, intradiol enzymes, such as catechol 1,2-dioxygenase or protocatechuate 3,4-dioxygenase, contain an Fe³⁺ atom in the catalytic center and cleave the aromatic substrate between two vicinal hydroxyl groups (7, 37, 38), whereas dioxygenases of the extradiol class, such as catechol 2,3-dioxygenase or protocatechuate 4,5-dioxygenase, contain Fe²⁺ and cleave the aromatic substrate adjacent to two vicinal hydroxyl groups (1, 13). Gentisate 1,2-dioxygenase cleaves aromatic rings containing hydroxyl groups situated para to one another. Although the mechanism of oxygen activation was proposed to be similar to that of enzymes of the extradiol dioxygenase class (20), and the active center contains Fe²⁺ (11, 21, 29, 49, 51), the Fe²⁺ is not bound to the enzyme by electron-donating ligands such as cysteine or tyrosine (21) as is the case for extradiol-cleaving dioxygenases (19). It is being assumed, therefore, that GDO represents a novel class of ring-cleaving dioxygenases.GDOs have been purified and characterized from gram-positive bacteria of the genera Bacillus and Rhodococcus (29, 50) and gram-negative bacteria of the genera Klebsiella, Comamonas, and Moraxella (11, 21, 49), and amino-terminal sequences of GDOs from Comamonas testosteroni and Comamonas acidovorans have been determined (21), but until now, no complete sequence of any GDO or of a gene encoding GDO has been reported. Here we describe the cloning and sequencing of the gene encoding the GDO from Sphingomonas sp. strain RW5 and its partial characterization. This GDO represents a novel class of dioxygenases with very low similarity to any other known ring-cleaving dioxygenases.     

Catechol 2,3-Dioxygenase from Pseudomonas sp. Strain ND6: Gene Sequence and Enzyme Characterization

The catechol 2,3-dioxygenase (C23O) gene in naphthalene catabolic plasmid pND6-1 of Pseudomonas sp. ND6 was cloned and sequenced. The C23O gene was consisted of 924 nucleotides and encoded a polypeptide of molecular weight 36 kDa containing 307 amino acid residues. The C23O of Pseudomonas sp. ND6 exhibited 93% and 89% identities in amino acid sequence with C23Os encoded by naphthalene catabolic plasmid NAH7 from Pseudomonas putida G7 and the chromosome of Pseudomonas stutzeri AN10 respectively. The Pseudomonas sp. ND6 C23O gene was overexpressed in Escherichia coli DH 5α using the lac promoter of pUC18, and its gene product was purified by DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography. The enzymology experiments indicated that the specific activity and thermostability of C23O from Pseudomonas sp. ND6 were better than those of C23O from Pseudomonas putida G7.     

Purification and Characterization of Hydroxyquinol 1,2-Dioxygenase from Azotobacter sp. Strain GP1

Hydroxyquinol 1,2-dioxygenase was purified from cells of the soil bacterium Azotobacter sp. strain GP1 grown with 2,4,6-trichlorophenol as the sole source of carbon. The presumable function of this dioxygenase enzyme in the degradative pathway of 2,4,6-trichlorophenol is discussed. The enzyme was highly specific for 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene) and hydroxyquinol (1,2,4-trihydroxybenzene) and was found to perform ortho cleavage of the hydroxyquinol compounds, yielding chloromaleylacetate and maleylacetate, respectively. With the conversion of 1 mol of 6-chlorohydroxyquinol, the consumption of 1 mol of O(inf2) and the formation of 1 mol of chloromaleylacetate were observed. Catechol was not accepted as a substrate. The enzyme has to be induced, and no activity was found in cells grown on succinate. The molecular weight of native hydroxyquinol 1,2-dioxygenase was estimated to 58,000, with a sedimentation coefficient of 4.32. The subunit molecular weight of 34,250 indicates a dimeric structure of the dioxygenase enzyme. The addition of Fe(sup2+) ions significantly activated enzyme activity, and metal-chelating agents inhibited it. Electron paramagnetic resonance data are consistent with high-spin iron(III) in a rhombic environment. The NH(inf2)-terminal amino acid sequence was determined for up to 40 amino acid residues and compared with sequences from literature data for other catechol and chlorocatechol dioxygenases.     

Haloalkylphosphorus Hydrolases Purified from Sphingomonas sp. Strain TDK1 and Sphingobium sp. Strain TCM1

Phosphotriesterases catalyze the first step of organophosphorus triester degradation. The bacterial phosphotriesterases purified and characterized to date hydrolyze mainly aryl dialkyl phosphates, such as parathion, paraoxon, and chlorpyrifos. In this study, we purified and cloned two novel phosphotriesterases from Sphingomonas sp. strain TDK1 and Sphingobium sp. strain TCM1 that hydrolyze tri(haloalkyl)phosphates, and we named these enzymes haloalkylphosphorus hydrolases (TDK-HAD and TCM-HAD, respectively). Both HADs are monomeric proteins with molecular masses of 59.6 (TDK-HAD) and 58.4 kDa (TCM-HAD). The enzyme activities were affected by the addition of divalent cations, and inductively coupled plasma mass spectrometry analysis suggested that zinc is a native cofactor for HADs. These enzymes hydrolyzed not only chlorinated organophosphates but also a brominated organophosphate [tris(2,3-dibromopropyl) phosphate], as well as triaryl phosphates (tricresyl and triphenyl phosphates). Paraoxon-methyl and paraoxon were efficiently degraded by TCM-HAD, whereas TDK-HAD showed weak activity toward these substrates. Dichlorvos was degraded only by TCM-HAD. The enzymes displayed weak or no activity against trialkyl phosphates and organophosphorothioates. The TCM-HAD and TDK-HAD genes were cloned and found to encode proteins of 583 and 574 amino acid residues, respectively. The primary structures of TCM-HAD and TDK-HAD were very similar, and the enzymes also shared sequence similarity with fenitrothion hydrolase (FedA) of Burkholderia sp. strain NF100 and organophosphorus hydrolase (OphB) of Burkholderia sp. strain JBA3. However, the substrate specificities and quaternary structures of the HADs were largely different from those of FedA and OphB. These results show that HADs from sphingomonads are novel members of the bacterial phosphotriesterase family.     

Possible Involvement of Toluene-2,3-Dioxygenase in Defluorination of 3-Fluoro-Substituted Benzenes by Toluene-Degrading Pseudomonas sp. Strain T-12

Pseudomonas sp. strain T-12 cells in which the toluene-degradative pathway enzymes have been induced can transform many 3-fluoro-substituted benzenes to the corresponding 2,3-catechols with simultaneous elimination of the fluorine substituent as inorganic fluoride. Substrates for this transformation included 3-fluorotoluene, 3-fluorotrifluorotoluene, 3-fluorohalobenzenes, 3-fluoroanisole, and 3-fluorobenzonitrile. While 3-fluorotoluene and 3-fluoroanisole produced only defluorinated catechols, other substrates generated catechol products with and without the fluorine substituent. The steric size of the C-1 substituent affected the ratio of defluorinated to fluorinated catechols formed from a substrate. A mechanism for the defluorination reaction involving toluene-2,3-dioxygenase is proposed.     

鞘氨醇单胞菌PY3菲降解基因的克隆及序列分析

将菲降解菌鞘氨醇单胞菌(Sphingomanas sp.)PY3的DNA片段与pUC119质粒连接后,转化大肠杆菌JM109,经筛选得到两个质粒,分别命名为pUp1(带有23kb外源DNA片段)和pUp2(带有39kb外源DNA片段)。pUp 1的DNA含有2个ORF。ORF 1由275个氨基酸组成,与恶臭假单胞菌(Pseudomonas putida)F1的甲苯水解酶及菌株Pseudomonas CF600的甲苯水解酶在氨基酸水平上有47%的同源性。ORF 2由327个氨基酸组成,与嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)的邻苯二酚双加氧酶(phe B)及紫红红球菌(Rhodococcus rhodochrous)CTM的邻苯二酚双加氧酶(C23O)在氨基酸水平上分别有57%和44%的同源性。     

Altering catalytic properties of 3-chlorocatechol-oxidizing extradiol dioxygenase from Sphingomonas xenophaga BN6 by random mutagenesis

The 2,3-dihydroxybiphenyl 1,2-dioxygenase from Sphingomonas xenophaga strain BN6 (BphC1) oxidizes 3-chlorocatechol by a rather unique distal ring cleavage mechanism. In an effort to improve the efficiency of this reaction, bphC1 was randomly mutated by error-prone PCR. Mutants which showed increased activities for 3-chlorocatechol were obtained, and the mutant forms of the enzyme were shown to contain two or three amino acid substitutions. Variant enzymes containing single substitutions were constructed, and the amino acid substitutions responsible for altered enzyme properties were identified. One variant enzyme, which contained an exchanged amino acid in the C-terminal part, revealed a higher level of stability during conversion of 3-chlorocatechol than the wild-type enzyme. Two other variant enzymes contained amino acid substitutions in a region of the enzyme that is considered to be involved in substrate binding. These two variant enzymes exhibited a significantly altered substrate specificity and an about fivefold-higher reaction rate for 3-chlorocatechol conversion than the wild-type enzyme. Furthermore, these variant enzymes showed the novel capability to oxidize 3-methylcatechol and 2,3-dihydroxybiphenyl by a distal cleavage mechanism.     

Characterization of a Novel Angular Dioxygenase from Fluorene-Degrading Sphingomonas sp. Strain LB126

In this study, the genes involved in the initial attack on fluorene by Sphingomonas sp. strain LB126 were investigated. The α and β subunits of a dioxygenase complex (FlnA1-FlnA2), showing 63 and 51% sequence identity, respectively, to the subunits of an angular dioxygenase from the gram-positive dibenzofuran degrader Terrabacter sp. strain DBF63, were identified. When overexpressed in Escherichia coli, FlnA1-FlnA2 was responsible for the angular oxidation of fluorene, 9-hydroxyfluorene, 9-fluorenone, dibenzofuran, and dibenzo-p-dioxin. Moreover, FlnA1-FlnA2 was able to oxidize polycyclic aromatic hydrocarbons and heteroaromatics, some of which were not oxidized by the dioxygenase from Terrabacter sp. strain DBF63. The quantification of resulting oxidation products showed that fluorene and phenanthrene were the preferred substrates of FlnA1-FlnA2.     

Genome Sequence of the Welan Gum-Producing Strain Sphingomonas sp. ATCC 31555

Sphingomonas sp. strain ATCC 31555 can produce an anionic heteropolysaccharide, welan gum, which shows excellent stability and viscosity retention even at high temperatures. Here we present a 4.0-Mb assembly of its genome sequence. We have annotated 10 coding sequences (CDSs) responsible for the welan gum biosynthesis and 55 CDSs related to monosaccharide metabolism.     

Reduction of azo dyes by redox mediators originating in the naphthalenesulfonic acid degradation pathway of Sphingomonas sp. strain BN6.

The anaerobic reduction of azo dyes by Sphingomonas sp. strain BN6 was analyzed. Aerobic conversion of 2-naphthalenesulfonate (2NS) by cells of strain BN6 stimulated the subsequent anaerobic reduction of the sulfonated azo dye amaranth at least 10-fold. In contrast, in crude extracts, the azo reductase activity was not stimulated. A mutant of strain BN6 which was not able to metabolize 2NS showed increased amaranth reduction rates only when the cells were resuspended in the culture supernatant of 2NS-grown BN6 wild-type cells. The same increase could be observed with different bacterial strains. This suggested the presence of an extracellular factor which was formed during the degradation of 2NS by strain BN6. The addition of 1,2-dihydroxynaphthalene, the first intermediate of the degradation pathway of 2NS, or its decomposition products to cell suspensions of the mutant of strain BN6 (2NS-) increased the activity of amaranth reduction. The presence of bacterial cells was needed to maintain the reduction process. Thus, the decomposition products of 1,2-dihydroxynaphthalene are suggested to act as redox mediators which are able to anaerobically shuttle reduction equivalents from the cells to the extracellular azo dye.     

Purification and Properties of Gentisate 1,2-Dioxygenase from Rhodococcus erythropolis S-1

Gentisate 1,2-dioxygenase, which participates in salicylate and m-hydroxybenzoate metabolism, was purified from cell-free extracts of Rhodococcus erythropolis S-1, a Gram-positive bacterium. The purified enzyme gave a single band on native PAGE and SDS–PAGE. The molecular mass of the enzyme was estimated to be 328 kDa. The structure of the enzyme appears to be an octamer of identical subunits. The enzyme from this bacterium was similar in general enzymatic properties to a gentisate 1,2-dioxygenase from a Gram-negative bacterium except for molecular mass and structure.     

Two kinds of chlorocatechol 1,2-dioxygenase from 2,4-dichlorophenoxyacetate-degrading Sphingomonas sp. strain TFD44

Two kinds of chlorocatechol 1,2-dioxygenase (CCD), TfdC and TfdC2 were detected in Sphingomonas sp. strain TFD44. These two CCDs could be simultaneously synthesized in TFD44 during its growth with 2,4-D as the sole carbon and energy sources. The apparent subunit molecular masses of TfdC and TfdC2 estimated by SDS-PAGE analysis were 33.8 and 33.1 kDa, respectively. The genes encoding the two CCDs were cloned and expressed in Escherichia coli. The two purified CCDs showed broad substrate specificities but had different specificity patterns. TfdC showed the highest specificity constant for 3-chlorocatechol and TfdC2 showed the highest specificity constant for 3,5-dichlorocatechol. The substrate specificity difference seemed to correlate with the alternation of amino acid supposed to be involved in the interaction with substrates. Whereas phylogenetic analysis indicated that the CCDs of Sphingomonas constitute a distinctive group among Gram-negative bacteria, TfdC and TfdC2 of TFD44 have divergently evolved in terms of their substrate specificity.     

Characterization of a 2,3-dihydroxybiphenyl dioxygenase from the naphthalenesulfonate-degrading bacterium strain BN6.

An extradiol dioxygenase was cloned from the naphthalenesulfonate-degrading bacterial strain BN6 by screening a gene bank for colonies with 2,3-dihydroxybiphenyl dioxygenase activity. DNA sequence analysis of a 1,358-bp fragment revealed an open reading frame of only 486 bp. This is the smallest gene encoding an extradiol dioxygenase found until now. Expression of the gene in a T7 expression vector enabled purification of the enzyme. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the protein was a dimer with a subunit size of 21.7 kDa. The enzyme oxidized 2,3-dihydroxybiphenyl, 3-isopropylcatechol, 3- and 4-chlorocatechol, and 3- and 4-methylcatechol. Since the ability to convert 3-chlorocatechol is an unusual characteristic for an extradiol-cleaving dioxygenase, this reaction was analyzed in more detail. The deduced amino-terminal amino acid sequence differed from the corresponding sequence of the 1,2-dihydroxynaphthalene dioxygenase, which had been determined earlier from the enzyme purified from this strain. This indicates that strain BN6 carries at least two different extradiol dioxygenases.     

Degradation of substituted naphthalenesulfonic acids by Sphingomonas xenophaga BN6

Sphingomonas xenophaga BN6 was isolated from the river Elbe as a member of a multispecies bacterial culture which mineralized 6-aminonaphthalene-2-sulfonate. Pure cultures of strain BN6 converted a wide range of amino- and hydroxynaphthalene-2-sulfonates via a catabolic pathway similar to that described for the metabolism of naphthalene to salicylate by Pseudomonas putida NAH7 or Pseudomonas sp NCIB 9816. In contrast to the naphthalene-degrading pseudomonads, S. xenophaga BN6 only partially degraded the naphthalenesulfonates and excreted the resulting amino- and hydroxysalicylates in almost stoichiometric amounts. Enzymes that take part in the degradative pathway of the naphthalenesulfonates by strain BN6 were purified, characterized and compared with the isofunctional enzymes from the naphthalene-degrading pseudomonads. According to the enzyme structures and the catalytic constants, no fundamental differences were found between the 1,2-dihydroxynaphthalene dioxygenase or the 2′-hydroxybenzalpyruvate aldolase from strain BN6 and the isofunctional enzymes from the naphthalene-degrading pseudomonads. The limited available sequence information about the enzymes from strain BN6 suggests that they show about 40–60% sequence identity to the isofunctional enzymes from the pseudomonads. In addition to the gene for the 1,2-dihydroxynaphthalene dioxygenase, the genes for two other extradiol dioxygenases were cloned and sequenced from strain BN6 and the corresponding gene products were studied. S. xenophaga BN6 has also been used as a model organism to study the mechanism of the non-specific reduction of azo dyes under anaerobic conditions and to establish combined anaerobic/aerobic treatment systems for the degradation of sulfonated azo dyes. Furthermore, the degradation of substituted naphthalenesulfonates by mixed cultures containing strain BN6 was studied in continuous cultures and was described by mathematical models. Received 02 April 1999/ Accepted in revised form 09 July 1999     

鞘氨醇单胞菌TP-3合成新型生物聚合物Ss的发酵条件优化

鞘氨醇单胞菌(Sphingomonas sp.)TP-3能合成一种具有增稠性、假塑性、成凝胶特性和乳化性能的新型生物聚合物Ss。运用单因素实验和均匀设计法对菌株TP-3合成聚合物Ss的发酵条件进行优化, 实验结果表明, 培养基组成为葡萄糖41.2 g/L, 豆饼粉2.0 g/L, NaCl 0.85 g/L, K2HPO4 1.46 g/L, MgSO4 0.12 g/L, MnCl2 0.0075 g/L, FeSO4 0.002 g/L, 初始pH为7.0, 在27°C, 180 r/min的条件下摇床培养60 h, 聚合物Ss的产量达到21.5 g/L。该聚合物生产成本低, 在油田开发中极具应用前景。     

Mineralization of 4-Chlorodibenzofuran by a Consortium Consisting of Sphingomonas sp. Strain RW1 and Burkholderia sp. Strain JWS

The dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1 (R.-M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) attacks 4-chlorodibenzofuran on the unsubstituted aromatic ring via distal dioxygenation adjacent to the ether bridge to produce 3(prm1)-chloro-2,2(prm1),3-trihydroxybiphenyl, which was identified by nuclear magnetic resonance spectroscopy and mass spectrometry. The compound is subsequently meta cleaved, and the respective intermediate is hydrolyzed to form a C-5 moiety, which is further degraded to Krebs cycle intermediates and to 3-chlorosalicylate. This dead-end product is released into the culture medium. A coculture of strain RW1 and the 3,5-dichlorosalicylate-degrading strain Burkholderia sp. strain JWS (A. Schindowski, R.-M. Wittich, and P. Fortnagel, FEMS Microbiol. Lett. 84:63-70, 1991) is able to completely degrade 4-chlorodibenzofuran with concomitant release of Cl(sup-) and formation of biomass.     

Purification and Characterization of a Three-Component Salicylate 1-Hydroxylase from Sphingomonas sp. Strain CHY-1

In the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs), salicylate hydroxylases catalyze essential reactions at the junction between the so-called upper and lower catabolic pathways. Unlike the salicylate 1-hydroxylase from pseudomonads, which is a well-characterized flavoprotein, the enzyme found in sphingomonads appears to be a three-component Fe-S protein complex, which so far has not been characterized. Here, the salicylate 1-hydroxylase from Sphingomonas sp. strain CHY-1 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component, designated PhnII, exhibited an α₃β₃ heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per α subunit. In the presence of purified reductase (PhnA4) and ferredoxin (PhnA3) components, PhnII catalyzed the hydroxylation of salicylate to catechol with a maximal specific activity of 0.89 U/mg and showed an apparent K_m for salicylate of 1.1 ± 0.2 μM. The hydroxylase exhibited similar activity levels with methylsalicylates and low activity with salicylate analogues bearing additional hydroxyl or electron-withdrawing substituents. PhnII converted anthranilate to 2-aminophenol and exhibited a relatively low affinity for this substrate (K_m, 28 ± 6 μM). 1-Hydroxy-2-naphthoate, which is an intermediate in phenanthrene degradation, was not hydroxylated by PhnII, but it induced a high rate of uncoupled oxidation of NADH. It also exerted strong competitive inhibition of salicylate hydroxylation, with a K_i of 0.68 μM. The properties of this three-component hydroxylase are compared with those of analogous bacterial hydroxylases and are discussed in light of our current knowledge of PAH degradation by sphingomonads.