Pentaerythritol Tetranitrate Reductase Research Articles

Transformation of TNT, 2,4-DNT, and PETN by Raoultella planticola M30b and Rhizobium radiobacter M109 and exploration of the associated enzymes.

The nitrated compounds 2,4-dinitrotoluene (2,4-DNT), 2,4,6-trinitrotoluene (TNT), and pentaerythritol tetranitrate (PETN) are toxic xenobiotics widely used in various industries. They often coexist as environmental contaminants. The aims of this study were to evaluate the transformation of 100mg L-1 of TNT, 2,4-DNT, and PETN by Raoultella planticola M30b and Rhizobium radiobacter M109c and identify enzymes that may participate in the transformation. These strains were selected from 34 TNT transforming bacteria. Cupriavidus metallidurans DNT was used as a reference strain for comparison purposes. Strains DNT, M30b and M109c transformed 2,4-DNT (100%), TNT (100, 94.7 and 63.6%, respectively), and PETN (72.7, 69.3 and 90.7%, respectively). However, the presence of TNT negatively affects 2,4-DNT and PETN transformation (inhibition > 40%) in strains DNT and M109c and fully inhibited (100% inhibition) 2,4-DNT transformation in R. planticola M30b.Genomes of R. planticola M30b and R. radiobacter M109c were sequenced to identify genes related with 2,4-DNT, TNT or PETN transformation. None of the tested strains presented DNT oxygenase, which has been previously reported in the transformation of 2,4-DNT. Thus, unidentified novel enzymes in these strains are involved in 2,4-DNT transformation. Genes encoding enzymes homologous to the previously reported TNT and PETN-transforming enzymes were identified in both genomes. R. planticola M30b have homologous genes of PETN reductase and xenobiotic reductase B, while R. radiobacter M109c have homologous genes to GTN reductase and PnrA nitroreductase. The ability of these strains to transform explosive mixtures has a potentially biotechnological application in the bioremediation of contaminated environments.

Covalent Attachment of Active Enzymes to Upconversion Phosphors Allows Ratiometric Detection of Substrates.

Upconverting phosphors (UCPs) convert multiple low energy photons into higher energy emission via the process of photon upconversion and offer an attractive alternative to organic fluorophores for use as luminescent probes. Here, UCPs were capped with functionalized silica in order to provide a surface to covalently conjugate proteins with surface‐accessible cysteines. Variants of green fluorescent protein (GFP) and the flavoenzyme pentaerythritol tetranitrate reductase (PETNR) were then attached via maleimide‐thiol coupling in order to allow energy transfer from the UCP to the GFP or flavin cofactor of PETNR, respectively. PETNR retains its activity when coupled to the UCPs, which allows reversible detection of enzyme substrates via ratiometric sensing of the enzyme redox state.

Selectivity through discriminatory induced fit enables switching of NAD(P)H coenzyme specificity in Old Yellow Enzyme ene-reductases.

Most ene‐reductases belong to the Old Yellow Enzyme (OYE) family of flavin‐dependent oxidoreductases. OYEs use nicotinamide coenzymes as hydride donors to catalyze the reduction of alkenes that contain an electron‐withdrawing group. There have been many investigations of the structures and catalytic mechanisms of OYEs. However, the origin of coenzyme specificity in the OYE family is unknown. Structural NMR and X‐ray crystallographic data were used to rationally design variants of two OYEs, pentaerythritol tetranitrate reductase (PETNR) and morphinone reductase (MR), to discover the basis of coenzyme selectivity. PETNR has dual‐specificity and reacts with NADH and NADPH; MR accepts only NADH as hydride donor. Variants of a β‐hairpin motif in an active site loop of both these enzymes were studied using stopped‐flow spectroscopy. Specific attention was placed on the potential role of arginine residues within the β‐hairpin motif. Mutagenesis demonstrated that Arg130 governs the preference of PETNR for NADPH, and that Arg142 interacts with the coenzyme pyrophosphate group. These observations were used to switch coenzyme specificity in MR by replacing either Glu134 or Leu146 with arginine residues. These variants had increased (~15‐fold) affinity for NADH. Mutagenesis enabled MR to accept NADPH as a hydride donor, with E134R MR showing a significant (55‐fold) increase in efficiency in the reductive half‐reaction, when compared to the essentially unreactive wild‐type enzyme. Insight into the question of coenzyme selectivity in OYEs has therefore been addressed through rational redesign. This should enable coenzyme selectivity to be improved and switched in other OYEs.

Isotopically labeled flavoenzymes and their uses in probing reaction mechanisms.

The incorporation of stable isotopes into proteins is beneficial or essential for a range of experiments, including NMR, neutron scattering and reflectometry, proteomic mass spectrometry, vibrational spectroscopy and "heavy" enzyme kinetic isotope effect (KIE) measurements. Here, we present detailed protocols for the stable isotopic labeling of pentaerythritol tetranitrate reductase (PETNR) via recombinant expression in E. coli. PETNR is an ene-reductase belonging to the Old Yellow Enzyme (OYE) family of flavoenzymes, and is regarded as a model system for studying hydride transfer reactions. Included is a discussion of how efficient back-exchange of amide protons in the protein core can be achieved and how the intrinsic flavin mononucleotide (FMN) cofactor can be exchanged, allowing the production of isotopologues with differentially labeled protein and cofactor. In addition to a thorough description of labeling strategies, we briefly exemplify how data analysis and interpretation of "heavy" enzyme KIEs can be performed.

Nonequivalence of Second Sphere "Noncatalytic" Residues in Pentaerythritol Tetranitrate Reductase in Relation to Local Dynamics Linked to H-Transfer in Reactions with NADH and NADPH Coenzymes.

Many enzymes that catalyze hydride transfer reactions work via a mechanism dominated by quantum mechanical tunneling. The involvement of fast vibrational modes of the reactive complex is often inferred in these reactions, as in the case of the NAD(P)H-dependent pentaerythritol tetranitrate reductase (PETNR). Herein, we interrogated the H-transfer mechanism in PETNR by designing conservative (L25I and I107L) and side chain shortening (L25A and I107A) PETNR variants and using a combination of experimental approaches (stopped-flow rapid kinetics, X-ray crystallography, isotope/temperature dependence studies of H-transfer and NMR spectroscopy). X-ray data show subtle changes in the local environment of the targeted side chains but no major structural perturbation caused by mutagenesis of these two second sphere active site residues. However, temperature dependence studies of H-transfer revealed a coenzyme-specific and complex thermodynamic equilibrium between different reactive configurations in PETNR–coenzyme complexes. We find that mutagenesis of these second sphere “noncatalytic” residues affects differently the reactivity of PETNR with NADPH and NADH coenzymes. We attribute this to subtle, dynamic structural changes in the PETNR active site, the effects of which impact differently in the nonequivalent reactive geometries of PETNR−NADH and PETNR−NADPH complexes. This inference is confirmed through changes observed in the NMR chemical shift data for PETNR complexes with unreactive 1,4,5,6-tetrahydro-NAD(P) analogues. We show that H-transfer rates can (to some extent) be buffered through entropy–enthalpy compensation, but that use of integrated experimental tools reveals hidden complexities that implicate a role for dynamics in this relatively simple H-transfer reaction. Similar approaches are likely to be informative in other enzymes to understand the relative importance of (distal) hydrophobic side chains and dynamics in controlling the rates of enzymatic H-transfer.

1H, 15N and 13C backbone resonance assignments of pentaerythritol tetranitrate reductase from Enterobacter cloacae 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.

Expanding the Scope of Biomolecule Monitoring with Ratiometric Signaling from Rare‐Earth Upconverting Phosphors

Upconversion (UC) is a powerful mulitphoton mechanism that converts low‐energy photons into higher energy emission. One of the most investigated UC systems is upconverting phosphors (UCPs). Here, a new, one‐pot synthetic procedure was used to prepare water dispersible, visibly emissive, rare‐earth doped UCPs that were capped with the functional groups oleic acid (OA), 6‐aminohexanoic acid (AHA), and 6‐maleimidohexanoic acid (MHA). These synthesized UCPs were characterized by UC luminescence, dynamic light scattering (DLS), transmission electron microscopy (TEM), and powder X‐ray diffraction (pXRD). This study expands upon our previous proof‐of‐principle work in demonstrating the use of UCPs (both synthesized and commercial) to detect on the level and function of biological analytes, from enzymes to key disease biomarkers (PETNR, glucose oxidase, vitamin B12, and cytochrome c). By tailoring the absorption profile of the biomolecule cofactors to the UCP emission, a wide‐range of analytes can be utilized. We also demonstrate the ability of our system to reversibly monitor the addition of enzyme substrates via repeat oxidation and reduction of pentaerythritol tetranitrate reductase (PETNR).

Drug design based on pentaerythritol tetranitrate reductase: synthesis and antibacterial activity of Pogostone derivatives

Our previous work showed that Pogostone exerts antibacterial effects by targeting pentaerythritol tetranitrate reductase (PETNR). In order to develop derivatives of Pogostone with potent antibacterial activity, we performed molecular docking studies of Pogostone with PETNR and analyzed structure-activity relationships, which guided the structure design and the subsequent facile organocatalytic synthesis of Pogostone derivatives under mild reaction conditions. Several of the synthesized compounds showed antibacterial activity in vitro, including one compound (3h) that was highly effective against methicillin-resistant Staphylococcus aureus. These results suggest that Pogostone derivatives bearing functional groups on the side chain may be good leads for antibacterial drug development.

Untangling Heavy Protein and Cofactor Isotope Effects on Enzyme-Catalyzed Hydride Transfer.

"Heavy" (isotopically labeled) enzyme isotope effects offer a direct experimental probe of the role of protein vibrations on enzyme-catalyzed reactions. Here we have developed a strategy to generate isotopologues of the flavoenzyme pentaerythritol tetranitrate reductase (PETNR) where the protein and/or intrinsic flavin mononucleotide (FMN) cofactor are isotopically labeled with 2H, 15N, and 13C. Both the protein and cofactor contribute to the enzyme isotope effect on the reductive hydride transfer reaction, but their contributions are not additive and may partially cancel each other out. However, the isotope effect specifically arising from the FMN suggests that vibrations local to the active site play a role in the hydride transfer chemistry, while the protein-only "heavy enzyme" effect demonstrates that protein vibrations contribute to catalysis in PETNR. In all cases, enthalpy-entropy compensation plays a major role in minimizing the magnitude of "heavy enzyme" isotope effects. Fluorescence lifetime measurements of the intrinsic flavin mononucleotide show marked differences between "light" and "heavy" enzymes on the nanosecond-picosecond time scale, suggesting relevant time scale(s) for those vibrations implicated in the "heavy enzyme" isotope effect on the PETNR reaction.

Fast protein motions are coupled to enzyme H-transfer reactions.

Coupling of fast protein dynamics to enzyme chemistry is controversial and has ignited considerable debate, especially over the past 15 years in relation to enzyme-catalyzed H-transfer. H-transfer can occur by quantum tunneling, and the temperature dependence of kinetic isotope effects (KIEs) has emerged as the "gold standard" descriptor of these reactions. The anomalous temperature dependence of KIEs is often rationalized by invoking fast motions to facilitate H-transfer, yet crucially, direct evidence for coupled motions is lacking. The fast motions hypothesis underpinning the temperature dependence of KIEs is based on inference. Here, we have perturbed vibrational motions in pentaerythritol tetranitrate reductase (PETNR) by isotopic substitution where all non-exchangeable atoms were replaced with the corresponding heavy isotope ((13)C, (15)N, and (2)H). The KIE temperature dependence is perturbed by heavy isotope labeling, demonstrating a direct link between (promoting) vibrations in the protein and the observed KIE. Further we show that temperature-independent KIEs do not necessarily rule out a role for fast dynamics coupled to reaction chemistry. We show causality between fast motions and enzyme chemistry and demonstrate how this impacts on experimental KIEs for enzyme reactions.

Biocatalytic Asymmetric Alkene Reduction: Crystal Structure and Characterization of a Double Bond Reductase from Nicotiana tabacum.

The application of biocatalysis for the asymmetric reduction of activated C=C is a powerful tool for the manufacture of high-value chemical commodities. The biocatalytic potential of “-ene” reductases from the Old Yellow Enzyme (OYE) family of oxidoreductases is well-known; however, the specificity of these enzymes toward mainly small molecule substrates has highlighted the need to discover “-ene” reductases from different enzymatic classes to broaden industrial applicability. Here, we describe the characterization of a flavin-free double bond reductase from Nicotiana tabacum (NtDBR), which belongs to the leukotriene B4 dehydrogenase (LTD) subfamily of the zinc-independent, medium chain dehydrogenase/reductase superfamily of enzymes. Using steady-state kinetics and biotransformation reactions, we have demonstrated the regio- and stereospecificity of NtDBR against a variety of α,β-unsaturated activated alkenes. In addition to catalyzing the reduction of typical LTD substrates and several classical OYE-like substrates, NtDBR also exhibited complementary activity by reducing non-OYE substrates (i.e., reducing the exocyclic C=C double bond of (R)-pulegone) and in some cases showing an opposite stereopreference in comparison with the OYE family member pentaerythritol tetranitrate (PETN) reductase. This serves to augment classical OYE “-ene” reductase activity and, coupled with its aerobic stability, emphasizes the potential industrial value of NtDBR. Furthermore, we also report the X-ray crystal structures of the holo-, binary NADP(H)-bound, and ternary [NADP+ and 4-hydroxy-3-methoxycinnamaldehyde (9a)-bound] NtDBR complexes. These will underpin structure-driven site-saturated mutagenesis studies aimed at enhancing the reactivity, stereochemistry, and specificity of this enzyme.

Electro-enzymatic viologen-mediated substrate reduction using pentaerythritol tetranitrate reductase and a parallel, segmented fluid flow system

Many redox enzymes require expensive reduced cofactors like NAD(P)H which need to be recycled during catalysis, presenting a major cost and technical barrier to industrial exploitation. An electrochemical biphasic microfluidic setup is presented here, in which these cofactors are replaced by a mediator (methyl viologen) that acts by feeding electrons into the active site of the enzyme pentaerythritol tetranitrate reductase (PETNR). In this microfluidic recirculation setup, both enzyme and mediator remain in the reactor for reuse, allowing easy product recovery. System optimisation studies were performed using 2-cyclohexen-1-one as a model substrate prior to the investigation of a variety of different substrates whose reduction rates were determined to be 15–70% of those obtained when NADPH was used as sole electron donor. Additional data obtained with a thermophilic ‘ene’ reductase (TOYE) support the potential universality of this device for possible industrial applications.

A surprising observation that oxygen can affect the product enantiopurity of an enzyme‐catalysed reaction

Enzymes are natural catalysts, controlling reactions with typically high stereospecificity and enantiospecificity in substrate selection and/or product formation. This makes them useful in the synthesis of industrially relevant compounds, particularly where highly enantiopure products are required. The flavoprotein pentaerythritol tetranitrate (PETN) reductase is a member of the Old Yellow Enzyme family, and catalyses the asymmetric reduction of β-alkyl-β-arylnitroalkenes. Under aerobic conditions, it additionally undergoes futile cycles of NAD(P)H reduction of flavin, followed by reoxidation by oxygen, which generates the reactive oxygen species (ROS) hydrogen peroxide and superoxide. Prior studies have shown that not all reactions catalysed by PETN reductase yield enantiopure products, such as the reduction of (E)-2-phenyl-1-nitroprop-1-ene (PNE) to produce (S)-2-phenyl-1-nitropropane (PNA) with variable enantiomeric excess (ee). Recent independent studies of (E)-PNE reduction by PETN reductase showed that the major product formed could be switched to (R)-PNA, depending on the reaction conditions. We investigated this phenomenon, and found that the presence of oxygen and ROS influenced the overall product enantiopurity. Anaerobic reactions produced consistently higher nitroalkane (S)-PNA product yields than aerobic reactions (64% versus 28%). The presence of oxygen dramatically increased the preference for (R)-PNA formation (up to 52% ee). Conversely, the presence of the ROS superoxide and hydrogen peroxide switched the preference to (S)-PNA product formation. Given that oxygen has no role in the natural catalytic cycle, these findings demonstrate a remarkable ability to manipulate product enantiopurity of this enzyme-catalysed reaction by simple manipulation of reaction conditions. Potential mechanisms of this unusual behaviour are discussed.

Inside Cover: Focused Directed Evolution of Pentaerythritol Tetranitrate Reductase by Using Automated Anaerobic Kinetic Screening of Site‐Saturated Libraries (ChemBioChem 17/2010)

The inside cover picture shows the crystal structure and high-throughput screening procedure of pentaerythritol tetranitrate reductase (PETNR) for generating biocatalysts with altered product identity and/or enantiopurities. The lower figure shows the active site and altered biocatalytic abilities of wild-type and mutant PETNR. For more information, see the paper by N. S. Scrutton et al. on p. 2433 ff.

Focused Directed Evolution of Pentaerythritol Tetranitrate Reductase by Using Automated Anaerobic Kinetic Screening of Site‐Saturated Libraries

This work describes the development of an automated robotic platform for the rapid screening of enzyme variants generated from directed evolution studies of pentraerythritol tetranitrate (PETN) reductase, a target for industrial biocatalysis. By using a 96-well format, near pure enzyme was recovered and was suitable for high throughput kinetic assays; this enabled rapid screening for improved and new activities from libraries of enzyme variants. Initial characterisation of several single site-saturation libraries targeted at active site residues of PETN reductase, are described. Two mutants (T26S and W102F) were shown to have switched in substrate enantiopreference against substrates (E)-2-aryl-1-nitropropene and α-methyl-trans-cinnamaldehyde, respectively, with an increase in ee (62 % (R) for W102F). In addition, the detection of mutants with weak activity against α,β-unsaturated carboxylic acid substrates showed progress in the expansion of the substrate range of PETN reductase. These methods can readily be adapted for rapid evolution of enzyme variants with other oxidoreductase enzymes.

Tunneling in enzymatic and nonenzymatic hydrogen transfer reactions

AbstractThis paper is a response to an invitation to share my viewpoint by writing an opinion piece (not a review) on proton tunneling, especially from the point of view of whether it has a greater importance in enzymatic reactions than in other reactions. The paper begins with a discussion of the emergence of a conceptual framework for including tunneling in reaction rate calculations; the framework is general enough to include not only transfer of protons but also transfer of hydrogen atoms and hydride ions and their isotopes, and not only enzymatically catalyzed reactions but also nonenzymatic reactions. Then the paper discusses the special issues that arise when the reaction rate under consideration is for an enzyme‐catalyzed reaction. The emphasis is on physical considerations in reaction rate calculations, not on system‐specific comparison of results for different modes of reaction. It is argued that enzymatic and nonenzymatic reactions may be treated within the same basic framework except that ensemble averaging, which is not usually required for gas‐phase reactions, is essential for treating enzyme reactions. Enzymes explicitly discussed include methylamine dehydrogenase, aromatic amine dehydrogenase, E. coli dihydrofolate reductase, hyperthermophilic dihydrofolate reductase, liver alcohol dehydrogenase, methylmalonyl‐CoA mutase, soybean lipoxygenase, copper amine oxidase, pentaerythritol tetranitrate reductase, morphinone reductase, enolase, xylose isomerase, and 4‐oxalocrotonate tautomerase. Copyright © 2010 John Wiley & Sons, Ltd.

The Substrate Spectra of Pentaerythritol Tetranitrate Reductase, Morphinone Reductase, N‐Ethylmaleimide Reductase and Estrogen‐Binding Protein in the Asymmetric Bioreduction of Activated Alkenes

AbstractFour flavoproteins from the old yellow enzyme (OYE) family, pentaerythritol tetranitrate (PETNR) reductase, N‐ethylmaleimide reductase (NEMR), morphinone reductase (MorR) and estrogen‐binding protein (EBP1), exhibited a broad substrate tolerance by accepting conjugated enals, enones, imides, dicarboxylic acids and esters, as well as a nitroalkene and therefore can be employed for the asymmetric bioreduction of carbon‐carbon double (CC) bonds. In particular, morphinone reductase and estrogen‐binding protein often showed a complementary stereochemical preference in comparison to that of previously investigated OYEs.

Biocatalysis with Thermostable Enzymes: Structure and Properties of a Thermophilic ‘ene’‐Reductase related to Old Yellow Enzyme

We report the crystal structure of a thermophilic "ene" reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T(m)>70 degrees C) and shows increased resistance to denaturation in water-miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene-reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water-miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.

Evidence To Support the Hypothesis That Promoting Vibrations Enhance the Rate of an Enzyme Catalyzed H-Tunneling Reaction

In recent years there has been a shift away from transition state theory models for H-transfer reactions. Models that incorporate tunneling as the mechanism of H-transfer are now recognized as a better description of such reactions. Central to many models of H-tunneling is the notion that specific vibrational modes of the protein and/or substrate can increase the probability of a H-tunneling reaction, modes that are termed promoting vibrations. Thus far there has been limited evidence that promoting vibrations can increase the rate of H-transfer. In the present communication we examine the single hydride transfer from both NADPH and NADH to FMN in the reductive half-reaction of pentaerythritol tetranitrate reductase (PETNR). We find that there is a significant promoting vibration with NADPH but not with NADH and that the observed rate of hydride transfer is significantly (approximately 15x) faster with NADPH. We rule out differences in rate due to variation in driving force and the donor-acceptor distance, suggesting it is the promoting vibration with NADPH that is the origin of the increased observed rate. This study therefore provides direct evidence that promoting vibrations can lead to an increase in rate.

Asymmetric Reduction of Activated Alkenes by Pentaerythritol Tetranitrate Reductase: Specificity and Control of Stereochemical Outcome by Reaction Optimisation.

We show that pentaerythritol tetranitrate reductase (PETNR), a member of the 'ene' reductase old yellow enzyme family, catalyses the asymmetric reduction of a variety of industrially relevant activated alpha,beta-unsaturated alkenes including enones, enals, maleimides and nitroalkenes. We have rationalised the broad substrate specificity and stereochemical outcome of these reductions by reference to molecular models of enzyme-substrate complexes based on the crystal complex of the PETNR with 2-cyclohexenone 4a. The optical purity of products is variable (49-99% ee), depending on the substrate type and nature of substituents. Generally, high enantioselectivity was observed for reaction products with stereogenic centres at Cbeta (>99% ee). However, for the substrates existing in two isomeric forms (e.g., citral 11a or nitroalkenes 18-19a), an enantiodivergent course of the reduction of E/Z-forms may lead to lower enantiopurities of the products. We also demonstrate that the poor optical purity obtained for products with stereogenic centres at Calpha is due to non-enzymatic racemisation. In reactions with ketoisophorone 3a we show that product racemisation is prevented through reaction optimisation, specifically by shortening reaction time and through control of solution pH. We suggest this as a general strategy for improved recovery of optically pure products with other biocatalytic conversions where there is potential for product racemisation.