Article pubs.acs.org/biochemistry
ATP-Dependent Electron Activation Module of Benzoyl-Coenzyme A Reductase from the Hyperthermophilic Archaeon Ferroglobus placidus Georg Schmid,† Hendrik Auerbach,‡ Antonio J. Pierik,§ Volker Schünemann,‡ and Matthias Boll*,† †
Fakultät für Biologie-Mikrobiologie, Institut für Biologie II, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany Fachbereich Physik, TU Kaiserslautern, 67663 Kaiserslautern, Germany § Fachbereich Chemie, TU Kaiserslautern, 67663 Kaiserslautern, Germany ‡
ABSTRACT: The class I benzoyl-coenzyme A (BzCoA) reductases (BCRs) are key enzymes in the anaerobic degradation of aromatic compounds that catalyze the ATPdependent dearomatization of their substrate to a cyclic dienoyl-CoA. The phylogenetically distinct Thauera- and Azoarcus-type BCR subclasses are iron−sulfur enzymes and consist of an ATP-hydrolyzing electron activation module and a BzCoA reduction module. More than 20 years after their initial identification, all biochemical information about class I BCRs derives from studies of the wild-type enzyme from the denitrifying bacterium Thauera aromatica (BCRTaro). Here, we describe the first heterologous production and purification of the ATP-hydrolyzing, electron-activating module of an Azoarcus-type BCR from the hyperthermophilic archaeon Ferroglobus placidus, BzdPQFpla. The Fe content, UV/vis spectroscopic, and Mössbauer spectroscopic properties of the 57Fe-enriched enzyme clearly identified a [4Fe-4S]+/2+ cluster with a redox potential (E°′) of −376 mV as a cofactor. ATP hydrolysis is required to overcome a redox barrier of ∼250 mV for stoichiometric electron transfer from the [4Fe-4S]+ cluster to the substrate benzene ring (E°′BzCoA/dienoyl‑CoA = −622 mV). BzdPQFpla exhibited ATPase activity (15 nmol min−1 mg−1; Km = 270 μM) at 75 °C, which was relatively stable in air in contrast to BCRTaro. The results obtained revealed high levels of functional and molecular similarity between Azoarcus-type BCRs and the homologous ATP-dependent activator components of 2-hydroxyacyl-CoA dehydratases involved in amino acid fermentations. Insights into the diversity and evolution of ATP-dependent electron-activating modules for catalytic or stoichiometric low-potential electron transfer processes are presented.
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are abundant in obligate anaerobes, and have so far only been isolated from the Fe(III)-respiring Deltaproteobacterium Geobacter metallireducens.9 They contain an active site tungstopterin cofactor in the BamB active site subunit that has recently been structurally characterized in a Bam(BC)2 complex.10 It has been hypothesized that class II BCRs drive endergonic BzCoA reduction by a flavin-based electron bifurcation.2 In the past 20 years, the kinetic, spectroscopic and molecular properties of the extremely oxygen-sensitive BCRTaro have been characterized to some detail, though a crystal structure is still not available. The redox potentials of the three [4Fe-4S] cofactors are all below −500 mV.11 Reduced but not oxidized BCRTaro exhibits BzCoA-independent ATPase activity.7 ATPhydrolysis in the BcrAD module induces conformational changes in the vicinity of a [4Fe-4S]+ cluster that possibly result in a lowering of the redox potential of the cluster.11 The ATP-dependently generated low-potential electrons are subsequently transferred to the BcrBC active site module (Figure
enzoyl-coenzyme A (BzCoA) reductases (BCRs) are key enzymes in the degradation of monocyclic aromatic compounds in the absence of oxygen and catalyze the twoelectron reduction of the substrate to cyclohexa-1,5-diene-1carboxyl-CoA (1,5-dienoyl-CoA) (for recent reviews, see refs 1−4) (Figure 1). This electron transfer reaction proceeds with an E°′ of −622 mV at an extremely low redox potential and requires a coupling to an exergonic reaction.5 There are two completely different classes of BCRs that both yield the identical 1,5-dienoyl-CoA product. The initially identified class I BCRs are found in facultatively anaerobic bacteria, and a class I BCR has so far only been isolated and studied in the denitrifying Betaproteobacterium Thauera aromatica (BCRTaro).6 Members of class I BCRs couple BzCoA reduction with reduced ferredoxin as an electron donor to a stoichiometric hydrolysis of two ATP molecules to two ADP molecules and two Pi.7,8 BCRTaro is composed of two modules: the BcrA and BcrD subunits have sequences that are significantly similar to each other and form the electronactivating, ATP-binding, and probably single-[4Fe-4S] clustercoordinating module; the BcrBC subunits form the BzCoAreducing active site module that probably binds two additional [4Fe-4S]+/2+ clusters (Figure 1).2 The nonrelated class II BCRs © 2016 American Chemical Society
Received: July 18, 2016 Revised: September 5, 2016 Published: September 5, 2016 5578
DOI: 10.1021/acs.biochem.6b00729 Biochemistry 2016, 55, 5578−5586
Article
Biochemistry
components (e.g., HgdAB in Ac. fermentans with two additional [4Fe-4S] clusters). Transfer of a single electron to the CoA ester substrate initiates the radical-based dehydration of the 2hydroxyacyl-CoA compound via ketyl and enoxy radical intermediates.17 In contrast to BCR, ATP-dependent electron transfer is only catalytically required for the redox-neutral dehydration reaction. The architecture of the HAD activators and the BCR electron activation modules resembles that of the ATP-dependent dinitrogenase reductase.16,18 Though the function in ATP-dependent electron transfer of dinitrogenase reductase and the activators of BCRs or HADs appears to be similar, they belong to two nonhomologous classes of ATPases. The strictly anaerobic hyperthermophilic Ferroglobus placidus is the only archaeon known to use a number of aromatic compounds as carbon and energy sources19 that are degraded via BzCoA.20,21 The genome of F. placidus contains the genes putatively encoding a class I BCR of the Azoarcus type (Ferp_1184−1187, termed BzdNOPQFpla), with BzdPQFpla putatively representing the two ATP-binding activation module subunits.20 Extracts from F. placidus grown with benzoate and Fe(III) as the electron acceptor catalyzed the Ti(III) citrateand ATP-dependent reduction of BzCoA to 1,5-dienoyl-CoA.21 Heterologous expression of the four putative BCRFpla genes followed by heat precipitation at 80 °C resulted in an enzyme preparation that exhibited a weak ATP-dependent BzCoA reducing activity at an optimum between 80 and 85 °C. In this preparation, the BzdNOFpla active site subunits were greatly lost most possibly because of thermal instability in the as-isolated state.21 In this work, we heterologously produced, isolated, and characterized the ATP-binding, electron-activating BzdPQFpla module of Strep-tagged BCRFpla. The first characterization of Azoarcus-type BCR components revealed a number of marked differences from the corresponding module from BCRTaro and suggests that Azoarcus-type BCRs and the activators of HADs are phylogenetically and functionally more related than the corresponding components of Thauera- and Azoarcus-type BCRs.
Figure 1. Molecular architecture and reaction catalyzed by class I BCR from T. aromatica, BCRTaro. The electron-activating, ATP-hydrolyzing module BcrAD is colored dark gray.
1). Enzymatic BzCoA reduction is considered to proceed in one-electron transfer steps via highly reactive radical intermediates according to a Birch-like reduction mechanism.3,12 Class I BCRs always contain four different subunits, and on the basis of amino acid sequence similarity, they can be divided into two different types: the Thauera type (e.g., in T. aromatica, termed BcrABCD) and the Azoarcus type (e.g., present in Azoarcus and Aromatoleum species, termed BzdNOPQ).1,3 In all organisms investigated so far, the bcrABCD and bzdNOPQ genes are located next to each other. They are part of benzoateinduced operon-containing genes encoding the electron donor ferredoxin, enzmyes involved in downstream reactions of the benzoyl-CoA degradation pathway, as well as transporters and transcriptional regulators. Class I BCRs have amino acid sequences that are similar to those of (R)-2-hydroxyacyl-CoA dehydratases (HADs) from amino acid-fermenting Firmicutes and Fusobacteria. HADs catalyze the mechanistically difficult syn dehydration of (R)-2-hydroxyacyl-CoA to (E)-2-enoyl-CoA (for reviews, see refs 3 and 13−15). Both class I BCRs and HADs catalyze ATP-driven single-electron transfer to a thioester substrate, and together, they form the BCR/HAD radical enzyme family.3,16 Similar to BCRs, HADs are composed of two separate modules, which, however, only transiently form a complex. The homodimeric activation components bind a subunit-bridging [4Fe-4S]+/2+ cluster (e.g., termed “component A” encoded by the hgdC gene in 2hydroxyglutaryl-CoA dehydratase of Acidaminococcus fermentans). They ATP-dependently transfer a low-potential single electron to the [4Fe-4S] cluster(s) of the dehydratase
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EXPERIMENTAL PROCEDURES Heterologous Gene Expression and Enzyme Production in Escherichia coli. The bzdNOPQ Fpla genes (Ferp_1184−87) were synthesized by Geneart (Regensburg, Germany) with a nucleotide sequence optimized for the codon usage of E. coli. The last gene in this operon, bzdQ, was elongated at its 3′ end with 30 nucleotides encoding the amino acids of a Strep tag (WSHPQFEK). Cloning and heterologous expression in E. coli C43 (DE) pRKISC were performed as described previously.21 57Fe-labeled BzdPQFpla for Mössbauer spectroscopy was obtained by adding 18 μM 57Fe to the E. coli growth medium.21 Briefly, 100 mg of 57Fe was dissolved in 3 mL of 6 M HCl at 80 °C. The 57Fe solution was mixed with 100 mL of 0.6 M nitrilotriacetic acid, and the pH was adjusted to 6.5 with 5 M NaOH. After being autoclaved, this solution was added to 100 L of sterile growth medium. Purification of Recombinant Proteins. E. coli cells with heterologously produced BzdNOPQFpla were suspended in lysis buffer (1 g of cells in 1.5 mL of lysis buffer) containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.3), 150 mM NaCl, 4 mM MgCl2, 10% (v/v) glycerol, 0.1 mg of DNase I, 1.0 mM dithiothreitol, and 50 μM Na2S2O4. Cells were lysed by being passed through a French pressure cell at 137 MPa, followed by centrifugation at 145000g for 1.5 h at 4 5579
DOI: 10.1021/acs.biochem.6b00729 Biochemistry 2016, 55, 5578−5586
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Biochemistry °C. The supernatant was loaded on a Strep-Tactin Superflow column (IBA, Göttingen, Germany). After the column was washed with 10−15 column volumes of washing buffer [50 mM HEPES (pH 8.0), 150 mM NaCl, 10% (v/v) glycerol, 1.0 mM dithiothreitol, and 50 μM Na2S2O4], column-bound proteins were eluted with 5 mM desthiobiotin. The elution fraction was concentrated with a Vivaspin Turbo 4 centrifugal concentrator (10000 MWCO) to a final concentration of 5−30 mg mL−1. Determination of Protein Concentrations and Iron Content. The protein concentration was determined by the method of Bradford using BSA as a standard. The iron content of BzdPQFpla was determined by a colorimetric assay based on the absorption of a phenanthroline−Fe(II) complex at 512 nm.22 For this purpose, 7.5 μL of 25% (v/v) HCl was added to 250 μL of a BzdPQFpla solution containing 350−550 μg of protein. After incubation at 80 °C for 10 min, the sample was centrifuged (10000g for 10 min). The supernatant was mixed with 750 μL of H2Odest, 50 μL of 10% (w/v) hydroxylamine, and 250 μL of 0.1% (w/v) phenanthroline. After incubation at 20 °C for 30 min, the absorption was measured at 512 nm. The iron concentration was quantified by comparison with a standard curve of 0−20 nmol of (NH4)2Fe(SO4)2. Gel Filtration. Gel filtration was conducted using a 24 mL Superdex 200 10/300 column (GE Healthcare). The column was loaded with 100 μL of a protein solution containing 3 mg mL−1 protein. The gel filtration buffer contained 50 mM HEPES (pH 7.5), 150 mM NaCl, and 4 mM MgCl2. The column was calibrated with thyroglobulin (Mr = 669000), ferritin (Mr = 440000), ovalbumin (Mr = 43000), and chymotrypsinogen A (Mr = 25000). ATPase Assays. All ATPase enzyme assays were performed anaerobically at 75 °C in 100 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 7.0) with 10 mM MgCl2 in the absence of benzoyl-CoA. The buffer containing 3 mM Na-ATP was preheated to 75 °C, and the assay was started by adding the preheated enzyme solution (final concentration of 0.6−1.2 mg mL−1). The ATPase activity of the oxidized enzyme was determined by adding 50 μM thionine acetate to 11 μM BzdPQFpla before the assay was started. At different time points, 40 μL subsamples were mixed with 40 μL of 5% trichloroacetic acid (TCA) to stop the reaction. After centrifugation (20800g for 15 min at 4 °C), the supernatant was analyzed for the formation of free phosphate by a malachite green assay.23 For this purpose, 30 μL of supernatant was mixed with 870 μL of H2Odest and 100 μL of the malachite green solution that was prepared by adding 2.5 mL of a 7.5% ammonium heptamolybdate solution and 0.2 mL of Tween 20 to 10 mL of 15% H2SO4 with 0.44 g of malachite green. After incubation at room temperature for 10 min, the absorption at 630 nm was determined. Quantification was achieved by comparison with a KH2PO4 standard curve. UV/Vis Spectroscopy. UV/vis spectra of purified BzdPQFpla were recorded under anaerobic conditions inside a glovebox. The enzyme solution (32 μM) was stepwise reduced by addition of sodium dithionite (800 μM stock solution). When the spectrum no longer changed, the solution was reoxidized with thionine (800 μM stock solution). Redox Titration. To determine the midpoint redox potential of the [4Fe-4S] cluster of BzdPQFpla, benzylviologen (E°′ = −359 mV) and diethylsafranin (E°′ = −251 mV)24 were used as redox dyes. Measurements were taken at 25 °C under anaerobic conditions in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.0), 50 mM KCl, and
4 mM MgCl2. A mixture of BzdPQFpla and redox dye was stepwise reduced with sodium dithionite until both were completely reduced. After each reduction step, an UV/vis spectrum (300−800 nm) was recorded to follow the reduction of the protein and of the redox dye. BzdPQFpla was applied at a concentration of 29 μM together with 100 μM benzylviologen. Reduction of BzdPQ was followed at 430 nm and reduction of benzylviologen at 670 nm. The redox potential after each addition of Na2S2O4 was calculated from the ratio of oxidized to reduced redox dye by the Nernst equation. Diethylsafranine measurements were conducted with a concentration of the redox dye of 50 μM and 13 μM BzdPQ. Here the reduction of BzdPQ was followed at 410 nm and that of diethylsafranine at 555 nm. The data from these measurements were fitted to a Nernst curve with n = 1 using GraphPad Prism 6. Phylogenetic Analysis. Amino acid sequences were aligned using ClustalW as implemented in MEGA version 5.2. These sequences were used to create a phylogenetic tree using the neighbor-joining algorithm as implemented in MEGA version 5.2. The following sequences were analyzed: Ac. fermentans HgdC (CAA42196.1), Clostridium symbiosum HgdC (AAD31675.1), Fusobacterium nucleatum HgdC (NP_603113.1), Clostridium sporogenes FldI (AAL18809.1), Clostridium dif f icile HadI (AAV40818.1), Azoarcus sp. CIB BzdP (AAQ08808.1) and BzdQ (AAQ08809.1), Aromatoleum aromaticum BzdP (WP_011236236.1) and BzdQ (WP_011236235.1), F. placidus BzdP (ADC65344.1) and BzdQ (ADC65345.1), T. aromatica BcrA (CAA12249.1) and BcrD (CAA12250.1), Rhodopseudomonas palustris BcrA (WP_013503789.1) and BcrD (WP_013503788.1), and Magnetospirillum magnetotacticum BcrA (WP_009868467.1) and BcrD (WP_009868466.1). Mö ssbauer Spectroscopy. Mö ssbauer spectra were recorded at 77 K using a conventional spectrometer in the constant-acceleration mode and a continuous flow cryostat (OptistatDN, Oxford Instruments). The measurements obtained at two different magnetic fields and low temperatures were taken with a closed cycle cryostat equipped with a superconducting magnet as described previously.25 Isomer shifts (δ) are given relative to α-Fe at room temperature. The spectra at 77 K were analyzed by least-squares fits using the Lorentzian line shape, and the magnetically split spectra were simulated on the basis of the spin Hamiltonian approximation26 using Vinda Add on for Excel 2003.27 EPR Spectroscopy. EPR spectra were recorded between 10 and 50 K with a Bruker ELEXSYS E580 X-band EPR spectrometer equipped with an ER4102ST cavity and an Oxford Instruments flow cryostat. Scans between 0 and 500 mT (with a modulation amplitude of 1.0 mT and a modulation frequency of 100 kHz at a resonant frequency of 9.46 GHz) did not reveal significant signals. Mössbauer samples (which were used to record the spectra of the [4Fe-4S]+ cluster in Figure 5A,B) were crushed in a liquid nitrogen-cooled mortar and cooled pestle and transferred as frozen ice chunks into liquid nitrogen-cooled wide-bore EPR tubes. To prevent accumulation of solid oxygen, EPR spectra were recorded directly after the transfer.
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RESULTS Heterologous Expression and Purification of BzdPQFpla. In a previous study, the genes encoding the four subunits of BCR from F. placidus, bzdNOPQ, were heterologously expressed and enriched by heat precipitation of E. coli 5580
DOI: 10.1021/acs.biochem.6b00729 Biochemistry 2016, 55, 5578−5586
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Biochemistry proteins.21 Though such BCR preparations showed some BzCoA activity below 1 nmol min−1 mg−1, the poor expression and the loss of subunits during the heat precipitation did not allow a biochemical characterization. In this work, we followed a different strategy and cloned and heterologously expressed bzdNOPQ in E. coli C43 (DE) pRKISC with a C-terminal Strep tag fused to the last protein of the operon, BzdQ. After Strep-Tactin affinity chromatography, two proteins at a nearly perfect 1:1 stoichiometry with molecular masses of 27 and 35 kDa were detected by SDS− PAGE (Figure 2). These proteins correspond to BzdP
(Ferp_1186, MWcalculated of 27.9 kDa) and BzdQ with a Strep tag (Ferp_1187, MWcalculated of 33.7 kDa), which together form the electron activation module of the BCRFpla. Obviously, the nontagged active site components were either poorly expressed or completely lost during Strep-Tactin affinity chromatography. The purified BzdPQFpla component was analyzed for its native molecular mass by analytical gel filtration on a Superdex 200 column. A major part of BzdPQFpla eluted according to a molecular weight of 73 kDa, indicating that the enzyme was mainly present as a heterodimer (MWcalculated of 62 kDa); a minor peak (
