Toluene and ethylbenzene oxidation by purified naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

Purified naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 oxidized toluene to benzyl alcohol and benzaldehyde by reactions involving benzylic monooxygenation and dioxygen-dependent alcohol oxidation, respectively. Xylene and nitrotoluene isomers were also oxidized to substituted benzyl alcohol and benzaldehyde derivatives. NDO oxidized ethylbenzene sequentially through (S)-1-phenethyl alcohol (77% enantiomeric excess) and acetophenone to 2-hydroxyacetophenone. In addition, NDO also oxidized ethylbenzene through styrene to (R)-1-phenyl-1,2-ethanediol (74% enantiomeric excess) by reactions involving desaturation and dihydroxylation, respectively. Isotope experiments with 18O2, H2 18O, and D2O suggest that 1-phenethyl alcohol is oxidized to acetophenone by a minor reaction involving desaturation followed by tautomerization. The major reaction in the conversion of 1-phenethyl alcohol and benzyl alcohol to acetophenone and benzaldehyde, respectively, probably involves monohydroxylation to form a gem-diol intermediate which stereospecifically loses the incoming hydroxyl group to leave the carbonyl product. These results are compared with similar reactions catalyzed by cytochrome P-450.     

Diverse reactions catalyzed by naphthalene dioxygenase fromPseudomonas sp strain NCIB 9816

Naphthalene dioxygenase (NDO) fromPseudomonas sp strain NCIB 9816 is a multicomponent enzyme system which initiates naphthalene catabolism by catalyzing the addition of both atoms of molecular oxygen and two hydrogen atoms to the substrate to yield enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. NDO has a relaxed substrate specificity and catalyzes the dioxygenation of many related 2- and 3-ring aromatic and hydroaromatic (benzocyclic) compounds to their respectivecis-diols. Biotransformations with a diol-accumulating mutant, recombinant strains and purified enzyme components have established that in addition tocis-dihydroxylation, NDO also catalyzes a variety of other oxidations which include monohydroxylation, desaturation (dehydrogenation),O-andN-dealkylation and sulfoxidation reactions. In several cases, the absolute stereochemistry of the oxidation products formed by NDO are opposite to those formed by toluene dioxygenase (TDO). The reactions catalyzed by NDO and other microbial dioxygenases can yield specific hydroxylated compounds which can serve as chiral synthons in the preparation of a variety of compounds of interest to pharmaceutical and specialty chemical industries. We present here recent work documenting the diverse array of oxidation reactions catalyzed by NDO. The trends observed in the oxidation of a series of benzocyclic aromatic compounds are compared to those observed with TDO and provide the basis for prediction of regio- and stereospecificity in the oxidation of related substrates. Based on the types of reactions catalyzed and the biochemical characteristics of NDO, a mechanism for oxygen activation by NDO is proposed.     

Purification and properties of ferredoxinNAP, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816.

One of the three components of the naphthalene dioxygenase occurring in induced cells of Pseudomonas sp. strain NCIB 9816 has been purified to homogeneity. The protein contained 2 g-atoms each of iron and acid-labile sulfur and had an apparent molecular weight of 13,600. The evidence indicates that it is a ferredoxin-type protein that functions as an intermediate electron transfer protein in naphthalene dioxygenase activity.     

Oxidation of biphenyl by a multicomponent enzyme system from Pseudomonas sp. strain LB400.

Pseudomonas sp. strain LB400 grows on biphenyl as the sole carbon and energy source. This organism also cooxidizes several chlorinated biphenyl congeners. Biphenyl dioxygenase activity in cell extract required addition of NAD(P)H as an electron donor for the conversion of biphenyl to cis-2,3-dihydroxy-2,3-dihydrobiphenyl. Incorporation of both atoms of molecular oxygen into the substrate was shown with 18O2. The nonlinear relationship between enzyme activity and protein concentration suggested that the enzyme is composed of multiple protein components. Ion-exchange chromatography of the cell extract gave three protein fractions that were required together to restore enzymatic activity. Similarities with other multicomponent aromatic hydrocarbon dioxygenases indicated that biphenyl dioxygenase may consist of a flavoprotein and iron-sulfur proteins that constitute a short electron transport chain involved in catalyzing the incorporation of both atoms of molecular oxygen into the aromatic ring.     

Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816.

Cells of Pseudomonas sp. strain NCIB 9816, after growth with naphthalene or salicylate, contain a multicomponent enzyme system that oxidizes naphthalene to cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. We purified one of these components to homogeneity and found it to be an iron-sulfur flavoprotein that loses the flavin cofactor during purification. Dialysis against flavin adenine dinucleotide (FAD) showed that the enzyme bound 1 mol of FAD per mol of enzyme protein. The enzyme consisted of a single polypeptide with an apparent molecular weight of 36,300. The purified protein contained 1.8 g-atoms of iron and 2.0 g-atoms of acid-labile sulfur and showed absorption maxima at 278, 340, 420, and 460 nm, with a broad shoulder at 540 nm. The purified enzyme catalyzed the reduction of cytochrome c, dichlorophenolindophenol, Nitro Blue Tetrazolium, and ferricyanide. These activities were enhanced in the presence of added FAD. The ability of the enzyme to catalyze the reduction of the ferredoxin involved in naphthalene reduction and other electron acceptors indicates that it functions as an NAD(P)H-oxidoreductase in the naphthalene dioxygenase system. The results suggest that naphthalene dioxygenase requires two proteins with three redox groups to transfer electrons from NADH to the terminal oxygenase.     

Stereospecific dihydroxylation of the styrene vinyl group by purified naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

Naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 adds both atoms of the dioxygen molecule to styrene to form (R)-l-phenyl-1,2-ethanediol. Product formation is tightly coupled to dioxygen consumption and NADH oxidation. NDO oxidizes styrene-d8 at almost the same initial rate as styrene. The results indicate that dioxygen activation by NDO is different from that by cytochrome P-450 and other monooxygenases, which oxidize styrene to styrene 1,2-oxide.     

Regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

The regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene was examined with mutant and recombinant strains expressing naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. The initial oxidation products were isolated and identified by gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry. Salicylate-induced cells of Pseudomonas sp. strain 9816/11 and isopropyl-beta-D-thiogalactopyranoside-induced cells of Escherichia coli JM109(DE3)(pDTG141) oxidized fluorene to (+)-(3S,4R)-cis-3,4-dihydroxy-3,4-dihydrofluorene (80 to 90% relative yield; > 95% enantiomeric excess [ee]) and 9-fluorenol (< 10% yield). The same cells oxidized dibenzofuran to (1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzofuran (60 to 70% yield; > 95% ee) and (3S,4R)-cis-3, 4-dihydroxy-3,4-dihydrodibenzofuran (30 to 40% yield; > 95% ee). Induced cells of both strains, as well as the purified dioxygenase, also oxidized dibenzothiophene to (+)-(1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzothiophene (84 to 87% yield; > 95% ee) and dibenzothiophene sulfoxide (< 15% yield). The major reaction catalyzed by naphthalene dioxygenase with each substrate was stereospecific dihydroxylation in which the cis-dihydrodiols were of identical regiochemistry and of R configuration at the benzylic center adjacent to the bridgehead carbon atom. The regiospecific oxidation of dibenzofuran differed from that of the other substrates in that a significant amount of the minor cis-3,4-dihydrodiol regioisomer was formed. The results indicate that although the absolute stereochemistry of the cis-diene diols was the same, the nature of the bridging atom or heteroatom influenced the regiospecificity of the reactions catalyzed by naphthalene dioxygenase.     

Stereospecific oxidation of (R)- and (S)-1-indanol by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

A recombinant Escherichia coli strain which expresses naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 oxidized (S)-1-indanol to trans-(1S,3S)-indan-1,3-diol (95.5%) and (R)-3-hydroxy-1-indanone (4.5%). The same cells oxidized (R)-1-indanol to cis-1,3-indandiol (71%), (R)-3-hydroxy-1-indanone (18.2%), and cis-1,2,3-indantriol (10.8%). Purified NDO oxidized (S)-1-indenol to both syn- and anti-2,3-dihydroxy-1-indanol.     

Amino acid substitutions in naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 result in regio- and stereo-specific hydroxylation of flavanone and isoflavanone

Wild-type naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 transforms relatively planar flavone and isoflavone to cis-dihydrodiols. However, this enzyme cannot catalyze the transformation of flavanone and isoflavanone in which a phenyl group bonds to the stereogenic C2 or C3 of the C-ring. Protein modeling suggested that Phe224 in the substrate binding site of NDO may play a key role in substrate specificity toward flavanone and isoflavanone. Site-directed mutants of NDO with substitution of Phe224 with Tyr biotransformed only the (S)-stereoisomers of flavanone and isoflavanone, producing an 8-OH group on the A-ring. In contrast, the Phe224Cys and Phe224Gln substitutions, which used (2S)-flavanone as a substrate, and Phe224Lys, which transformed (2S)-flavanone and (3S)-isoflavanone, each showed lower activity than the Phe224Tyr substitution. The remainder of the tested mutants had no activity with flavanone and isoflavanone. Protein docking studies of flavanone and isoflavanone to the modeled mutant enzyme structures revealed that an expanded substrate binding site, due to mutation at 224, as well as appropriate hydrophobic interaction with the residue at 224, are critical for successful binding of the substrates. Results of this study also suggested that in addition to the previously known Phe352, the Phe224 site of NDO appears to be important site for expanding the substrate range of NDO and bringing regiospecific and stereospecific hydroxylation reactions to C8 of the flavanone and isoflavanone A-rings.     

Cloning,nucleotide sequence and characterization of genes encoding naphthalene dioxygenase of Pseudomonas putida strain NCIB9816

We have cloned the naphthalene dioxygenase(ND)-coding genes from Pseudomonas putida strain NCIB9816 based on their ability to convert indole to indigo. The region coding for this enzyme activity was sequenced and three successive open reading frames were found. The corresponding gene products were identified using the T7 polymerase/promoter system. All of them are necessary for the ND activity. A comparison of the ND-coding genes with the ones coding for benzene dioxygenase revealed significant homology which was more pronounced at the nucleotide level than at the amino acid level.     

Desaturation, dioxygenation, and monooxygenation reactions catalyzed by naphthalene dioxygenase from Pseudomonas sp. strain 9816-4.

The stereospecific oxidation of indan and indene was examined with mutant and recombinant strains expressing naphthalene dioxygenase of Pseudomonas sp. strain 9816-4. Pseudomonas sp. strain 9816/11 and Escherichia coli JM109(DE3)[pDTG141] oxidized indan to (+)-(1S)-indanol, (+)-cis-(1R,2S)-indandiol, (+)-(1S)-indenol, and 1-indanone. The same strains oxidized indene to (+)-cis-(1R,2S)-indandiol and (+)-(1S)-indenol. Purified naphthalene dioxygenase oxidized indan to the same four products formed by strains 9816/11 and JM109(DE3)[pDTG141]. In addition, indene was identified as an intermediate in indan oxidation. The major products formed from indene by purified naphthalene dioxygenase were (+)-(1S)-indenol and (+)-(1R,2S)-indandiol. The results show that naphthalene dioxygenase catalyzes the enantiospecific monooxygenation of indan to (+)-(1S)-indanol and the desaturation of indan to indene, which then serves as a substrate for the formation of (+)-(1R,2S)-indandiol and (+)-(1S)-indenol. The relationship of the desaturase, monooxygenase, and dioxygenase activities of naphthalene dioxygenase is discussed with reference to reactions catalyzed by toluene dioxygenase, plant desaturases, cytochrome P-450, methane monooxygenase, and other bacterial monooxygenases.     

Oxidation and dehalogenation of 4-chlorophenylacetate by a two-component enzyme system from Pseudomonas sp. strain CBS3

In cell-free extracts from Pseudomonas sp. strain CBS3 the conversion of 4-chlorophenylacetate to 3,4-dihydroxyphenylacetate was demonstrated. By Sephacryl S-200 chromatography two protein fractions, A and B, were obtained which both were essential for enzyme activity. Fe2+ and NADH were cofactors of the reaction. NADPH also activated the enzyme, but less effectively than NADH. FAD had no influence on enzyme activity. 4-Hydroxyphenylacetate, 4-chloro-3-hydroxyphenylacetate, and 3-chloro-4-hydroxyphenylacetate were poor substrates for the enzyme, suggesting that these substances are not intermediates of the reaction. We therefore suggest that the reaction proceeds via a dioxygenated intermediate.     

Metabolism of naphthalene, fluorene, and phenanthrene: preliminary characterization of a cloned gene cluster from Pseudomonas putida NCIB 9816.

A modified cloning procedure was used to obtain large DNA insertions (20 to 30 kb) from Pseudomonas putida NCIB 9816 that expressed polycyclic aromatic hydrocarbon (PAH) transformation activity in Escherichia coli HB101. Four subclones (16 [in both orientations], 12, and 8.5 kb in size) were constructed from the initial clones. Naphthalene, fluorene, and phenanthrene transformations were investigated in these eight NCIB 9816 clones by a simple agar plate assay method, which was developed to detect and identify potential PAH metabolites. Results indicated that the necessary genes encoding the initial ring fission of the three PAHs in E. coli cells are located in an 8.5-kb EcoRI-XhoI portion, but the lower-pathway genes are not present in a 38-kb neighborhood region. These NCIB 9816 clones could transform naphthalene and phenanthrene to salicylic acid and 1-hydroxy-2-naphthoic acid, respectively. With the same clones, fluorene was degraded to 9-hydroxyfluorene, 9-fluorenone, and two unidentified compounds. Genetic similarity between the NAH7 upper-pathway genes and the cloned NCIB 9816 genes was confirmed by Southern blot DNA-DNA hybridization. In spite of this genetic similarity, the abilities of the two clusters to transform multiple PAHs were different. Under our experimental conditions, only the metabolites from naphthalene transformation by the NAH7 clone (pE317) were detected, whereas the NCIB 9816 clones produced metabolites from all three PAHs.     

Complete sequence and genetic organization of pDTG1, the 83 kilobase naphthalene degradation plasmid from Pseudomonas putida strain NCIB 9816-4

The complete 83,042 bp sequence of the circular naphthalene degradation plasmid pDTG1 from Pseudomonas putida strain NCIB 9816-4 was determined in order to examine the process by which the nah and sal operons may have been compiled and distributed in nature. Eighty-nine open reading frames were predicted using computer analyses, comprising 80.0% of the pDTG1 DNA sequence. The most distinctive feature of the plasmid is the upper and lower naphthalene degradation operons, which occupy 9.5 kb and 13.4 kb regions, respectively, bordered by numerous defective mobile genetic element fragments. Identified on this plasmid were homologues of genes required for large plasmid replication, maintenance, and conjugation, as well as transposases, resolvases, and integrases, suggesting an evolution that involved the lateral transfer of DNA between bacterial species. Also found were genes that contain a high degree of sequence similarity to other known degradation genes, as well as genes involved in chemotaxis. Although the incompatibility group designation of pDTG1 remains unresolved, striking sequence organization and homology exists between the plasmid backbones of pDTG1 and the IncP-9 toluene-degradation plasmid pWW0, which suggests a divergent evolution from a progenitor plasmid prior to degradative gene incorporation.     

Catabolism of biphenyl by Pseudomonas sp. NCIB 10643 and Nocardia sp. NCIB 10503

Summary The metabolism of biphenyl by Pseudomonas sp. NCIB 10643 is reported in detail; that of Nocardia sp. NCIB 10503 is briefly investigated. Both organisms dissimilate biphenyl by the same route via oxidation to 2,3-dihydroxybiphenyl, meta cleavage to a product identified as 2-hydroxy-6-oxo-phenylhexa-2,4-dienoate which is then cleaved to give benzoate. Benzoate is a deadend metabolite in the pseudomonad but in the nocardia is further catabolised to catechol and thence to cis, cis-muconate. The enzymes involved in the individual steps of the proposed pathway have been assayed. The proposed pathway differs from that previously suggested for Pseudomonas sp. NCIB 10643 but is the same as found in other pseudomonads. This is the first report of catabolism of biphenyl in an actinomycete.     

Isolation and preliminary characterization of the subunits of the terminal component of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4.

The terminal oxygenase component (ISPNAP) of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4 was purified to homogeneity. The protein contained approximately 4 g-atoms each of iron and acid-labile sulfide per mol of ISPNAP, and enzyme activity was stimulated significantly by addition of exogenous iron. The large (alpha) and small (beta) subunits of ISPNAP were isolated by two different procedures. The NH2-terminal amino acid sequences of the alpha and beta subunits were identical to the deduced amino acid sequences reported for the ndoB and ndoC genes from P. putida NCIB 9816 and almost identical to the NH2-terminal amino acid sequences determined for the large and small subunits of ISPNAP from P. putida G7. Gel filtration in the presence of 6 M urea gave an alpha subunit with an absorption maximum at 325 nm and broad absorption between 420 and 450 nm. The alpha subunit contained approximately 2 g-atoms each of iron and acid-labile sulfide per mol of the subunit. The beta subunit did not contain iron or acid-labile sulfide. These results, taken in conjunction with the deduced amino acid sequences of the large subunits from several iron-sulfur oxygenases, indicate that each alpha subunit of ISPNAP contains a Rieske [2Fe-2S] center.     

Oxidative release of nitrite from 2-nitrotoluene by a three-component enzyme system from Pseudomonas sp. strain JS42.

Pseudomonas sp. strain JS42 utilizes 2-nitrotoluene (2NT) as the sole source of carbon and energy for growth. Intact cells catalyze the oxidation of 2NT to 3-methylcatechol and nitrite in a reaction that requires molecular oxygen. Cell extracts oxidized 2NT to 3-methylcatechol and nitrite in the presence of NAD(P)H and ferrous iron. Ion-exchange chromatography yielded three protein fractions (A, B, and C) which were all required for the oxidation of 2NT to 3-methylcatechol and nitrite. Component B (reductase2NT) catalyzed a NAD(P)H-dependent reduction of cytochrome c. Solutions of component A (ISP2NT) were brown and showed absorption maxima at 458 and 324 nm. Two major bands with M(r)s 52,500 and 28,000 were observed when ISP2NT was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Component C could be replaced by ferredoxin NAP from the Pseudomonas putida NCIB 9816-4 naphthalene dioxygenase system and was given the designation ferredoxin2NT. Experiments with 18O2 showed that both oxygen atoms were added to the aromatic ring of 2NT to yield 3-methylcatechol. The enzyme is a new multicomponent enzyme system which we have designated 2NT 2,3-dioxygenase.     

Catabolism of alkylbenzenes by Pseudomonas sp. NCIB 10643

Summary Pseudomonas sp. NCIB 10643 grew on a range of n-alkylbenzenes (C2-C7) and on several branched species within this chain size (isopropylbenzene, isobutylbenzene, sec-butylbenzene, tert-butylbenzene and tert-amylbenzene). All of the alkylbenzenes were catabolized via ring attack, rather than side-chain attack, proceeding via initial dioxygenase activity resulting in the corresponding 2,3-dihydro-2,3-dihydroxyalkylbenzene, which underwent reduction to the corresponding 2,3-dihydroxyl-intermediate (3-alkyl-substituted catechols). The 3-substituted catechols were ring-cleaved by an extra-diol type enzyme between C1 and C2 resulting in characteristic meta ring-fission products. Further catabolism was by hydrolytic attack to give alkyl-chain dependent carboxylic acids and, presumably, 2-oxopenta-4-enoate. Details of the intermediates and enzymes involved in alkylbenzene catabolism are given. This is the most versatile aromatic, ring-cleaving, alkylbenzene-utilizing bacterium thus far reported.Offprint requests to: C. Ratledge