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

Parales, Rebecca E.; Lee, Kyung Ho; Resnick, Sol M.; Jiang, Haiyan; Lessner, Daniel J.; Gibson, David T.

doi:10.1128/jb.182.6.1641-1649.2000

Cited by 182 publications

(143 citation statements)

References 60 publications

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“…The crystal structure also showed that the alternative NDO substrate indole binds near the mononuclear iron, suggesting that this is the oxygen-activating site (12). In support of this proposal, site-directed mutagenesis studies on NDO demonstrated that substitution of the hydrophobic residues lining the substrate binding pocket alter both the stereo-and regioselectivity of NDO (13). Also, the ligands of the mononuclear iron are one solvent, two histidines and one bidentate aspartic acid forming a 2-His 1-carboxylate "facial triad," as observed in many other O 2 -activating iron-containing enzymes (14,15).…”

mentioning

confidence: 53%

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

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174

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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mentioning

confidence: 53%

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

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174

311

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“…This amino acid corresponds to Thr-351 in the large subunit of naphthalene dioxygenase. However, the change of Thr-351 to Asn in naphthalene dioxygenase had a minor effect on product formation (32).…”

Section: Discussionmentioning

confidence: 99%

Alteration of Regiospecificity in Biphenyl Dioxygenase by Active-Site Engineering

et al. 2002

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Structure 6:571-586, 1998), we developed a three-dimensional model of KF707 BphA1 of Pseudomonas pseudoalcaligenes KF707. Based on structural information about the amino acids which coordinate the catalytic nonheme iron center, we constructed 12 site-directed BphA1 mutants with changes at positions 227, 332, 335, 376, 377, and 383 and expressed these enzymes in Escherichia coli. The Ile335Phe, Thr376Asn, and Phe377Leu Bph Dox mutants exhibited altered regiospecificities for various PCBs compared with wild-type Bph Dox. In particular, the Ile335Phe mutant acquired the ability to degrade 2,5,2,5-tetrachlorobiphenyl by 3,4-dioxygenation and showed bifunctional 2,3-dioxygenase and 3,4-dioxygenase activities for 2,5,2-trichlorobiphenyl and 2,5,4-trichlorobiphenyl. Furthermore, two mutants, the Phe227Val and Phe377Ala mutants, introduced molecular oxygen at the 2,3 position, forming 3-chloro-2,3-dihydroxy biphenyl with concomitant dechlorination.

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“…Substitiution of valine or leucine for Phe-352 near the active site iron in the ␣-subunit of NDO altered the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene and also changed the region of oxidation of biphenyl and phenanthrene (473,475).…”

Section: Enzymatic Upgrading Of Petroleum Fractions and Pure Hydrocarmentioning

confidence: 99%

Recent Advances in Petroleum Microbiology

Hamme¹,

Singh

Ward

2003

Microbiol Mol Biol Rev

1,201

640

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Recent advances in molecular biology have extended our understanding of the metabolic processes related to microbial transformation of petroleum hydrocarbons. The physiological responses of microorganisms to the presence of hydrocarbons, including cell surface alterations and adaptive mechanisms for uptake and efflux of these substrates, have been characterized. New molecular techniques have enhanced our ability to investigate the dynamics of microbial communities in petroleum-impacted ecosystems. By establishing conditions which maximize rates and extents of microbial growth, hydrocarbon access, and transformation, highly accelerated and bioreactor-based petroleum waste degradation processes have been implemented. Biofilters capable of removing and biodegrading volatile petroleum contaminants in air streams with short substrate-microbe contact times (<60 s) are being used effectively. Microbes are being injected into partially spent petroleum reservoirs to enhance oil recovery. However, these microbial processes have not exhibited consistent and effective performance, primarily because of our inability to control conditions in the subsurface environment. Microbes may be exploited to break stable oilfield emulsions to produce pipeline quality oil. There is interest in replacing physical oil desulfurization processes with biodesulfurization methods through promotion of selective sulfur removal without degradation of associated carbon moieties. However, since microbes require an environment containing some water, a two-phase oil-water system must be established to optimize contact between the microbes and the hydrocarbon, and such an emulsion is not easily created with viscous crude oil. This challenge may be circumvented by application of the technology to more refined gasoline and diesel substrates, where aqueous-hydrocarbon emulsions are more easily generated. Molecular approaches are being used to broaden the substrate specificity and increase the rates and extents of desulfurization. Bacterial processes are being commercialized for removal of H2S and sulfoxides from petrochemical waste streams. Microbes also have potential for use in removal of nitrogen from crude oil leading to reduced nitric oxide emissions provided that technical problems similar to those experienced in biodesulfurization can be solved. Enzymes are being exploited to produce added-value products from petroleum substrates, and bacterial biosensors are being used to analyze petroleum-contaminated environments

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

Cited by 182 publications

References 60 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

Alteration of Regiospecificity in Biphenyl Dioxygenase by Active-Site Engineering

Recent Advances in Petroleum Microbiology

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