p-hydroxylation reactions catalyzed by naphthalene dioxygenase

In this study, naphthalene dioxygenase is shown to catalyze the oxidation of methylphenols and chlorophenols by p- and/or o-hydroxylation reactions. For instance, m-cresol was oxidized to methylhydroquinone with formation of 3- and 4-methylcatechol as minor products. 2-Chlorophenol was exclusively oxidized to chlorohydroquinone, which is an important building block for pharmaceutical products and other organic compounds. The oxygen incorporated in the p-hydroxylation reaction from m-cresol is derived from water with consumption of O2.     

Episodic positive selection during the evolution of naphthalene dioxygenase to nitroarene dioxygenase

Using different maximum-likelihood models of adaptive evolution, signatures of natural selective pressure, operating across the naphthalene family of dioxygenases, were examined. A lineage- and branch-site specific combined analysis revealed that purifying selection pressure dominated the evolutionary history of the enzyme family. Specifically, episodic positive Darwinian selection pressure, affecting only a few sites in a subset of lineages, was found to be responsible for the evolution of nitroarene dioxygenases (NArDO) from naphthalene dioxygenase (NDO). Site-specific analysis confirmed the absence of diversifying selection pressure at any particular site. Different sets of positively selected residues, obtained from branch-site specific analysis, were detected for the evolution of each NArDO. They were mainly located around the active site, the catalytic pocket and their adjacent regions, when mapped onto the 3D structure of the α-subunit of NDO. The present analysis enriches the current understanding of adaptive evolution and also broadens the scope for rational alteration of substrate specificity of enzyme by directed evolution.     

Production of dyestuffs from indole derivatives by naphthalene dioxygenase and toluene dioxygenase

AIMS: To isolate and characterize the phorate [O,O-diethyl-S-(ethylthio)methyl phosphoradiothioate] degrading bacteria from agricultural soil, and their assessment for multifarious biological activities of environmental and agronomic significance. METHODS AND RESULTS: Based on their morphological and biochemical characteristics, the selected isolates PS-1, PS-2 and PS-3 were presumptively identified as Rhizobium, Pseudomonas and Proteus species, respectively. The HPLC analysis of phorate in bioaugmented soil revealed its complete disappearance within 40 days. The degradation isotherms of the isolates PS-1, PS-2 and PS-3 suggested time-dependent disappearance of phorate following the first-order rate kinetics at the corresponding rate constants of 0.04, 0.05 and 0.04 d-1. Besides, the isolates concurrently exhibited substantial phosphate solubilization, indole acetic acid (IAA) and siderophore production, as well as limited biocontrol activity against fungal phytopathogens. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The data revealed the potential of isolates for collateral plant growth promotion, biocontrol and bioremediation. The selected strains may serve as an important bioresource for development of effective super-bioinoculants.     

Similar enzymes, different structures: phthalate dioxygenase is an alpha3alpha3 stacked hexamer, not an alpha3beta3 trimer like "normal" Rieske oxygenases

Phthalate dioxygenase (PDO) is a member of a class of bacterial oxygenases that contain both Rieske [2Fe-2S] and Fe(II) mononuclear centers. Recent crystal structures of several Rieske dioxygenases showed that they exist as alpha(3)beta(3) multimers with subunits arranged head-to-tail in alpha and beta stacked planar rings. The structure of PDO, which consists of only alpha-subunits, remains to be solved. Although similar to other Rieske dioxygenases in many aspects, PDO was shown to differ in the mechanism of catalysis. Gel filtration and analytical centrifugation experiments, supplemented with mass spectrometric analysis (both ESI-MS and ESI-GEMMA), in this work showed a hexameric arrangement of subunits in the PDO multimer. Our proposed model for the subunit arrangement in PDO postulates two alpha(3) planar rings one on top the other, similar to the alpha(3)beta(3) arrangement in other Rieske dioxygenases. Unlike other Rieske dioxygenases, this arrangement brings two Rieske and two mononuclear centers, all on separate subunits, into proximity, allowing their cooperation for catalysis. Potential reasons necessitating this unusual structural arrangement are discussed.     

Structural insight into the dioxygenation of nitroarene compounds: the crystal structure of nitrobenzene dioxygenase

Nitroaromatic compounds are used extensively in many industrial processes and have been released into the environment where they are considered environmental pollutants. Nitroaromatic compounds, in general, are resistant to oxidative attack due to the electron-withdrawing nature of the nitro groups and the stability of the benzene ring. However, the bacterium Comamonas sp. strain JS765 can grow with nitrobenzene as a sole source of carbon, nitrogen and energy. Biodegradation is initiated by the nitrobenzene dioxygenase (NBDO) system. We have determined the structure of NBDO, which has a hetero-hexameric structure similar to that of several other Rieske non-heme iron dioxygenases. The catalytic subunit contains a Rieske iron-sulfur center and an active-site mononuclear iron atom. The structures of complexes with substrates nitrobenzene and 3-nitrotoluene reveal the structural basis for its activity with nitroarenes. The substrate pocket contains an asparagine residue that forms a hydrogen bond to the nitro-group of the substrate, and orients the substrate in relation to the active-site mononuclear iron atom, positioning the molecule for oxidation at the nitro-substituted carbon.     

The role of active-site residues in naphthalene dioxygenase

The three-component naphthalene dioxygenase enzyme system catalyzes the first step in the degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. A member of a large family of bacterial Rieske non-heme iron oxygenases, naphthalene dioxygenase is known to oxidize over 60 different aromatic compounds, and many of the products are enantiomerically pure. The crystal structure of the oxygenase component revealed the enzyme to be an α₃β₃ hexamer and identified the amino acids located near the active site. Site-directed mutagenesis studies have identified the residues involved in electron transfer and those responsible for controlling the regioselectivity and enantioselectivity of the enzyme. The results of these studies suggest that naphthalene dioxygenase can be engineered to catalyze a new and extended range of useful reactions.     

Choice of microbial host for the naphthalene dioxygenase bioconversion

The use of whole cell biotransformations for single and multistep enzyme conversions is gaining widespread application. In this study the naphthalene dioxygenasenah A gene was transferred intoPseudomonas aeruginosa PAC 1R,Escherichia coli JM107 andPseudomonas putida PpG 277. The effect of ethanol on these genetically engineered Gram-negative bacteria was studied by measurement of enzyme activity, stability and cell integrity. Ethanol has been used in biotransformations as a co-substrate carbon source for co-factor recycling and as a co-solvent increasing dissolved substrate and product levels. Ethanol increased the dissolved substrate (naphthalene) concentration slightly and dissolved product ((+)-cis-(1R, 2S)-dihydroxy-1,2-dihydronaphthalene) by approximately 30% at 4% (w/v) ethanol. BothP. aeruginosa PAC 1R andP. putida PpG 277 showed decreased activity with increasing ethanol concentration whilstE. coli enzyme activity increased with increasing ethanol concentration being comparable to that when glucose was used as a carbon source. This project highlighted the many factors involved in the selection of microbial hosts for whole cell biotransformation processes.     

Nitric oxide binding to cationic and anionic ferric porphyrins in aqueous solution: reversible formation of ferrous NO species of the cationic porphyrin

Two water-soluble ferric porphyrins, sodium 5α,10β,15α,20β-tetrakis(2-(sulfonatoacetamido)phenyl)porphyrinatoiron(III) (Fe^IIITanP) and 5α,10β,15α,20β-tetrakis(2-(N,N,N-trimethylammoniumacetamido)phenyl)porphyrinatoiron(III) chloride (Fe^IIITcatP), were synthesized. The pK_a values of the coordinated H₂O of Fe^IIITanP and Fe^IIITcatP were evaluated to be 8.0 and 4.1, respectively. Reactions of NO with the ferric porphyrins were examined spectrophotometrically in aqueous solution. Porphyrin Fe^IIITanP binds NO reversibly to give the corresponding ferric NO species at pH 1.3 and pH 3.0, and Fe^IIITcatP reacts similarly with NO at pH 1.3. The thermodynamic data for the NO binding were estimated from van't Hoff plots. At pH 3.0, visible and ESR spectral data indicated that Fe^IIITcatP binds NO reversibly to produce ferrous NO species depending on NO partial pressures. These results were discussed based on through-space intramolecular interactions between the coordinated H₂O or NO and the ionic substituents of the porphyrins.     

High levels of expression of the iron-sulfur proteins phthalate dioxygenase and phthalate dioxygenase reductase in Escherichia coli

Phthalate dioxygenase (PDO), a hexamer with one Rieske-type [2Fe-2S] and one Fe (II)-mononuclear center per monomer, and its reductase (PDR), which contains flavin mononucleotide and a plant-type ferredoxin [2Fe-2S] center, are expressed by Burkholderia cepacia at approximately 30mg of crude PDO and approximately 1mg of crude PDR per liter of cell culture when grown with phthalate as the main carbon source. A high level expression system in Escherichia coli was developed for PDO and PDR. Optimization relative to E. coli cell line, growth parameters, time of induction, media composition, and iron-sulfur additives resulted in yields of about 1g/L for PDO and about 0.2g/L for PDR. Protein expression was correlated to the increase in pH of the cell culture and exhibited a pronounced (variable from 5 to 20h) lag after the induction. The specific activity of purified PDO did not depend on the pH of the cell culture when harvested. However, when the pH of the culture reached 8.5-9, a large fraction of the PDR that was expressed lacked its ferredoxin domain, presumably because of proteolysis. Termination of growth while the pH of the cell culture was <8 decreased the fraction of proteolyzed enzyme, whereas yields of the unclipped PDR were only marginally lower. Overall, changes in pH of the cell culture were found to be an excellent indicator of the overall level of native protein expression. Its monitoring allowed the real time tracking of the protein expression and made it possible to tailor the expression times to achieve a combination of high quality and high yield of protein.     

Involvement of naphthalene dioxygenase in indole-3-acetic acid biosynthesis by Pseudomonas putida

Two variants of plant growth-promoting strain Pseudomonas putida BS1380 harboring the naphthalene degradative plasmid pBS2 and the recombinant plasmid pNAU64 that contains the genes encoding for naphthalene dioxygenase were constructed by conjugation. The ability of this strain to produce phytohormone indole-3-acetic acid from different carbon sources was studied. Indole-3-acetic acid synthesis by these transconjugants was 15-30 times as much in contrast to a wild-type strain with glucose as the sole carbon source. No difference was observed in other carbon or nitrogen sources. It is suggested that naphthalene dioxygenase is involved in the conversion of indole-3-pyruvic acid to indole-3-acetic acid.     

Mass-spectrometric characterization of two posttranslational modifications of cysteine dioxygenase

Recent crystal structures of cysteine dioxygenase (CDO) suggest the presence of two posttranslational modifications adjacent to the catalytic iron center: a thioether cross-link between Cys93 and Tyr157 and extra electron density at Cys164 which was variously explained as cystine or cysteine sulfinic acid. Purification of recombinant rat CDO yields “mature” and “immature” forms with distinct electrophoretic mobilities. We have positively identified and characterized the two modifications in the products of three sequential proteolytic digestions using liquid chromatography coupled with tandem mass spectrometry. The cross-link is unique to the mature form and was identified in an ion of m/z 3,225.403, consistent with a Tyr-Cys cross-link of peptides Gly80-Phe94 with His155-Phe167. The cross-link is liable to cleavage by in-source decay and the resulting separate peptides were sequenced by collision-induced dissociation tandem mass spectrometry. Mass-spectrometric analysis of these same and overlapping peptides in the presence or absence of reductants and alkylating agents identified the second modification to be a cystine formed between Cys164 and exogenous cysteine as proposed earlier. Both modifications have been shown to form in the presence of high levels of cysteine and iron. This and the presence of small amounts of an apparently off-pathway cystine at position Cys93 suggest that although these conditions promote CDO maturation, they may actually arise via nonenzymatic, nonphysiological processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular insights into substrate recognition and catalysis by phthalate dioxygenase from Comamonas testosteroni

Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDO_KF1 from Comamonas testosteroni KF1 and found that it had an apparent k_cat/K_m for phthalate of 0.58 ± 0.09 μM⁻¹s⁻¹, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 Å resolution revealed that it is a hexamer comprising two stacked α₃ trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDO_KF1 larger than that of other characterized ROs. Complexes of PDO_KF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.     

Multiple mutations at the active site of naphthalene dioxygenase affect regioselectivity and enantioselectivity

The importance of five amino acids at the active site of the multicomponent naphthalene dioxygenase (NDO) system was determined by generating site-directed mutations in various combinations. The substrate specificities of the mutant enzymes were tested with the substrates indole, indoline, 2-nitrotoluene (2NT), naphthalene, biphenyl, and phenanthrene. Transformation of these substrates measured the ability of the mutant enzymes to catalyze dioxygenation, monooxygenation, and desaturation reactions. In addition, the position of oxidation and the enantiomeric composition of products were characterized. All enzymes with up to three amino acid substitutions were able to catalyze dioxygenation reactions. A subset of these enzymes could also catalyze the monooxygenation of 2NT and desaturation of indoline. Single amino acid substitutions at positions 352 and 206 had the most profound effects on product formation. Of the single mutations made, only changes at position 352 affected the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene, but in the presence of the F352I mutation, changes at positions 206 and 295 also affected enantioselectivity. Major shifts in regioselectivity with biphenyl and phenanthrene resulted with several of the singly, doubly, and triply mutated enzymes. A new product not formed by the wild-type enzyme, phenanthrene cis-9,10-dihydrodiol, was formed as a major product from phenanthrene by enzymes with two (A206I/F352I) or three amino acid substitutions (A206I/F352I/H295I). The results indicate that a variety of amino acid substitutions are tolerated at the active site of NDO. Journal of Industrial Microbiology & Biotechnology (2001) 27, 94–103. Received 25 September 2000/ Accepted in revised form 29 June 2001     

Active site residues controlling substrate specificity in 2-nitrotoluene dioxygenase from Acidovorax sp. strain JS42

Acidovorax (formerly Pseudomonas) sp. strain JS42 utilizes 2-nitrotoluene as sole carbon, nitrogen, and energy source. 2-Nitrotoluene 2,3-dioxygenase (2NTDO) catalyzes the initial step in 2-nitrotoluene degradation by converting 2-nitrotoluene to 3-methylcatechol. In this study, we identified specific amino acids at the active site that control specificity. The residue at position 350 was found to be critical in determining both the enantiospecificity of 2NTDO with naphthalene and the ability to oxidize the ring of mononitrotoluenes. Substitution of Ile350 by phenylalanine resulted in an enzyme that produced 97% (+)-(1R, 2S)-cis-naphthalene dihydrodiol, in contrast to the wild type, which produced 72% (+)-(1R, 2S)-cis-naphthalene dihydrodiol. This substitution also severely reduced the ability of the enzyme to produce methylcatechols from nitrotoluenes. Instead, the methyl group of each nitrotoluene isomer was preferentially oxidized to form the corresponding nitrobenzyl alcohol. Substitution of a valine at position 258 significantly changed the enantiospecificity of 2NTDO (54% (−)-(1S, 2R)-cis-naphthalene dihydrodiol formed from naphthalene) and the ability of the enzyme to oxidize the aromatic ring of nitrotoluenes. Based on active site modeling using the crystal structure of nitrobenzene 1,2 dioxygenase from Comamonas sp. JS765, Asn258 appears to contribute to substrate specificity through hydrogen bonding to the nitro group of nitrotoluenes.     

Expression,purification and substrate specificities of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2

3-Nitotoluene dioxygenase (3-NTDO) is the first enzyme in the degradation pathway of 3-nitrotoluene (3-NT) by Diaphorobacter sp. strain DS2. The complete gene sequences of 3-NTDO were PCR amplified from genomic DNA of Diaphorobacter sp., cloned, sequenced and expressed. The 3-NTDO gene revealed a multi component structure having a reductase, a ferredoxin and two oxygenase subunits. Clones expressing the different subunits were constructed in pET21a expression vector system and overexpressed in E. coli BL21(DE3) host. Each subunit was individually purified separately to homogeneity. The active recombinant enzyme was reconstituted in vitro by mixing all three purified subunits. The reconstituted recombinant enzyme could catalyse biotransformations on a variety of organic aromatics.     

Synthetic indole Mannich bases: Their ability to modulate in vitro cellular immunity

The synthetic indole Mannich bases 1–13 have been investigated for their ability to modulate immune responses measured in vitro. These activities were based on monitoring their affects on T-lymphocyte proliferation, reactive oxygen species (ROS), IL (interleukin)-2, IL-4, and nitric oxide production. Compound 5 was found to be the most potent immunomodulator in this context. Four of the synthesized compounds, 5, 11, 12, and 13, have significant potent inhibitory effects on T-cell proliferation, IL-4, and nitric oxide production. However, none of the thirteen indole compounds exerted any activity against ROS production.     

Oxidation of carbazole to 3-hydroxycarbazole by naphthalene 1,2-dioxygenase and biphenyl 2,3-dioxygenase

Abstract Naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4 and biphenyl dioxygenase from Beijerinckia sp. B8/36 oxidized the aromatic N-heterocycle carbazole to 3-hydroxycarbazole. Toluene dioxygenase from Pseudomonas putida F39/D did not oxidize carbazole. Transformations were carried out by mutant strains which oxidize naphthalene and biphenyl to cis -dihydrodiols, and with a recombinant E. coli strain expressing the structural genes of naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4. 3-Hydroxycarbazole is presumed to result from the dehydration of an unstable cis -dihydrodiol.     

Detection of basal NO production in rat tissues using iron–dithiocarbamate complexes

We probe endogenous NO production in WKY rats by trapping NO with iron–dithiocarbamate complexes. The aim was to detect non-stimulated NO production in small organs like kidneys of juvenile rats. The yields of mononitrosyl Fe–dithiocarbamate complexes are small and difficult to quantify in the presence of strong contaminating signals from Cu²⁺–DETC complexes. We evaluate four methods to improve the detection of mononitrosyl Fe–dithiocarbamate adducts: progressive microwave saturation, tissue perfusion, spectral subtraction, and finally, reduction of the tissue with sodium dithionite. While the first three were only moderately useful, reduction was very helpful for quantification of the mononitrosyl Fe–dithiocarbamate yield. The increase in sensitivity allows the detection of non-stimulated NO release in small organs of juvenile rats.     

Distal end of 105-125 loop - A putative reductase binding domain of phthalate dioxygenase

The phthalate dioxygenase system consists of the dioxygenase, PDO, which contains a Rieske [2Fe-2S] center and a Fe(II)-mononuclear center, and the reductase, PDR. Involvement of the distal end of the 105-125 loop of PDO in its interaction with PDR was tested by substituting charged residues in the loop with alanines and by replacing the conserved tryptophan-94. Compared to wild-type PDO, all variants had lower catalytic activity and the Rieske centers were reduced more slowly by reduced PDR. The rates of oxidation of the Rieske centers by oxygen, which represent electron transfer between the Rieske and mononuclear centers, were essentially unaffected. These results suggest that positively charged residues of the distal end of the 105-125 loop are collectively involved in PDR binding with the PDO. Contrary to expectations, Trp94 variants were not directly involved in electron transfer between PDR and PDO. The tryptophan appears to have mainly a structural role, apparently preserving the hydrophilic environment of the Rieske center.     

Heteroalicyclic carboxamidines as inhibitors of inducible nitric oxide synthase; the identification of (2R)-2-pyrrolidinecarboxamidine as a potent and selective haem-co-ordinating inhibitor

Heteroalicyclic carboxamidines were synthesised and evaluated as inhibitors of nitric oxide synthases. (2R)-2-Pyrrolidinecarboxamidine, in particular, was shown to be a highly potent in vitro (IC₅₀ = 0.12 μM) and selective iNOS inhibitor (>100-fold vs both eNOS and nNOS), with probable binding to the key anchoring glutamate residue and co-ordination to the haem iron.