Initial reactions in the oxidation of naphthalene by Pseudomonas putida

Jeffrey, Alan M.; Yeh, HJ; Jerina, Donald M.; Patel, Thakor R.; Davey, JF; Gibson, David T.

doi:10.1021/bi00674a018

Cited by 245 publications

(160 citation statements)

References 32 publications

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“…(ϩ)-cis-(1R,2S)-Dihydroxy-1,2-dihydronaphthalene (naphthalene cis-dihydrodiol) was prepared as described previously (26). Water was distilled and further purified using a Millipore reverse osmosis system.…”

Section: Methodsmentioning

confidence: 99%

Single Turnover Chemistry and Regulation of O2Activation by the Oxygenase Component of Naphthalene 1,2-Dioxygenase

Wolfe¹,

Parales²,

Gibson³

et al. 2001

Journal of Biological Chemistry

173

311

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Naphthalene 1,2-dioxygenase (NDOS) is a three-component enzyme that catalyzes cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene formation from naphthalene, O 2 , and NADH. We have determined the conditions for a single turnover of NDOS for the first time and studied the regulation of catalysis. As isolated, the ␣ 3 ␤ 3 oxygenase component (NDO) has up to three catalytic pairs of metal centers (one mononuclear Fe 2؉ and one diferric Rieske iron-sulfur cluster). This form of NDO is unreactive with O 2 . However, upon reduction of the Rieske cluster and exposure to naphthalene and O 2 , ϳ0.85 cisdiol product per occupied mononuclear iron site rapidly forms. Substrate binding is required for oxygen reactivity. Stopped-flow and chemical quench analyses indicate that the rate constant of the single turnover product-forming reaction significantly exceeds the NDOS turnover number. UV-visible and electron paramagnetic resonance spectroscopies show that during catalysis, one mononuclear iron and one Rieske cluster are oxidized per product formed, satisfying the two-electron reaction stoichiometry. The addition of oxidized or reduced NDOS ferredoxin component (NDF) increases both the product yield and rate of oxidation of formerly unreactive Rieske clusters. The results show that NDO alone catalyzes dioxygenase chemistry, whereas NDF appears to serve only an electron transport role, in this case redistributing electrons to competent active sites.Rieske nonheme iron dioxygenases catalyze a remarkable reaction in which dioxygen is cleaved and both atoms are inserted across a double bond of an unactivated aromatic nucleus to yield a cis-dihydrodiol (1-4). These enzymes initiate the biodegradation of some of the most recalcitrant aromatic compounds that enter the environment from both natural and industrial sources, making their study of great importance for progress in bioremediation practices (5, 6). In addition, the inherent enantio-and regiospecificity of these enzymes make them useful for synthetic applications (7,8). Several of these multicomponent dioxygenases are known that differ in the number of components and the number and type of subunits in the oxygenase component that contains the active site. However, all of the enzyme systems share the common features of a reductase component that can accept and pass on two electrons from reduced pyridine nucleotide, an electron transport system that may be encompassed into the reductase, and an oxygenase that has both Rieske [2Fe-2S] clusters and mononuclear iron centers.One of the most thoroughly studied of the Rieske nonheme iron dioxygenases is naphthalene 1,2-dioxygenase (NDOS) 1 isolated from Pseudomonas sp. NCIB 9816-4, which catalyzes the reaction shown in Scheme 1 to yield (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (9).The complete NDOS consists of three protein components: the 36-kDa reductase protein (NDR) that contains a FAD molecule and a plant-type [2Fe-2S] cluster, the 14-kDa ferredoxin electron transfer protein (NDF) that contains a [2Fe-2S] Rieske cluster, an...

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Section: Methodsmentioning

confidence: 99%

Single Turnover Chemistry and Regulation of O2Activation by the Oxygenase Component of Naphthalene 1,2-Dioxygenase

Wolfe¹,

Parales²,

Gibson³

et al. 2001

Journal of Biological Chemistry

173

311

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“…Wildtype NDO converts naphthalene to cis-naphthalene 1,2-dihydrodiol (30,31). Biotransformations with naphthalene resulted in the formation of cis-naphthalene 1,2-dihydrodiol by all NDO variants except those carrying the F202L and D362A substitutions, which formed no product.…”

Section: Constructionmentioning

confidence: 98%

Substrate Specificity of Naphthalene Dioxygenase: Effect of Specific Amino Acids at the Active Site of the Enzyme

et al. 2000

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The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydrophobic, which is consistent with the enzyme's preference for aromatic hydrocarbon substrates. Although NDO catalyzes cis-dihydroxylation of a wide range of substrates, it is highly regio-and enantioselective. Site-directed mutagenesis was used to determine the contributions of several active-site residues to these aspects of catalysis. Amino acid substitutions at Asn-201, Phe-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no effect on product formation with naphthalene or biphenyl as substrates and had slight but significant effects on product formation from phenanthrene. The naphthalene dioxygenase (NDO) enzyme system (EC 1.14.12.12) from Pseudomonas sp. strain NCIB 9816-4 catalyzes the first step in the aerobic degradation of naphthalene. In this reaction ( Fig. 1), NDO adds both atoms of oxygen to the aromatic nucleus of naphthalene, forming homochiral (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (cis-naphthalene dihydrodiol) (30, 31). In addition, NDO catalyzes the oxidation of a wide variety of aromatic compounds to enantiomerically pure chiral products (8, 56). The NDO system consists of three components, each of which has been purified and characterized. An iron-sulfur flavoprotein reductase and an iron-sulfur ferredoxin transfer electrons from NAD(P)H to the catalytic oxygenase component (14,15,23,24). The oxygenase is composed of large and small subunits, ␣ and ␤, respectively, that are in an ␣ 3 ␤ 3 configuration (35). NDO is a member of a large family of oxygenases whose ␣ subunits contain a Rieske [2Fe-2S] center and mononuclear nonheme iron (10). In the NDO system, electrons are transferred from the Rieske center of the ferredoxin to the Rieske center of the oxygenase ␣ subunit. The reduced Rieske center in one ␣ subunit transfers an electron to mononuclear iron at the active site in an adjacent ␣ subunit (35, 50). His-208, His-213, and Asp-362 coordinate the active-site iron, forming a 2-His-1-carboxylate facial triad. This structural motif is found in other mononuclear nonheme iron enzymes, including tyrosine hydroxylase, isopenicillin synthetase, and 2,3-dihydroxybiphenyl 1,2-dioxygenase (26, 40). Asp-205 in the catalytic domain of the NDO ␣ subunit is hydrogen bonded to His-208 and to His-104 in the adjacent ␣ subunit (Fig. 2). His-104 is one of the Rieske center ligands. Asp-205 has been shown to be required for efficient electron transfer from the Rieske center to the active-site iron (50).Recent studies have shown that the oxygenase ␣ subunits are responsible for determining the substrate specificities of NDO and the related enzymes 2-nitrotoluene dioxygenase (2NTDO) from Pseudomonas sp. strain JS42 and 2,4-dinitrotoluene dioxygenase (DNTDO) from Burkholderia sp. strain DNT (48...

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“…The initial reaction is catalyzed by a multicomponent enzyme system designated naphthalene dioxygenase (NDO) (11,12,23,24). NDO catalyzes the NAD(P)H-dependent enantiospecific incorporation of dioxygen into naphthalene to form (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (cis-naphthalene dihydrodiol) (26,27) (Fig. 1).…”

mentioning

confidence: 99%

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

et al. 1995

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Pseudomonas sp. strain 9816-4 grows with naphthalene as the sole source of carbon and energy (9). The initial reaction is catalyzed by a multicomponent enzyme system designated naphthalene dioxygenase (NDO) (11,12,23,24). NDO catalyzes the NAD(P)H-dependent enantiospecific incorporation of dioxygen into naphthalene to form (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (cis-naphthalene dihydrodiol) (26, 27) ( Fig. 1). An analogous reaction is catalyzed by toluene dioxygenase (TDO) from Pseudomonas putida F1, where enantiomerically pure (ϩ)-cis-(1S,2R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol) is the first detectable oxidation product (17,31,60). TDO also catalyzes the enantiospecific oxidation of naphthalene to (ϩ)-cis-naphthalene dihydrodiol (18,39).In addition to the enantiospecific oxidation of naphthalene and toluene, NDO and TDO from the above strains oxidize many related aromatic compounds to optically active dihydrodiols (10,18,28,30). Other bacterial dioxygenases show similar properties, and more than 130 chiral arene cis-dihydrodiols have been produced from a small number of strains (7,35,48). The high enantiomeric purity of these compounds has led to their use as chiral synthons in the enantiospecific synthesis of a wide variety of biologically active natural products (7,8,46,57). The present studies focus on another facet of this interesting group of dioxygenases, that is, their ability to catalyze reactions other than the formation of arene cis-dihydrodiols. For example, the TDO expressed by P. putida F39/D oxidizes indan to (1R)-indanol and oxidizes indene to cis-(1S,2R)-indandiol and (1S)-indenol (55). Similar reactions have been reported for TDO from P. putida UV4, although the 1-indenol produced by this strain is the (1R)-enantiomer (3, 5).We now report the identification and absolute stereochemistry of the products formed from indan and indene by NDO from Pseudomonas sp. strain 9816-4 and confirm earlier observations on the desaturation of indan to indene by NDO (22). MATERIALS AND METHODSOrganisms. Pseudomonas sp. strain 9816/11 is a mutant which oxidizes naphthalene stoichiometrically to (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (40). This organism is a derivative of Pseudomonas sp. strain 9816-4 (9, 59), which harbors the genes for naphthalene catabolism on an 83-kb NAH plasmid designated pDTG1 (45). Pseudomonas sp. strain 9816/C84, a cured strain, was used as a control in experiments with strain 9816/11. Escherichia coli strain JM109 (DE3)[pDTG141] contains the structural genes (nahAaAbAcAd) for NDO in plasmid pT7-5 (50). Expression of NDO in this strain is inducible by the addition of isopropylthiogalactopyranoside (IPTG). E. coli JM109(DE3)[pT7-5] was used as a control in experiments with strain JM109(DE3) [pDTG141].Biotransformation experiments. Strain 9816/11 was grown at 30ЊC in mineral salts basal medium (MSB) (49) with 0.2% (wt/vol) pyruvate as a carbon source in the presence of 0.05% (wt/vol) salicylate or anthranilate. These aromatic acids induce the s...

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Initial reactions in the oxidation of naphthalene by Pseudomonas putida

Cited by 245 publications

References 32 publications

Single Turnover Chemistry and Regulation of O2Activation by the Oxygenase Component of Naphthalene 1,2-Dioxygenase

Single Turnover Chemistry and Regulation of O2Activation by the Oxygenase Component of Naphthalene 1,2-Dioxygenase

Substrate Specificity of Naphthalene Dioxygenase: Effect of Specific Amino Acids at the Active Site of the Enzyme

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

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