Proton transfer in the oxidative half-reaction of pentaerythritol tetranitrate reductase. Structure of the reduced enzyme-progesterone complex and the roles of residues Tyr186, His181, His184

The roles of His181, His184 and Tyr186 in PETN reductase have been examined by mutagenesis, spectroscopic and stopped-flow kinetics, and by determination of crystallographic structures for the Y186F PETN reductase and reduced wild-type enzyme-progesterone complex. Residues His181 and His184 are important in the binding of coenzyme, steroids, nitroaromatic ligands and the substrate 2-cyclohexen-1-one. The H181A and H184A enzymes retain activity in reductive and oxidative half-reactions, and thus do not play an essential role in catalysis. Ligand binding and catalysis is not substantially impaired in Y186F PETN reductase, which contrasts with data for the equivalent mutation (Y196F) in Old Yellow Enzyme. The structure of Y186F PETN reductase is identical to wild-type enzyme, with the obvious exception of the mutation. We show in PETN reductase that Tyr186 is not a key proton donor in the reduction of alpha/beta unsaturated carbonyl compounds. The structure of two electron-reduced PETN reductase bound to the inhibitor progesterone mimics the catalytic enzyme-steroid substrate complex and is similar to the structure of the oxidized enzyme-inhibitor complex. The reactive C1-C2 unsaturated bond of the steroid is inappropriately orientated with the flavin N5 atom for hydride transfer. With steroid substrates, the productive conformation is achieved by orientating the steroid through flipping by 180 degrees , consistent with known geometries for hydride transfer in flavoenzymes. Our data highlight mechanistic differences between Old Yellow Enzyme and PETN reductase and indicate that catalysis requires a metastable enzyme-steroid complex and not the most stable complex observed in crystallographic studies.     

H-tunneling in the multiple H-transfers of the catalytic cycle of morphinone reductase and in the reductive half-reaction of the homologous pentaerythritol tetranitrate reductase

The mechanism of flavin reduction in morphinone reductase (MR) and pentaerythritol tetranitrate (PETN) reductase, and flavin oxidation in MR, has been studied by stopped-flow and steady-state kinetic methods. The temperature dependence of the primary kinetic isotope effect for flavin reduction in MR and PETN reductase by nicotinamide coenzyme indicates that quantum mechanical tunneling plays a major role in hydride transfer. In PETN reductase, the kinetic isotope effect (KIE) is essentially independent of temperature in the experimentally accessible range, contrasting with strongly temperature-dependent reaction rates, consistent with a tunneling mechanism from the vibrational ground state of the reactive C-H/D bond. In MR, both the reaction rates and the KIE are dependent on temperature, and analysis using the Eyring equation suggests that hydride transfer has a major tunneling component, which, unlike PETN reductase, is gated by thermally induced vibrations in the protein. The oxidative half-reaction of MR is fully rate-limiting in steady-state turnover with the substrate 2-cyclohexenone and NADH at saturating concentrations. The KIE for hydride transfer from reduced flavin to the alpha/beta unsaturated bond of 2-cyclohexenone is independent of temperature, contrasting with strongly temperature-dependent reaction rates, again consistent with ground-state tunneling. A large solvent isotope effect (SIE) accompanies the oxidative half-reaction, which is also independent of temperature in the experimentally accessible range. Double isotope effects indicate that hydride transfer from the flavin N5 atom to 2-cyclohexenone, and the protonation of 2-cyclohexenone, are concerted and both the temperature-independent KIE and SIE suggest that this reaction also proceeds by ground-state quantum tunneling. Our results demonstrate the importance of quantum tunneling in the reduction of flavins by nicotinamide coenzymes. This is the first observation of (i) three H-nuclei in an enzymic reaction being transferred by tunneling and (ii) the utilization of both passive and active dynamics within the same native enzyme.     

Type II Hydride Transferases from Different Microorganisms Yield Nitrite and Diarylamines from Polynitroaromatic Compounds

Homogenous preparations of XenB of Pseudomonas putida, pentaerythritol tetranitrate reductase of Enterobacter cloacae, and N-ethylmaleimide reductase of Escherichia coli, all type II hydride transferases of the Old Yellow Enzyme family of flavoproteins, are shown to reduce the polynitroaromatic compound 2,4,6-trinitrotoluene (TNT). The reduction of this compound yields hydroxylaminodinitrotoluenes and Meisenheimer dihydride complexes, which, upon condensation, yield stoichiometric amounts of nitrite and diarylamines, implying that type II hydride transferases are responsible for TNT denitration, a process with important environmental implications for TNT remediation.     

Atomic resolution structures and solution behavior of enzyme-substrate complexes of Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase. Multiple conformational states and implications for the mechanism of nitroaromatic explosive degradation

The structure of pentaerythritol tetranitrate (PETN) reductase in complex with the nitroaromatic substrate picric acid determined previously at 1.55 A resolution indicated additional electron density between the indole ring of residue Trp-102 and the nitro group at C-6 of picrate. The data suggested the presence of an unusual bond between substrate and the tryptophan side chain. Herein, we have extended the resolution of the PETN reductase-picric acid complex to 0.9 A. This high-resolution analysis indicates that the active site is partially occupied with picric acid and that the anomalous density seen in the original study is attributed to the population of multiple conformational states of Trp-102 and not a formal covalent bond between the indole ring of Trp-102 and picric acid. The significance of any interaction between Trp-102 and nitroaromatic substrates was probed further in solution and crystal complexes with wild-type and mutant (W102Y and W102F) enzymes. Unlike with wild-type enzyme, in the crystalline form picric acid was bound at full occupancy in the mutant enzymes, and there was no evidence for multiple conformations of active site residues. Solution studies indicate tighter binding of picric acid in the active sites of the W102Y and W102F enzymes. Mutation of Trp-102 does not impair significantly enzyme reduction by NADPH, but the kinetics of decay of the hydride-Meisenheimer complex are accelerated in the mutant enzymes. The data reveal that decay of the hydride-Meisenheimer complex is enzyme catalyzed and that the final distribution of reaction products for the mutant enzymes is substantially different from wild-type enzyme. Implications for the mechanism of high explosive degradation by PETN reductase are discussed.     

Degradation of explosives by nitrate ester reductases

Explosive-contaminated land poses a hazard both to the environment and to human health. Microbial enzymes, either in their native or heterologous hosts, are a powerful and low-cost tool for eliminating this environmental hazard. As many explosives have only been present in the environment for 10 years, and with similar molecules not known in Nature, the origin of enzymes specialized for the breakdown of explosives is of particular interest. Screening of environmental isolates resulted in the discovery of flavoproteins capable of denitrating the explosives pentaerythritol tetranitrate (PETN) and glycerol trinitrate. These nitrate ester reductases are related in sequence and structure to Old Yellow Enzyme from Saccharomyces carlsbergenisis. All the members of this family have alpha/beta barrel structures and FMN as a prosthetic group, and reduce various electrophilic substrates. The nitrate ester reductases are, however, unusual in that they display activity towards the highly recalcitrant, aromatic explosive 2,4,6-trinitrotoluene, via a reductive pathway resulting in nitrogen liberation. We have embarked on a detailed study of the structure and mechanism of PETN reductase from a strain of Enterobacter cloacae. Work is focused currently on relating structure and function within this growing family of enzymes, with a view to engineering novel enzymes exhibiting useful characteristics.     

Crystal structure of bacterial morphinone reductase and properties of the C191A mutant enzyme

The crystal structure of the NADH-dependent bacterial flavoenzyme morphinone reductase (MR) has been determined at 2.2-A resolution in complex with the oxidizing substrate codeinone. The structure reveals a dimeric enzyme comprising two 8-fold beta/alpha barrel domains, each bound to FMN, and a subunit folding topology and mode of flavin-binding similar to that found in Old Yellow Enzyme (OYE) and pentaerythritol tetranitrate (PETN) reductase. The subunit interface of MR is formed by interactions from an N-terminal beta strand and helices 2 and 8 of the barrel domain and is different to that seen in OYE. The active site structures of MR, OYE, and PETN reductase are highly conserved reflecting the ability of these enzymes to catalyze "generic" reactions such as the reduction of 2-cyclohexenone. A region of polypeptide presumed to define the reducing coenzyme specificity is identified by comparison of the MR structure (NADH-dependent) with that of PETN reductase (NADPH-dependent). The active site acid identified in OYE (Tyr-196) and conserved in PETN reductase (Tyr-186) is replaced by Cys-191 in MR. Mutagenesis studies have established that Cys-191 does not act as a crucial acid in the mechanism of reduction of the olefinic bond found in 2-cyclohexenone and codeinone.     

Aerobic Degradation of 2,4,6-Trinitrotoluene by Enterobacter cloacae PB2 and by Pentaerythritol Tetranitrate Reductase

Enterobacter cloacae PB2 was originally isolated on the basis of its ability to utilize nitrate esters, such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate, as the sole nitrogen source for growth. The enzyme responsible is an NADPH-dependent reductase designated PETN reductase. E. cloacae PB2 was found to be capable of slow aerobic growth with 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. Dinitrotoluenes were not produced and could not be used as nitrogen sources. Purified PETN reductase was found to reduce TNT to its hydride-Meisenheimer complex, which was further reduced to the dihydride-Meisenheimer complex. Purified PETN reductase and recombinant Escherichia coli expressing PETN reductase were able to liberate nitrogen as nitrite from TNT. The ability to remove nitrogen from TNT suggests that PB2 or recombinant organisms expressing PETN reductase may be useful for bioremediation of TNT-contaminated soil and water.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C backbone resonance assignments of pentaerythritol tetranitrate reductase from <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> PB2

Pentaerythritol tetranitrate reductase (PETNR) is a flavoenzyme possessing a broad substrate specificity and is a member of the Old Yellow Enzyme family of oxidoreductases. As well as having high potential as an industrial biocatalyst, PETNR is an excellent model system for studying hydrogen transfer reactions. Mechanistic studies performed with PETNR using stopped-flow methods have shown that tunneling contributes towards hydride transfer from the NAD(P)H coenzyme to the flavin mononucleotide (FMN) cofactor and fast protein dynamics have been inferred to facilitate this catalytic step. Herein, we report the near-complete ¹H, ¹⁵N and ¹³C backbone resonance assignments of PETNR in a stoichiometric complex with the FMN cofactor in its native oxidized form, which were obtained using heteronuclear multidimensional NMR spectroscopy. A total of 97% of all backbone resonances were assigned, with 333 out of a possible 344 residues assigned in the ¹H–¹⁵N TROSY spectrum. This is the first report of an NMR structural study of a flavoenzyme from the Old Yellow Enzyme family and it lays the foundation for future investigations of functional dynamics in hydride transfer catalytic mechanism.     

Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2.

A mixed microbial culture capable of metabolizing the explosive pentaerythritol tetranitrate (PETN) was obtained from soil enrichments under aerobic and nitrogen-limiting conditions. A strain of Enterobacter cloacae, designated PB2, was isolated from this culture and was found to use PETN as a sole source of nitrogen for growth. Growth yields suggested that 2 to 3 mol of nitrogen was utilized per mol of PETN. The metabolites pentaerythritol dinitrate, 3-hydroxy-2,2-bis-[(nitrooxy)methyl]propanal, and 2,2-bis-[(nitrooxy)methyl]-propanedial were identified by mass spectrometry and 1H-nuclear magnetic resonance. An NADPH-dependent PETN reductase was isolated from cell extracts and shown to liberate nitrite from PETN, producing pentaerythritol tri- and dinitrates which were identified by mass spectrometry. PETN reductase was purified to apparent homogeneity by ion-exchange and affinity chromatography. The purified enzyme was found to be a monomeric flavoprotein with a M(r) of approximately 40,000, binding flavin mononucleotide noncovalently.     

Crystal structure of pentaerythritol tetranitrate reductase: "flipped" binding geometries for steroid substrates in different redox states of the enzyme.

Pentaerythritol tetranitrate reductase (PETN reductase) degrades high explosive molecules including nitrate esters, nitroaromatics and cyclic triazine compounds. The enzyme also binds a variety of cyclic enones, including steroids; some steroids act as substrates whilst others are inhibitors. Understanding the basis of reactivity with cyclic enones requires structural information for the enzyme and key complexes formed with steroid substrates and inhibitors. The crystal structure of oxidised and reduced PETN reductase at 1.5 A resolution establishes a close structural similarity to the beta/alpha-barrel flavoenzyme, old yellow enzyme. In complexes of oxidised PETN reductase with progesterone (an inhibitor), 1,4-androstadiene-3,17-dione and prednisone (both substrates) the steroids are stacked over the si-face of the flavin in an orientation different from that reported for old yellow enzyme. The specifically reducible 1,2 unsaturated bonds in 1,4-androstadiene-3,17-dione and prednisone are not optimally aligned with the flavin N5 in oxidised enzyme complexes. These structures suggest either relative "flipping" or shifting of the steroid with respect to the flavin when bound in different redox forms of the enzyme. Deuterium transfer from nicotinamide coenzyme to 1,4-androstadiene-3,17-dione via the enzyme bound FMN indicates 1alpha addition at the steroid C2 atom. These studies rule out lateral motion of the steroid and indicate that the steroid orientation is "flipped" in different redox states of the enzyme.     

Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter.

Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (Km = 23 microM) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35 degrees C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the alpha/beta barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively.     

Soapwort oxidoreductase is involved in trinitrotoluene detoxification

Plant enzymes participating in degradation of nitroaromatic compounds have not been biochemically characterized in details so far. From suspension culture of soapwort (Saponaria officinalis L.) we isolated a novel plant oxidoreductase involved in degradation of trinitrotoluene (TNT). The enzyme catalyses first steps of reduction of TNT nitro groups in the presence of NAD(P)H under anaerobic conditions. The enzyme is monomeric with molecular mass 29 kDa, its two isoforms have pI 4.8 and 5.1. According to the spectral and activation analysis the enzyme contains flavinmono-nucleotide as a prosthetic group. The structure properties suggest an evolutional relationship to oxophytodienoate reductase. The N-terminal amino acid sequence shows homology to family of Old Yellow Enzyme (E.C. 1.6.99.1).     

Function of coenzyme F420 in aerobic catabolism of 2,4, 6-trinitrophenol and 2,4-dinitrophenol by Nocardioides simplex FJ2-1A

2,4,6-Trinitrophenol (picric acid) and 2,4-dinitrophenol were readily biodegraded by the strain Nocardioides simplex FJ2-1A. Aerobic bacterial degradation of these pi-electron-deficient aromatic compounds is initiated by hydrogenation at the aromatic ring. A two-component enzyme system was identified which catalyzes hydride transfer to picric acid and 2,4-dinitrophenol. Enzymatic activity was dependent on NADPH and coenzyme F420. The latter could be replaced by an authentic preparation of coenzyme F420 from Methanobacterium thermoautotrophicum. One of the protein components functions as a NADPH-dependent F420 reductase. A second component is a hydride transferase which transfers hydride from reduced coenzyme F420 to the aromatic system of the nitrophenols. The N-terminal sequence of the F420 reductase showed high homology with an F420-dependent NADP reductase found in archaea. In contrast, no N-terminal similarity to any known protein was found for the hydride-transferring enzyme.     

Formation of an Olfactory Search Image for Explosives Odours in Sniffer Dogs*

The term ‘search image’ refers to an animal's heightened ability to detect a specific cryptic prey, after experience with that prey. Formulated in 1960 by Tinbergen (Archives Neerlandaises de zoologie, vol. 13, pp. 265–343), the search image concept has been the subject of much research, albeit almost entirely based on visual search cues in birds of prey. Given the theoretical and practical importance of this concept for foraging or searching in general, we set out to investigate whether dogs could form olfactory search images for explosive odours. Seven experienced explosives detector dogs were first tested for their ability to detect 30 g of the explosives 2,4,6‐trinitrotoluene (TNT), RDX + polyisobutylene + di(2‐ethylhexyl)sebacate + fuel oil (C4) or pentaerythritol tetranitrate (PETN) placed in various containers randomly distributed along a 300‐m limestone track. In consecutive stages of the experiment, we altered the relative percentages of the three explosives. The results showed that the percentage of TNT containers detected by the dogs increased in trials that followed placing of a high percentage of TNT relative to other explosives and decreased in trials that follwed placing of a relatively low percentage of TNT containers. The percentage detection of C4 and PETN was not influenced by their relative abundance at any stage. Overall, these results suggest that dogs can form an olfactory search image that might aid in prey/target detection.     

Engineering plants for the phytodetoxification of explosives

Summary  Widespread contaimination of the environment by explosives resulting from the manufacture, disposal and testing of munitions is becoming a matter of increasing concern. Most explosives are considered to be a major hazard to biological systems due to their toxic and mutagenic effects. Interest on the bioremediation of land contaminated with explosives has recently been focused on phytoremediation. Unfortunately., whilst plants have many advantages for the remediation of contaminated land and water, they lack the catabolic versatility which enables microorganisms to mineralize such a wide diversity of xenobiotic compounds. This raised the interesting question as to whether the impressive biodegradative capabilities of soil bacteria could be combined with the high biomass and stability of plants to yield an optimal system for in situ bioremediation of explosive residues in soil. Our investigation into the degradation of explosive residues by soil bacteria resulted in the isolation of Enterobacter cloacae PB2, which is capable of utilizing nitrate ester explosives such as pentaerythritol tetranitrate (PETN) and nitroglycerin as the sole source of nitrogen for growth. We have successfully introduced PETN reductase, the enzyme initiating explosive degradation in this organism, into plants to create transgenic plants that degrade explosives. Since the bacterial degradative pathways for many classes of organic pollutant have been elucidated, this may be a generally applicable method of achieving bioremediation of contaminated soil in the environment.     

X-ray structure of 12-oxophytodienoate reductase 1 provides structural insight into substrate binding and specificity within the family of OYE

BACKGROUND: 12-Oxophytodienoate reductase (OPR) is a flavin mononucleotide (FMN)-dependent oxidoreductase in plants that belongs to the family of Old Yellow Enzyme (OYE). It was initially characterized as an enzyme involved in the biosynthesis of the plant hormone jasmonic acid, where it catalyzes the reduction of the cyclic fatty acid derivative 9S,13S-12-oxophytodienoate (9S,13S-OPDA) to 1S,2S-3-oxo-2(2'[Z]-pentenyl)-cyclopentane-1-octanoate. Several isozymes of OPR are now known that show different stereoselectivities with regard to the four stereoisomers of OPDA. RESULTS: Here, we report the high-resolution crystal structure of OPR1 from Lycopersicon esculentum and its complex structures with the substrate 9R,13R-OPDA and with polyethylene glycol 400. OPR1 crystallizes as a monomer and folds into a (betaalpha)(8) barrel with an overall structure similar to OYE. The cyclopentenone ring of 9R,13R-OPDA is stacked above the flavin and activated by two hydrogen bonds to His187 and His190. The olefinic bond is properly positioned for hydride transfer from the FMN N(5) and proton transfer from Tyr192 to Cbeta and Calpha, respectively. Comparison of the OPR1 and OYE structures reveals striking differences in the loops responsible for binding 9R,13R-OPDA in OPR1. CONCLUSIONS: Despite extensive biochemical characterization, the physiological function of OYE still remains unknown. The similar catalytic cavity structures and the substrate binding mode in OPR1 strongly support the assumption that alpha,beta-unsaturated carbonyl compounds are physiological substrates of the OYE family. The specific binding of 9R,13R-OPDA by OPR1 explains the experimentally observed stereoselectivity and argues in favor of 9R,13R-OPDA or a structurally related oxylipin as natural substrate of OPR1.     

Subfunctionality of Hydride Transferases of the Old Yellow Enzyme Family of Flavoproteins of Pseudomonas putida

To investigate potential complementary activities of multiple enzymes belonging to the same family within a single microorganism, we chose a set of Old Yellow Enzyme (OYE) homologs of Pseudomonas putida. The physiological function of these enzymes is not well established; however, an activity associated with OYE family members from different microorganisms is their ability to reduce nitroaromatic compounds. Using an in silico approach, we identified six OYE homologs in P. putida KT2440. Each gene was subcloned into an expression vector, and each corresponding gene product was purified to homogeneity prior to in vitro analysis for its catalytic activity against 2,4,6-trinitrotoluene (TNT). One of the enzymes, called XenD, lacked in vitro activity, whereas the other five enzymes demonstrated type I hydride transferase activity and reduced the nitro groups of TNT to hydroxylaminodinitrotoluene derivatives. XenB has the additional ability to reduce the aromatic ring of TNT to produce Meisenheimer complexes, defined as type II hydride transferase activity. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite; the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo.     

Biodegradation of explosives by transgenic plants expressing pentaerythritol tetranitrate reductase.

Plants offer many advantages over bacteria as agents for bioremediation; however, they typically lack the degradative capabilities of specially selected bacterial strains. Transgenic plants expressing microbial degradative enzymes could combine the advantages of both systems. To investigate this possibility in the context of bioremediation of explosive residues, we generated transgenic tobacco plants expressing pentaerythritol tetranitrate reductase, an enzyme derived from an explosive-degrading bacterium that enables degradation of nitrate ester and nitroaromatic explosives. Seeds from transgenic plants were able to germinate and grow in the presence of 1 mM glycerol trinitrate (GTN) or 0.05 mM trinitrotoluene, at concentrations that inhibited germination and growth of wild-type seeds. Transgenic seedlings grown in liquid medium with 1 mM GTN showed more rapid and complete denitration of GTN than wild-type seedlings. This example suggests that transgenic plants expressing microbial degradative genes may provide a generally applicable strategy for bioremediation of organic pollutants in soil.     

The structure of Escherichia coli nitroreductase complexed with nicotinic acid: three crystal forms at 1.7 A, 1.8 A and 2.4 A resolution

Escherichia coli nitroreductase is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Its ability to convert relatively non-toxic prodrugs such as CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide) into highly cytotoxic derivatives has led to interest in its potential for cancer gene therapy. We have determined the structure of the enzyme bound to a substrate analogue, nicotinic acid, from three crystal forms at resolutions of 1.7 A, 1.8 A and 2.4 A, representing ten non-crystallographically related monomers. The enzyme is dimeric, and has a large hydrophobic core; each half of the molecule consists of a five-stranded beta-sheet surrounded by alpha-helices. Helices F and F protrude from the core region of each monomer. There is an extensive dimer interface, and the 15 C-terminal residues extend around the opposing monomer, contributing the fifth beta-strand. The active sites lie on opposite sides of the molecule, in solvent-exposed clefts at the dimer interface. The FMN forms hydrogen bonds to one monomer and hydrophobic contacts to both; its si face is buried. The nicotinic acid stacks between the re face of the FMN and Phe124 in helix F, with only one hydrogen bond to the protein. If the nicotinamide ring of the coenzyme NAD(P)H were in the same position as that of the nicotinic acid ligand, its C4 atom would be optimally positioned for direct hydride transfer to flavin N5. Comparison of the structure with unliganded flavin reductase and NTR suggests reduced mobility of helices E and F upon ligand binding. Analysis of the structure explains the broad substrate specificity of the enzyme, and provides the basis for rational design of novel prodrugs and for site-directed mutagenesis for improved enzyme activity.     

The Role of Oxophytodienoate Reductases in the Detoxification of the Explosive 2,4,6-Trinitrotoluene by Arabidopsis

The explosive 2,4,6-trinitrotoluene (TNT) is a significant environmental pollutant that is both toxic and recalcitrant to degradation. Phytoremediation is being increasingly proposed as a viable alternative to conventional remediation technologies to clean up explosives-contaminated sites. Despite the potential of this technology, relatively little is known about the innate enzymology of TNT detoxification in plants. To further elucidate this, we used microarray analysis to identify Arabidopsis (Arabidopsis thaliana) genes up-regulated by exposure to TNT and found that the expression of oxophytodienoate reductases (OPRs) increased in response to TNT. The OPRs share similarity with the Old Yellow Enzyme family, bacterial members of which have been shown to transform explosives. The three predominantly expressed forms, OPR1, OPR2, and OPR3, were recombinantly expressed and affinity purified. Subsequent biochemical characterization revealed that all three OPRs are able to transform TNT to yield nitro-reduced TNT derivatives, with OPR1 additionally producing the aromatic ring-reduced products hydride and dihydride Meisenheimer complexes. Arabidopsis plants overexpressing OPR1 removed TNT more quickly from liquid culture, produced increased levels of transformation products, and maintained higher fresh weight biomasses than wild-type plants. In contrast, OPR1,2 RNA interference lines removed less TNT, produced fewer transformation products, and had lower biomasses. When grown on solid medium, two of the three OPR1 lines and all of the OPR2-overexpressing lines exhibited significantly enhanced tolerance to TNT. These data suggest that, in concert with other detoxification mechanisms, OPRs play a physiological role in xenobiotic detoxification.Large amounts of land and water are heavily contaminated by explosives, mainly as a result of the manufacture and military use of munitions. The high financial cost associated with cleaning up these contaminated sites largely precludes the use of many existing remediation technologies, such as soil excavation and incineration or disposal to landfill. There is a great deal of work documenting the global contamination, general toxicity, and microbial metabolism of 2,4,6-trinitrotoluene (TNT) in the environment; however, relatively little is known about the enzymes mediating the detoxification of TNT in plants (for review, see Rylott and Bruce, 2009).Phytoremediation, the use of plants to remove environmental pollutants, offers a low-cost, sustainable alternative to conventional remediation technologies and is attracting considerable attention as a means to clean up sites contaminated with explosives. While TNT is a potent phytotoxin, plants are able to detoxify low levels of TNT. In an effort to determine how plant tolerance could be further improved, we are investigating the biochemistry and enzymology underlying the innate ability of plants to detoxify TNT. The detoxification of xenobiotics has been loosely categorized into three phases (Sandermann, 1992): activation, conjugation, and compartmentation. The proposed route of TNT detoxification follows these phases. The electron-withdrawing properties of the nitro groups of TNT make the aromatic ring electron deficient. This favors reductive transformation reactions in plants, and the TNT molecule is most commonly activated by the reduction of a nitro group to give hydroxylamino and then amino derivatives (Fig. 1, pathway A). Following the introduction of a functional group, more hydrophilic molecules such as Glc are conjugated to the activated TNT molecule (Gandia-Herrero et al., 2008), facilitating transport and subsequent compartmentation or sequestration.Open in a separate windowFigure 1.The transformation of TNT by pentaerythritol tetranitrate reductase. Pathway A shows the transformation of the nitro group to nitroso-dinitrotoluene (NODNT), HADNT, and then ADNT products. Pathway B shows the reduction of the aromatic ring to form hydride and dihydride Meisenheimer complexes, then chemical condensation with HADNT to form diarylamines.Data from both our microarray experiments (Gandia-Herrero et al., 2008) and other expression studies (Ekman et al., 2003; Mezzari et al., 2005) have found that members of the small gene family of oxophytodienoate reductases (OPRs) in Arabidopsis (Arabidopsis thaliana) are up-regulated following exposure to TNT. The Arabidopsis genome contains three characterized OPRs: OPR1, OPR2, and OPR3. In addition, there are three as yet uncharacterized putative OPRs, named here as OPR4, OPR5, and OPR6, with OPR4 and OPR5 being identical. The physiological functions of the OPRs remain obscure, with the exception of OPR3, which is involved in jasmonic acid biosynthesis, converting (9S,13S)-12-oxophytodienoic acid to 3-2(2′(Z)-pentyl)cyclopentane-1-octanoic acid in the peroxisome (Sanders et al., 2000; Stintzi and Browse, 2000; for review, see Wasternack, 2007). OPR3 is located on chromosome II within the Arabidopsis genome and contains a C-terminal Ser-Arg-Leu type 1 peroxisome-targeting sequence. The remaining OPRs are all located on chromosome I and do not possess any known organelle-targeting sequences. The spatial expression of OPR1 and OPR2 across root cells where TNT accumulates (Biesgen and Weiler, 1999; Baerenfaller et al., 2008) favors a role in detoxification.The OPRs share similarity with the Old Yellow Enzyme family, a group of flavoenzymes that has been repeatedly associated with the transformation of explosives (Binks et al., 1996; Schaller and Weiler, 1997; Snape et al., 1997; Basran et al., 1998; French et al., 1998; Blehert et al., 1999; Pak et al., 2000; Fitzpatrick et al., 2003; Williams et al., 2004). Studies also indicate that Old Yellow Enzyme homologs function as antioxidants, detoxifying the breakdown products of lipid peroxidation and other toxic electrophilic compounds (Kohli and Massey, 1998; Williams and Bruce, 2002; Fitzpatrick et al., 2003; Trotter et al., 2006). This oxidative stress could result from exposure to xenobiotics including TNT, wounding, or pathogen attack.Pentaerythritol tetranitrate reductase, an Old Yellow Enzyme homolog isolated from Enterobacter cloacae (Binks et al., 1996), possesses two catalytic activities toward TNT (Fig. 1): nitroreduction of TNT to form hydroxylamino-dinitrotoluene (HADNT) and then amino-dinitrotoluene (ADNT), and aromatic ring reduction of TNT to yield hydride and dihydride (2H⁻-TNT) Meisenheimer TNT adducts (French et al., 1998; Williams et al., 2004). The TNT ring-reduced compounds condense via a nonenzymatic reaction with HADNTs to form diarylamines, with the liberation of nitrite (Wittich et al., 2008). Expression of pentaerythritol tetranitrate reductase in tobacco (Nicotiana tabacum) confers both resistance to, and the ability to transform, TNT (French et al., 1999). OPR1, OPR2, and OPR3 share 43%, 44%, and 36% identity, respectively, with pentaerythritol tetranitrate reductase, and all possess the conserved active site amino acids crucial for TNT transformation by pentaerythritol tetranitrate reductase and other members of the Old Yellow Enzyme family (Snape et al., 1997; French et al., 1998; Khan et al., 2004), suggesting that they are capable of transforming TNT.The OPR4/5 protein is predicted to have reduced activity toward TNT, compared with the other OPRs, owing to a C-terminal truncation that removes residues thought to be important in binding the cofactor NADH, Thus, we investigated OPR1, -2, and -3 as likely candidates for the TNT nitroreduction activity in Arabidopsis.