2,3-Dihydroxybenzoate pathway in Pseudomonas putida. 1H n.m.r. study on the ring-cleavage site.

1. Ring cleavage of 2,3-dihydroxybenzoate by cell-free extracts of Pseudomonas putida leads to 2-hydroxy-6-oxo-(2Z,4E)-hexa-2,4-dienoic acid and CO2. 2. The 1H n.m.r. spectrum of the ring-fission product obtained in a 2H2O solution suggests that the extra-diol cleavage occurs between C-3 and C-4.     

3-(2-hydroxyphenyl)catechol as substrate for proximal meta ring cleavage in dibenzofuran degradation by Brevibacterium sp. strain DPO 1361.

Brevibacterium sp. strain DPO 1361 oxygenates dibenzofuran in the unusual angular position. The 3-(2-hydroxyphenyl)catechol thus generated is subject to meta ring cleavage in the proximal position, yielding 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid, which is hydrolyzed to 2-oxo-4-pentenoate and salicylate by 2-hydroxy-6-oxo-6-phenyl-2,4-hexadienoic acid hydrolase. The proximal mode of ring cleavage is definitely established by isolation and unequivocal structural characterization of a cyclization product of 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid, i.e., 3-(chroman-4-on-2-yl)pyruvate.     

Degradation of 2-sec-butylphenol: 3-sec-butylcatechol,2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid,and 2-methylbutyric acid as intermediates

Pseudomonas sp. strain HBP1 Prp, a mutant of strain HBP1 that was originally isolated on 2-hydroxybiphenyl, was able to grow on 2-sec-butylphenol as the sole carbon and energy source. During growth on 2-sec-butylphenol, 2-methylbutyric acid transiently accumulated in the culture medium. Its concentration reached a maximum after 20 hours and was below detection limit at the end of the growth experiment. The first three enzymes of the degradation pathway — a NADH-dependent monooxygenase, a metapyrocatechase, and ameta-fission product hydrolase — were partially purified. The product of the the monooxygenase reaction was identified as 3-sec-butylcatechol by mass spectrometry. This compound was a substrate for the metapyrocatechase and was converted to 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid which was identified by gas chromatography-mass spectrometry of its trimethylsilyl-derivative. The cofactor independentmeta-cleavage product hydrolase used 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid as a substrate. All three enzymes showed highest activities for 2-hydroxybiphenyl and its metabolites, respectively, indicating that 2-sec-butylphenol is metabolized via the same pathway as 2-hydroxybiphenyl.     

Purification and properties of 2-hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoic acid hydrolase involved in microbial degradation of carbazole

Hydrolysis following meta-ring cleavage by a dioxygenase is a well-known step in aromatic compound metabolism. The 2-hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoic acid hydrolase from Pseudomonas LD2 is a new member of the small group of characterized aromatic hydrolases that catalyze the cleavage of C-C bonds. In this study, the His(6)-tagged 2-hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoic acid (HOPDA) hydrolase was purified from a recombinant Escherichia coli strain utilizing immobilized metal affinity chromatography. 2-Hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoic acid hydrolase is a colorless homodimer with no cofactor requirement. The enzyme actively converted HOPDA into benzoic acid and 2-hydroxypenta-2,4-dienoic acid. The enzyme exhibited activity between pH 6.5 and 10.5 with a maximum activity at pH 7.0. The optimum temperature at pH 7.0 was 60 degrees C. The calculated K'(m) for HOPDA was 4.6 microM, the V(max) was 3.3 micromol min(-1), and the K(s) was 70.0 microM. This corresponds to a maximum specific turnover rate of 1300 HOPDAs(-1)dimer(-1). The deduced amino acid sequence of CarC showed 30.3, 31.3, and 31.8% identity with TodF (P. putida F1), XylF (P. putida), and DmpD (Pseudomonas sp. CF600), respectively, which are meta-cleavage compound hydrolases from other Pseudomonads. The amino acid sequence Gly-X-Ser-X-Gly, which is highly conserved in these hydrolases, is also found in CarC. Lysates from a strain expressing enzyme in which the putative active site serine is mutated to alanine showed a significant reduction in activity.     

Metabolism of biphenyl. Structure and physicochemical properties of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the meta-cleavage product from 2,3-dihydroxybiphenyl by Pseudomonas putida

The structure of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid for the meta-cleavage product of 2,3-dihydroxybiphenyl by a Pseudomonas putida strain was demonstrated on the basis of its chemical and physicochemical properties and those of its derivatives.     

Metabolism of biphenyl. 2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate: the meta-cleavage product from 2,3-dihydroxybiphenyl by Pseudomonas putida

1. 2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid was isolated and identified from washed suspensions of Pseudomonas putida incubated in the presence of 2,3-dihydroxybiphenyl. 2. Benzoic acid was isolated from reaction mixtures of crude cell-free extracts incubated with 2,3-dihydroxybiphenyl. 3. The presence in the same reaction mixtures of either 4-hydroxy-2-oxovalerate or 2-hydroxypenta-2,4-dienoate was suggested by mass spectrometry. 4. The degradative pathway of biphenyl is discussed.     

Analysis of cumene (isopropylbenzene) degradation genes from Pseudomonas fluorescens IP01.

We obtained the DNA fragments encoding 2-hydroxy-6-oxo-7-methylocta-2,4-dienoic acid (HOMODA) hydrolase in the cumene (isopropylbenzene) degrader Pseudomonas fluorescens strain IP01 via PCR using two synthesized oligonucleotides corresponding to the conserved regions within known meta-cleavage compound hydrolases. Following colony hybridization using the amplified DNA as a probe, a 4.5-kb HindIII fragment was isolated from P. fluorescens IP01. After determining the nucleotide sequence of this fragment, three open reading frames (ORF11 [cumH], ORF12 [cumD], and ORF13) were identified. The deduced amino acid sequence of ORF12 showed homology with meta-cleavage compound hydrolases encoded by the tod, dmp, xyl, and bph operons. Although the product of ORF12 was found to exhibit HOMODA and 2-hydroxy-6-oxohepta-2,4-dienoic acid (HOHDA) hydrolase activities, it did not exhibit 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase activity. The deduced amino acid sequence of ORF11 showed 40.4% homology with the sequence of todX in Pseudomonas putida F1 (Y. Wang, M. Ralings, D. T. Gibson, D. Labbé, H. Bergeron, R. Brousseau, and P. C. K. Lau, Mol. Gen. Genet. 246:570-579, 1995). The nucleotide sequence of ORF13 and its flanking region showed strong homology (91.0%) with IS52 from Pseudomonas savastanoi (Y. Yamada, P.-D. Lee, and T. Kosuge, Proc. Natl. Acad. Sci. USA 83:8263-8267, 1982). By characterization of cumH and cumD, the entire cum gene cluster from the cumene-degrader P. fluorescens IP01 (cumA1A2A3A4BCEGFHD) has been identified.     

Purification and characterization of meta-cleavage compound hydrolase from a carbazole degrader Pseudomonas resinovorans strain CA10

2-Hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4dienoic acid [6-(2'-aminophenyl)-HODA] hydrolase, involved in carbazole degradation by Pseudomonas resinovorans strain CA10, was purified to near homogeneity from an overexpressing Escherichia coli strain. The enzyme was dimeric, and its optimum pH was 7.0-7.5. Phylogenetic analysis showed the close relationship of this enzyme to other hydrolases involved in the degradation of monocyclic aromatic compounds, and this enzyme was specific for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (6-phenyl-HODA), having little activity toward 2-hydroxy-6-oxohepta-2,4-dienoic acid and 2-hydroxymuconic semialdehyde. The enzyme had a Km of 2.51 microM and k(cat) of 2.14 (s(-1)) for 6-phenyl-HODA (50 mM sodium phosphate, pH 7.5, 25 degrees C). The effect of the presence of an amino group or hydroxyl group at the 2'-position of phenyl moiety of 6-phenyl-HODA on the enzyme activity was found to be small; the activity decreased only in the order of 6-(2'-aminophenyl)-HODA (2.44 U/mg) > 6-phenyl-HODA (1.99 U / mg) > 2-hydroxy-6-oxo-6-(2'-hydroxyphenyl)-hexa-2,4-dienoic acid (1.05 U/mg). The effects of 2'-substitution on the activity were in accordance with the predicted reactivity based on the calculated lowest unoccupied molecular orbital energy for these substrates.     

A New Metabolic Pathway for meta Ring-fission Compounds of Biphenyl

The double bonds of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) were stabilized by methylation to establish which of the double bonds of the meta ring-fission compound of biphenyl was reduced by the HOPDA reducing enzyme. HOPDA reducing enzyme III converted 2-methoxy-6-oxo-6-phenylhexa-2,4-dienoic acid methyl ester into 2-methoxy-6-oxo-6-phenylhexa-2-enoic acid methyl ester. To discover the metabolic pathway of HOPDA, partially purified enzyme fractions were used. The eluate from a 2nd column of DEAE-cellulose transformed HOPDA to γ-benzoylbutyric acid, 2,6-dioxo-6-phenylhexanoic acid, and γ-benzoylbutyraldehyde. Fractions passed through the 1st column of DEAE-cellulose formed γ-benzoylbutyric acid and 2-hydroxy-6-oxo-6-phenylhexanoic acid from HOPDA. Based on these data and previous reports, a new metabolic divergence of biphenyl and related compounds was proposed.     

Rhodococcus sp. R04 BphD催化C-C断裂的动力学分析

2-羟基-6-氧-6-苯基己-2,4-二烯酸水解酶（BphD）是一种多氯联苯微生物降解途径中的关键酶. 本文通过紫外-可见光光谱分别对突变酶S110A和H265A催化过程中酶-底物复合物进行检测,同时利用停流光谱技术对BphD及其突变酶（S110A、H265A和W266A）催化底物2-羟基-6-氧-6-苯基己-2,4-二烯酸（HOPDA）前稳态动力学进行了研究.结果表明,在BphD催化C-C断裂过程中,产物2-羟基戊-2,4-二烯酸（HPD）迅速生成,其速率常数为22 S-1. 底物的消耗（速率常数,22022 S-1和803 S-1）及酶-底物复合物的变化（速率常数,55556 S-1和664 S-1）表明该酶催化过程包括2个动力学阶段：快速底物酮基化作用和C-C键断裂过程.紫外-可见光光谱扫描结果显示,在突变酶S110A的催化过程中,酶-底物复合物在492 nm及510 nm处有最大光吸收,而在突变酶H265A催化中,却没有相似的光吸收,只是在480 nm产生1个新肩峰. BphD及其突变酶S110A、H265A和W266A动力学分析表明,Ser-110主要负责底物C-C键断裂;His-265负责底物由烯醇式向酮式转变,并且与Ser-110和Trp-266共同参与了随后的C-C键断裂过程. 结果揭示,除了传统的催化三联体（Ser-110,Asp-237,His-265）外,Trp-266在该水解酶催化反应中也发挥非常重要的作用,这一发现丰富了C-C水解酶的反应动力学机制.     

Identification of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, 4-hydroxy-2-oxohexanoic acid, and 2-hydroxyhexa-2,4-dienoic acid and related enzymes involved in testosterone degradation in Comamonas testosteroni TA441

Comamonas testosteroni TA441 utilizes testosterone via aromatization of the A ring followed by meta-cleavage of the ring. The product of the meta-cleavage reaction, 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid, is degraded by a hydrolase, TesD. We directly isolated and identified two products of TesD as 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and (2Z,4Z)-2-hydroxyhexa-2,4-dienoic acid. The latter was a pure 4Z isomer. 2-Hydroxyhexa-2,4-dienoic acid was converted by a hydratase, TesE, and the product isolated from the reaction solution was identified as 2-hydroxy-4-hex-2-enolactone, indicating the direct product of TesE to be 4-hydroxy-2-oxohexanoic acid.     

Biodegradation of 2,4-Dichlorophenol through a Distal meta-Fission Pathway

Alcaligenes eutrophus JMP222, a derivative of A. eutrophus JMP134 which has lost plasmid pJP4 (encoding the tfd genes for the ortho fission pathway), was induced for the meta fission pathway when grown on o-cresol. Resting cell suspensions, grown on o-cresol, oxidized 2,4-dichlorophenol (2,4-DCP), a degradation product of 2,4-dichlorophenoxyacetic acid, to 3,5-dichlorocatechol. Further degradation of 3,5-dichlorocatechol was observed by the production of a yellow ring fission product with liberation of chloride. Oxidation of 2,4-DCP (305 (mu)M) in 47 hs resulted in 69% dehalogenation through this pathway. The ring fission product was characterized as 2-hydroxy-3,5-dichloro-6-oxo-hexa-2,4-dienoic acid by gas chromatography-mass spectrometry and gas chromatography-Fourier transform infrared spectroscopy. These data indicate that 2,4-DCP is degraded through a distal meta ring fission pathway, in contrast to either a suicidal proximal fission or the standard ortho fission pathway.     

Metabolism of 2,2'-dihydroxybiphenyl by Pseudomonas sp. strain HBP1: production and consumption of 2,2',3-trihydroxybiphenyl.

Cells of Pseudomonas sp. strain HBP1 grown on 2-hydroxy- or 2,2'-dihydroxybiphenyl contain NADH-dependent monooxygenase activity that hydroxylates 2,2'-dihydroxybiphenyl. The product of this reaction was identified as 2,2',3-trihydroxybiphenyl by 1H nuclear magnetic resonance and mass spectrometry. Furthermore, the monooxygenase activity also hydroxylates 2,2',3-trihydroxybiphenyl at the C-3' position, yielding 2,2',3,3'-tetrahydroxybiphenyl as a product. An estradiol ring cleavage dioxygenase activity that acts on both 2,2',3-tri- and 2,2',3,3'-tetrahydroxybiphenyl was partially purified. Both substrates yielded yellow meta-cleavage compounds that were identified as 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid and 2-hydroxy-6-(2,3-dihydroxyphenyl)-6-oxo-2,4-hexadienoic acid, respectively, by gas chromatography-mass spectrometry analysis of their respective trimethylsilyl derivatives. The meta-cleavage products were not stable in aqueous incubation mixtures but gave rise to their cyclization products, 3-(chroman-4-on-2-yl)pyruvate and 3-(8-hydroxychroman-4-on-2-yl)pyruvate, respectively. In contrast to the meta-cleavage compounds, which were turned over to salicylic acid and 2,3-dihydroxybenzoic acid, the cyclization products are not substrates to the meta-cleavage product hydrolase activity. NADH-dependent salicylate monooxygenase activity catalyzed the conversions of salicylic acid and 2,3-dihydroxybenzoic acid to catechol and pyrogallol, respectively. The partially purified estradiol ring cleavage dioxygenase activity that acted on the hydroxybiphenyls also produced 2-hydroxymuconic semialdehyde and 2-hydroxymuconic acid from catechol and pyrogallol, respectively.     

Identification of 9,17-Dioxo-1,2,3,4,10,19-Hexanorandrostan-5-oic Acid, 4-Hydroxy-2-Oxohexanoic Acid, and 2-Hydroxyhexa-2,4-Dienoic Acid and Related Enzymes Involved in Testosterone Degradation in Comamonas testosteroni TA441

Comamonas testosteroni TA441 utilizes testosterone via aromatization of the A ring followed by meta-cleavage of the ring. The product of the meta-cleavage reaction, 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid, is degraded by a hydrolase, TesD. We directly isolated and identified two products of TesD as 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and (2Z,4Z)-2-hydroxyhexa-2,4-dienoic acid. The latter was a pure 4Z isomer. 2-Hydroxyhexa-2,4-dienoic acid was converted by a hydratase, TesE, and the product isolated from the reaction solution was identified as 2-hydroxy-4-hex-2-enolactone, indicating the direct product of TesE to be 4-hydroxy-2-oxohexanoic acid.     

The microbial degradation of phenylalkanes. 2-Phenylbutane, 3-phenylpentane, 3-phenyldodecane and 4-phenylheptane

1. Two Pseudomonas strains capable of utilizing 2-phenylbutane, 3-phenylpentane and 4-phenylheptane as the sole carbon and energy source were isolated. 2. Two Nocardia strains capable of utilizing only 3-phenyldodecane as the sole carbon and energy source were isolated. 3. All the isolated strains were unable to grow on the corresponding phenylalkane-p-sulphonates. 4. From liquid cultures of Pseudomonas strains utilizing 2-phenylbutane, 2-(2,3-dihydro-2,3-dihydroxyphenyl)butane was isolated and identified. Evidence for a meta cleavage of the benzene ring was also obtained. 5. From liquid cultures of Pseudomonas strains utilizing 3-phenylpentane, 3-(2,3-dihydro-2,3-dihydroxyphenyl)pentane and 2-hydroxy-7-ethyl-6-oxonona-2,4-dienoic acid were isolated and identified. 6. Evidence for the formation of both a diol and a meta-cleavage compound was obtained from liquid cultures of both Pseudomonas strains utilizing 4-phenylheptane. 7. Liquid cultures of both Nocardia strains utilizing 3-phenyldodecane never formed a diol or a semialdehyde-related compound. 2-Phenylbutyric acid, 3-phenylvaleric acid and 4-phenylhexanoic acid were shown to be present in these cultures.     

Genetic structures of the genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase from biphenyl- and 4-chlorobiphenyl-degrading Pseudomonas sp. strain DJ-12.

The pcbC and pcbD genes of Pseudomonas sp. strain DJ-12, a natural isolate degrading biphenyl and 4-chlorobiphenyl, encode the 2,3-dihydroxybiphenyl 1,2-dioxygenase and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase, respectively. The two genes were sequenced and appear to be present in the order pcbD-pcbC as an operon.     

Metabolism of dibenzo-p-dioxin by Sphingomonas sp. strain RW1.

In the course of our screening for dibenzo-p-dioxin-utilizing bacteria, a Sphingomonas sp. strain was isolated from enrichment cultures inoculated with water samples from the river Elbe. The isolate grew with both the biaryl ethers dibenzo-p-dioxin and dibenzofuran (DF) as the sole sources of carbon and energy, showing doubling times of about 8 and 5 h, respectively. Biodegradation of the two aromatic compounds initially proceeded after an oxygenolytic attack at the angular position adjacent to the ether bridge, producing 2,2',3-trihydroxydiphenyl ether or 2,2',3-trihydroxybiphenyl from the initially formed dihydrodiols, which represent extremely unstable hemiacetals. Results obtained from determinations of enzyme activities and oxygen consumption suggest meta cleavage of the trihydroxy compounds. During dibenzofuran degradation, hydrolysis of 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)-hexa-2,4-dienoate yielded salicylate, which was branched into the catechol meta cleavage pathway and the gentisate pathway. Catechol obtained from the product of meta ring fission of 2,2',3-trihydroxydiphenyl ether was both ortho and meta cleaved by Sphingomonas sp. strain RW1 when this organism was grown with dibenzo-p-dioxin.     

Metabolism of dibenzo-p-dioxin by Sphingomonas sp. strain RW1.

In the course of our screening for dibenzo-p-dioxin-utilizing bacteria, a Sphingomonas sp. strain was isolated from enrichment cultures inoculated with water samples from the river Elbe. The isolate grew with both the biaryl ethers dibenzo-p-dioxin and dibenzofuran (DF) as the sole sources of carbon and energy, showing doubling times of about 8 and 5 h, respectively. Biodegradation of the two aromatic compounds initially proceeded after an oxygenolytic attack at the angular position adjacent to the ether bridge, producing 2,2',3-trihydroxydiphenyl ether or 2,2',3-trihydroxybiphenyl from the initially formed dihydrodiols, which represent extremely unstable hemiacetals. Results obtained from determinations of enzyme activities and oxygen consumption suggest meta cleavage of the trihydroxy compounds. During dibenzofuran degradation, hydrolysis of 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)-hexa-2,4-dienoate yielded salicylate, which was branched into the catechol meta cleavage pathway and the gentisate pathway. Catechol obtained from the product of meta ring fission of 2,2',3-trihydroxydiphenyl ether was both ortho and meta cleaved by Sphingomonas sp. strain RW1 when this organism was grown with dibenzo-p-dioxin.     

Bacterial metabolism of side-chain-fluorinated aromatics: unproductive meta-cleavage of 3-trifluoromethylcatechol

Summary Sixteen bacterial strains capable of degrading alkylbenzenes and alkylphenols were directly isolated from soil and water. The degradation pathways are discussed. Alkylcatechols are almost exclusively cleaved via meta-ring fission. Meta-cleavage of 3-trifluoromethyl-(TFM)-catechol was observed with all strains at different rates although the reaction rates compared to catechol as a substrate varied considerably. All 2-hydroxy-6-oxohepta-2,4-dienoic acid hydrolases investigated showed strong binding of 7,7,7-trifluoro-2-hydroxy-6-oxohepta-2,4-dienoic acid, the ring fission product of 3-TFM-catechol. Turnover rates, however, were negligible indicating this compound to be a general dead-end metabolite during metabolism of TFM-substituted compounds via meta-cleavage pathways.Offprint requests to: K.-H. Engesser     

Purification of two isofunctional hydrolases (EC 3.7.1.8) in the degradative pathway for dibenzofuran inSphingomonas sp. strain RW1

Sphingomonas sp. strain RW1, when grown in salicylate-salts medium, synthesized the enzymes for the degradation of dibenzofuran. The reaction subsequent tometa cleavage of the first benzene ring was found to be catalyzed by two isofunctional hydrolases, H1 and H2, which were purified by chromatography on anion exchange, hydrophobic interaction and gel filtration media. Each enzyme was able to hydrolze 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)hexa-2,4-dienoate and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate to produce salicylate and benzoate, respectively. SDS/PAGE of each purified enzyme showed a single band ofM _r 31 000 (H1) or 29 000 (H2). The N-terminal amino acid sequences of the two proteins showed 50% homology.Abbreviations DHB 2,3-dihydroxybiphenyl - DSM German Culture Collection (Braunschweig) - FPLC protein liquid chromatograph(y) - HOHPDA 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)hexa-2,4-dienoate - HOPDA 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate - THB 2,2,3-trihydroxybiphenyl