RESUMEN
Soybean (Glycine max (L.) Merrill) isoflavones are among the most important secondary metabolites, with functional benefits for human health. Soybeans accumulate three aglycone forms of isoflavones: genistein, daidzein, and glycitein. Soybean landrace Kumachi-1 does not accumulate malonylglycitin at all. Gene structure analysis indicated that Glyma.11G108300 (F6H4) of Kumachi-1 has a 3.8-kbp insertion, resulting in a truncated flavonoid 6-hydroxylase (F6H) sequence compared to the wild-type sequence in Fukuyutaka. Mapping experiments using a mutant line (MUT1246) with a phenotype similar to that of Kumachi-1, with a single-nucleotide polymorphism (SNP) in F6H4, revealed co-segregation of this mutation and the absence of glycitein isoflavones. We also identified a mutant line (K01) that exhibited a change in the HPLC retention time of glycitein isoflavones, accumulating glycoside and malonylglycoside forms of 6-hydroxydaidzein. K01 contains an SNP that produces a premature stop codon in Glyma.01G004200 (IOMT3), a novel soybean isoflavone O-methyltransferase (IOMT) gene. We further analyzed transgenic hairy roots of soybeans expressing Glyma.11G108300 (F6H4) and Glyma.01G004200 (IOMT3). Those overexpressing F6H4 accumulated malonylglycoside forms of 6-hydroxydaidzein (M_6HD), and co-expression of F6H4 and IOMT3 increased the level of malonylglycitin but not of M_6HD. These results indicate that F6H4 and IOMT3 are responsible for glycitein biosynthesis in soybean seed hypocotyl.
RESUMEN
The scaly-foot gastropod (Chrysomallon squamiferum), which lives in the deep-sea zone of oceans around thermal vents, has a black shell and scales on the foot. Both the black shell and scales contain iron sulfide minerals such as greigite (Fe3S4) and pyrite (FeS2). Although pyrite nanoparticles can be used as materials for solar panels, it is difficult to synthesize stable and spherical nanoparticles in vitro. In this study, we extracted organic molecules that interact with nano-pyrite from the shell of the scaly-foot gastropod to develop a low-cost, eco-friendly method for pyrite nanoparticles synthesis. Myoglobin (csMG), a heme protein, was identified in the iron sulfide layer of the shell. We purified recombinant csMG (r-csMG) and demonstrated that r-csMG helped in the conversion of ferric ions, sulfide ions and sulfur into spherical shaped pyrite nanoparticles at 80°C. To reduce the effort and cost of production, we showed that commercially available myoglobin from Equus caballus (ecMG) also induced the in vitro synthesis of pyrite nanoparticles. Using structure-function experiments with digested peptides, we highlighted that the amino acid sequence of r-csMG peptides controlled the spherical shape of the nanoparticle while the hemin molecules, which the peptides interacted with, maintained the size of nanoparticles. Synthesized pyrite nanoparticles exhibited strong photoluminescence in the visible wavelength region, suggesting its potential application as a photovoltaic solar cell material. These results suggest that materials for solar cells can be produced at low cost and energy under eco-friendly conditions. STATEMENT OF SIGNIFICANCE: Pyrite is a highly promising material for photovoltaic devices because of its excellent optical, electrical, magnetic, and transport properties and high optical absorption coefficient. Almost all current pyrite synthesis methods use organic solvents at high temperature and pressure under reducing conditions. Synthesized pyrite nanoparticles are unstable and are difficult to use in devices. The scaly-foot gastropod can synthesize pyrite nanoparticles in vivo, meaning that pyrite nanoparticles can be generated in an aqueous environment at low temperature. In this study, we demonstrated the synthesis of pyrite nanoparticles using a heme protein identified in the iron sulfide layer of the scaly-foot gastropod shell. These results exemplify how natural products in organisms can inspire the innovation of new technology.
Asunto(s)
Gastrópodos , Nanopartículas , Animales , Caballos , Mioglobina , Sulfuros/químicaRESUMEN
Characterization of short-lived reaction intermediates is essential for elucidating the mechanism of the reaction catalyzed by metalloenzymes. Here, we demonstrated that the photolysis of a caged compound under cryogenic temperature followed by thermal annealing is an invaluable technique for trapping of short-lived reaction intermediates of metalloenzymes through the study of membrane-integrated nitric oxide reductase (NOR) that catalyzes reductive coupling of two NO molecules to N2O at its heme/nonheme FeB binuclear center. Although NO produced by the photolysis of caged NO did not react with NOR under cryogenic temperature, annealing to â¼160 K allowed NO to diffuse and react with NOR, which was evident from the appearance of EPR signals assignable to the S = 3/2 state. This indicates that the nonheme FeB-NO species can be trapped as the intermediate. Time-resolved IR spectroscopy with the use of the photolysis of caged NO as a reaction trigger showed that the intermediate formed at 10 µs gave the NO stretching frequency at 1683 cm-1 typical of nonheme Fe-NO, confirming that the combination of the cryo-photolysis of caged NO and annealing enabled us to trap the reaction intermediate. Thus, the cryo-photolysis of the caged compound has great potential for the characterization of short-lived reaction intermediates.
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Metaloproteínas , Óxido Nítrico , Óxido Nítrico/química , Fotólisis , Oxidorreductasas/químicaRESUMEN
Iron-sulfur (Fe-S) clusters are essential cofactors for enzyme activity. These Fe-S clusters are present in structurally diverse forms, including [4Fe-4S] and [3Fe-4S]. Type-identification of the Fe-S cluster is indispensable in understanding the catalytic mechanism of enzymes. However, identifying [4Fe-4S] and [3Fe-4S] clusters in particular is challenging because of their rapid transformation in response to oxidation-reduction events. In this study, we focused on the relationship between the Fe-S cluster type and the catalytic activity of a tRNA-thiolation enzyme (TtuA). We reconstituted [4Fe-4S]-TtuA, prepared [3Fe-4S]-TtuA by oxidizing [4Fe-4S]-TtuA under strictly anaerobic conditions, and then observed changes in the Fe-S clusters in the samples and the enzymatic activity in the time-course experiments. Electron paramagnetic resonance analysis revealed that [3Fe-4S]-TtuA spontaneously transforms into [4Fe-4S]-TtuA in minutes to one hour without an additional free Fe source in the solution. Although the TtuA immediately after oxidation of [4Fe-4S]-TtuA was inactive [3Fe-4S]-TtuA, its activity recovered to a significant level compared to [4Fe-4S]-TtuA after one hour, corresponding to an increase of [4Fe-4S]-TtuA in the solution. Our findings reveal that [3Fe-4S]-TtuA is highly inactive and unstable. Moreover, time-course analysis of structural changes and activity under strictly anaerobic conditions further unraveled the Fe-S cluster type used by the tRNA-thiolation enzyme.
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Proteínas Hierro-Azufre , Hierro , Hierro/química , Espectroscopía de Resonancia por Spin del Electrón , Oxidación-Reducción , Azufre/química , ARN de Transferencia/química , Proteínas Hierro-Azufre/metabolismoRESUMEN
Nitric oxide synthase (NOS) is a cytochrome P450-type monooxygenase that catalyzes the oxidation of L-arginine to nitric oxide. We previously observed that intramolecular electron transfer from biopterin to Fe2+-O2 in Deinococcus radiodurans NOS (DrNOS) using pulse radiolysis. However, the rate of electron transfer in DrNOS (2.2 × 103 s-1) contrasts with a reported corresponding rate (11 s-1) in a mammalian NOS determined using rapid freeze-quench (RFQ) EPR. We applied pulse radiolysis to Bacillus subtilis NOS (bsNOS) and to rat neural NOS oxygenase domain NOS (mNOS). Concurrently, RFQ EPR was used to trap a pterin radical during single-turnover enzyme reactions of the enzymes. By using the pulse radiolysis method, hydrated electrons (eaq-) reduced the heme iron of NOS enzymes. Subsequently, ferrous heme reacted with O2 to form a Fe2+-O2 intermediate. In the presence of pterin, the intermediate of bsNOS was found to convert to other intermediate in the time range of milliseconds. A similar process was determined to have occurred after pulse radiolysis of the pterin-bound mNOS, though the rate was much slower. The intermediates of all of the NOS enzymes further converted to the original ferric form in the time range of seconds. When using the RFQ method, pterin radicals were formed very rapidly in both DrNOS and bsNOS in the time range of milliseconds. In contrast, the pterin radical in mNOS was observed to form slowly, at a rate of â¼20 s-1.
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Biopterinas , Óxido Nítrico , Animales , Ratas , Arginina/metabolismo , Biopterinas/metabolismo , Electrones , Compuestos Ferrosos , Hemo/metabolismo , Hierro , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Oxidación-Reducción , Pterinas , Bacillus subtilis/enzimologíaRESUMEN
Free fatty acid receptor 4 (FFAR4)/GPR120 comprises a receptor for medium- and long-chain fatty acids. We previously identified phytosphingosine (PHS) as a novel ligand of FFAR4. Although many natural FFAR4 ligands have carboxyl groups, PHS does not, thus suggesting that binding to FFAR4 is driven by a completely different mechanism than other natural ligands such as α-linolenic acid (ALA). To test this hypothesis, we performed docking simulation analysis using a FFAR4 homology model based on a protein model derived from the crystal structure of activated turkey beta-1 adrenoceptor. The docking simulation revealed that the probable hydrogen bonds to FFAR4 differ between various ligands. In particular, binding was predicted between R264 of the FFAR4 and the oxygen of the carboxylate group in ALA, as well as between E249 of the FFAR4 and the oxygen of the hydroxy group at the C4-position in PHS. Alanine substitution at E249 (E249A) dramatically reduced PHS-induced FFAR4 activation but demonstrated a weaker effect on ALA-induced FFAR4 activation. Kinetic analysis and Km values clearly demonstrated that the E249A substitution resulted in reduced affinity for PHS but not for ALA. Additionally, we observed that sphingosine, lacking a hydroxyl group at C4-position, could not activate FFAR4. Our data show that E249 of the FFAR4 receptor is crucial for binding to the hydroxy group at the C4-position in PHS, and this is a completely different molecular mechanism of binding from ALA. Because GPR120 agonists have attracted attention as treatments for type 2 diabetes, our findings may provide new insights into their development.
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Esfingosina/análogos & derivados , Esfingosina/metabolismo , Comunicación Celular , Ácidos Grasos , Células HEK293 , Humanos , Cinética , Ligandos , Simulación del Acoplamiento Molecular/métodos , Unión Proteica , Receptores Acoplados a Proteínas G , Esfingosina/fisiologíaRESUMEN
Multicopper oxidases have a wide range of substrate specificity to be involved in various physiological reactions. Pseudomonas syringae, a plant pathogenic bacterium, has a multicopper oxidase, CumA. Multicopper oxidases have ability to degrade plant cell wall component, lignin. Once P. syringae enter apoplast and colonize, they start to disrupt plant immunity. Therefore, deeper understanding of multicopper oxidases from plant pathogens helps to invent measures to prevent invasion into plant cell, which brings agricultural benefits. Several biochemical studies have reported lower activity of CumA compared with other multicopper oxidase called CotA. However, the mechanisms underlying the difference in activity have not yet been revealed. In order to acquire insight into them, we conducted a biophysical characterization of PsCumA. Our results show that PsCumA has weak type I copper EPR signal, which is essential for oxidation activity. We propose that difference in the coordination of copper ions may decrease reaction frequency.
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Proteínas Bacterianas/metabolismo , Cobre/metabolismo , Oxidorreductasas/metabolismo , Plantas/microbiología , Pseudomonas syringae/enzimología , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Oxidorreductasas/clasificación , FilogeniaRESUMEN
TtuA and TtuB are the sulfurtransferase and sulfur donor proteins, respectively, for biosynthesis of 2-thioribothymidine (s2T) at position 54 of transfer RNA (tRNA), which is responsible for adaptation to high temperature environments in Thermus thermophilus. The enzymatic activity of TtuA requires an iron-sulfur (Fe-S) cluster, by which a sulfur atom supplied by TtuB is transferred to the tRNA substrate. Here, we demonstrate that the Fe-S cluster directly receives sulfur from TtuB through its inherent coordination ability. TtuB forms a [4Fe-4S]-TtuB intermediate, but that sulfur is not immediately released from TtuB. Further desulfurization assays and mutation studies demonstrated that the release of sulfur from the thiocarboxylated C-terminus of TtuB is dependent on adenylation of the substrate tRNA, and the essential residue for TtuB desulfurization was identified. Based on these findings, the molecular mechanism of sulfur transfer from TtuB to Fe-S cluster is proposed.
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Proteínas Bacterianas/metabolismo , Proteínas Hierro-Azufre/metabolismo , ARN de Transferencia/metabolismo , Sulfurtransferasas/metabolismo , Thermus thermophilus/enzimología , Tiouridina/análogos & derivados , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Modelos Moleculares , Familia de Multigenes , Mutación , Unión Proteica , Conformación Proteica , ARN de Transferencia/genética , Relación Estructura-Actividad , Especificidad por Sustrato , Sulfurtransferasas/química , Sulfurtransferasas/genética , Thermus thermophilus/genética , Tiouridina/metabolismoRESUMEN
Inorganic pyrophosphatase (PPase) catalyses the hydrolysis reaction of inorganic pyrophosphate to phosphates. Our previous studies showed that manganese (Mn) activated PPase from the psychrophilic bacterium Shewanella sp. AS-11 (Mn-Sh-PPase) has a characteristic temperature dependence of the activity with an optimum at 5 °C. Here we report the X-ray crystallography and electron paramagnetic resonance (EPR) spectroscopy structural analyses of Sh-PPase in the absence and presence of substrate analogues. We successfully determined the crystal structure of Mn-Sh-PPase without substrate and Mg-activated Sh-PPase (Mg-Sh-PPase) complexed with substrate analogue (imidodiphosphate; PNP). Crystallographic studies revealed a bridged water placed at a distance from the di-Mn centre in Mn-Sh-PPase without substrate. The water came closer to the metal centre when PNP bound. EPR analysis of Mn-Sh-PPase without substrate revealed considerably weak exchange coupling, whose magnitude was increased by binding of substrate analogues. The data indicate that the bridged molecule has weak bonds with the di-Mn centre, which suggests a 'loose' structure, whereas it comes closer to di-Mn centre by substrate binding, which suggests a 'well-tuned' structure for catalysis. Thus, we propose that Sh-PPase can rearrange the active site and that the 'loose' structure plays an important role in the cold adaptation mechanism.
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Dominio Catalítico , Pirofosfatasa Inorgánica/química , Modelos Moleculares , Sitios de Unión , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Pirofosfatasa Inorgánica/metabolismo , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Transfer RNA (tRNA) is an adaptor molecule indispensable for assigning amino acids to codons on mRNA during protein synthesis. 2-thiouridine (s2U) derivatives in the anticodons (position 34) of tRNAs for glutamate, glutamine, and lysine are post-transcriptional modifications essential for precise and efficient codon recognition in all organisms. s2U34 is introduced either by (i) bacterial MnmA/eukaryote mitochondrial Mtu1 or (ii) eukaryote cytosolic Ncs6/archaeal NcsA, and the latter enzymes possess iron-sulfur (Fe-S) cluster. Here, we report the identification of novel-type MnmA homologs containing three conserved Cys residues, which could support Fe-S cluster binding and catalysis, in a broad range of bacteria, including thermophiles, Cyanobacteria, Mycobacteria, Actinomyces, Clostridium, and Helicobacter Using EPR spectroscopy, we revealed that Thermus thermophilus MnmA (TtMnmA) contains an oxygen-sensitive [4Fe-4S]-type cluster. Efficient in vitro formation of s2U34 in tRNALys and tRNAGln by holo-TtMnmA occurred only under anaerobic conditions. Mutational analysis of TtMnmA suggested that the Fe-S cluster is coordinated by the three conserved Cys residues (Cys105, Cys108, and Cys200), and is essential for its activity. Evolutionary scenarios for the sulfurtransferases, including the Fe-S cluster containing Ncs6/NcsA s2U thiouridylases and several distantly related sulfurtransferases, are proposed.
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Anticodón/genética , Proteínas de Escherichia coli/genética , ARN de Transferencia/genética , Sulfurtransferasas/genética , Codón/genética , Cianobacterias/genética , Escherichia coli/genética , Ácido Glutámico/genética , Glutamina/genética , Hierro/metabolismo , Lisina/genética , Mycobacterium/genética , Azufre/metabolismo , Sulfurtransferasas/química , Tiouridina/análogos & derivados , Tiouridina/metabolismoRESUMEN
A bio-organometallic intermediate, denoted PA, was previously trapped during the reduction of propargyl alcohol to allyl alcohol (AA) by nitrogenase, and a similar one was trapped during acetylene reduction, representing foundational examples of alkene binding to a metal center in biology. ENDOR spectroscopy led to the conclusion that these intermediates have η2 binding of the alkene, with the hydrogens on the terminal carbon structurally/magnetically equivalent and related by local mirror symmetry. However, our understanding of both the PA intermediate, and of the dependability of the ENDOR analysis on which this understanding was based, was constrained by the absence of reference iron-alkene complexes for EPR/ENDOR comparison. Here, we report an ENDOR study of the crystallographically characterized biomimetic iron(i) complex 1, which exhibits η2 coordination of styrene, thus connecting hyperfine and structural parameters of an Fe-bound alkene fragment for the first time. A tilt of the alkene plane of 1 from normal to the crystallographic Fe-C2-C1 plane causes substantial differences in the dipolar couplings of the two terminal vinylic protons. Comparison of the hyperfine couplings of 1 and PA confirms the proposed symmetry of PA, and that the η2 interaction forms a scalene Fe-C-C triangle, rather than an isosceles triangle. This spectroscopic study of a structurally characterized complex thus shows the exceptional sensitivity of ENDOR spectroscopy to structural details, while enhancing our understanding of the geometry of a key nitrogenase adduct.
RESUMEN
Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B12 enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na+ as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by 23Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M+ ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron-sulfur cluster. ENDOR studies of the PFL-AE/[13C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little-no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li+ to Cs+, PFL-AE activity sharply maximizes at K+, with NH4+ closely matching the efficacy of K+. PFL-AE is thus a type I M+-activated enzyme whose M+ controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron-sulfur cluster.
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Enzimas/metabolismo , Escherichia coli/enzimología , S-Adenosilmetionina/metabolismo , Acetiltransferasas , Secuencia de Aminoácidos , Sitios de Unión , Cationes Monovalentes/química , Cationes Monovalentes/metabolismo , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática , Enzimas/química , Escherichia coli/química , Escherichia coli/metabolismo , Modelos Moleculares , S-Adenosilmetionina/químicaRESUMEN
S-Adenosylmethionine (SAM) has a sulfonium ion with three distinct C-S bonds. Conventional radical SAM enzymes use a [4Fe-4S] cluster to cleave homolytically the C5',adenosine-S bond of SAM to generate a 5'-deoxyadenosyl radical, which catalyzes various downstream chemical reactions. Radical SAM enzymes involved in diphthamide biosynthesis, such as Pyrococcus horikoshii Dph2 (PhDph2) and yeast Dph1-Dph2 instead cleave the Cγ,Met-S bond of methionine to generate a 3-amino-3-carboxylpropyl radical. We here show radical SAM enzymes can be tuned to cleave the third C-S bond to the sulfonium sulfur by changing the structure of SAM. With a decarboxyl SAM analogue (dc-SAM), PhDph2 cleaves the Cmethyl-S bond, forming 5'-deoxy-5'-(3-aminopropylthio) adenosine (dAPTA, 1). The methyl cleavage activity, like the cleavage of the other two C-S bonds, is dependent on the presence of a [4Fe-4S]+ cluster. Electron-nuclear double resonance and mass spectroscopy data suggests that mechanistically one of the S atoms in the [4Fe-4S] cluster captures the methyl group from dc-SAM, forming a distinct EPR-active intermediate, which can transfer the methyl group to nucleophiles such as dithiothreitol. This reveals the [4Fe-4S] cluster in a radical SAM enzyme can be tuned to cleave any one of the three bonds to the sulfonium sulfur of SAM or analogues, and is the first demonstration a radical SAM enzyme could switch from an Fe-based one electron transfer reaction to a S-based two electron transfer reaction in a substrate-dependent manner. This study provides an illustration of the versatile reactivity of Fe-S clusters.
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Histidina/análogos & derivados , Proteínas Hierro-Azufre/metabolismo , S-Adenosilmetionina/metabolismo , Radicales Libres/química , Radicales Libres/metabolismo , Histidina/biosíntesis , Histidina/química , Proteínas Hierro-Azufre/química , Estructura Molecular , Pyrococcus horikoshii/enzimología , S-Adenosilmetionina/química , Saccharomyces cerevisiae/enzimología , Especificidad por SustratoRESUMEN
In enzymatic C-H activation by hydrogen tunneling, reduced barrier width is important for efficient hydrogen wave function overlap during catalysis. For native enzymes displaying nonadiabatic tunneling, the dominant reactive hydrogen donor-acceptor distance (DAD) is typically ca. 2.7 Å, considerably shorter than normal van der Waals distances. Without a ground state substrate-bound structure for the prototypical nonadiabatic tunneling system, soybean lipoxygenase (SLO), it has remained unclear whether the requisite close tunneling distance occurs through an unusual ground state active site arrangement or by thermally sampling conformational substates. Herein, we introduce Mn2+ as a spin-probe surrogate for the SLO Fe ion; X-ray diffraction shows Mn-SLO is structurally faithful to the native enzyme. 13C ENDOR then reveals the locations of 13C10 and reactive 13C11 of linoleic acid relative to the metal; 1H ENDOR and molecular dynamics simulations of the fully solvated SLO model using ENDOR-derived restraints give additional metrical information. The resulting three-dimensional representation of the SLO active site ground state contains a reactive (a) conformer with hydrogen DAD of â¼3.1 Å, approximately van der Waals contact, plus an inactive (b) conformer with even longer DAD, establishing that stochastic conformational sampling is required to achieve reactive tunneling geometries. Tunneling-impaired SLO variants show increased DADs and variations in substrate positioning and rigidity, confirming previous kinetic and theoretical predictions of such behavior. Overall, this investigation highlights the (i) predictive power of nonadiabatic quantum treatments of proton-coupled electron transfer in SLO and (ii) sensitivity of ENDOR probes to test, detect, and corroborate kinetically predicted trends in active site reactivity and to reveal unexpected features of active site architecture.
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Hidrógeno/metabolismo , Lipooxigenasa/química , Resonancia Magnética Nuclear Biomolecular , Sitios de Unión , Biocatálisis , Isótopos de Carbono , Cristalografía por Rayos X , Hidrógeno/química , Cinética , Lipooxigenasa/aislamiento & purificación , Lipooxigenasa/metabolismo , Simulación de Dinámica Molecular , Estructura Molecular , Especificidad por SustratoRESUMEN
Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen (N2) to two ammonia (NH3) molecules through the participation of its two protein components, the MoFe and Fe proteins. Electron transfer (ET) from the Fe protein to the catalytic MoFe protein involves a series of synchronized events requiring the transient association of one Fe protein with each αß half of the α2ß2 MoFe protein. This process is referred to as the Fe protein cycle and includes binding of two ATP to an Fe protein, association of an Fe protein with the MoFe protein, ET from the Fe protein to the MoFe protein, hydrolysis of the two ATP to two ADP and two Pi for each ET, Pi release, and dissociation of oxidized Fe protein-(ADP)2 from the MoFe protein. Because the MoFe protein tetramer has two separate αß active units, it participates in two distinct Fe protein cycles. Quantitative kinetic measurements of ET, ATP hydrolysis, and Pi release during the presteady-state phase of electron delivery demonstrate that the two halves of the ternary complex between the MoFe protein and two reduced Fe protein-(ATP)2 do not undergo the Fe protein cycle independently. Instead, the data are globally fit with a two-branch negative-cooperativity kinetic model in which ET in one-half of the complex partially suppresses this process in the other. A possible mechanism for communication between the two halves of the nitrogenase complex is suggested by normal-mode calculations showing correlated and anticorrelated motions between the two halves.
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Adenosina Trifosfato/química , Molibdoferredoxina/química , Complejos Multiproteicos/química , Oxidorreductasas/química , Adenosina Trifosfato/metabolismo , Animales , Transporte de Electrón , Hidrólisis , Cinética , Molibdoferredoxina/metabolismo , Complejos Multiproteicos/metabolismo , Fijación del Nitrógeno , Oxidorreductasas/metabolismo , Unión Proteica , Salmón/metabolismoRESUMEN
Pyrococcus horikoshii Dph2 (PhDph2) is an unusual radical S-adenosylmethionine (SAM) enzyme involved in the first step of diphthamide biosynthesis. It catalyzes the reaction by cleaving SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. To probe the reaction mechanism, we synthesized a SAM analogue (SAMCA), in which the ACP group of SAM is replaced with a 3-carboxyallyl group. SAMCA is cleaved by PhDph2, yielding a paramagnetic (S = 1/2) species, which is assigned to a complex formed between the reaction product, α-sulfinyl-3-butenoic acid, and the [4Fe-4S] cluster. Electron-nuclear double resonance (ENDOR) measurements with (13)C and (2)H isotopically labeled SAMCA support a π-complex between the CâC double bond of α-sulfinyl-3-butenoic acid and the unique iron of the [4Fe-4S] cluster. This is the first example of a radical SAM-related [4Fe-4S](+) cluster forming an organometallic complex with an alkene, shedding additional light on the mechanism of PhDph2 and expanding our current notions for the reactivity of [4Fe-4S] clusters in radical SAM enzymes.
Asunto(s)
Enzimas/química , Proteínas Hierro-Azufre/química , Compuestos Organometálicos/química , Pyrococcus horikoshii/enzimología , S-Adenosilmetionina/química , Alquenos/química , Anisotropía , Butiratos/química , Carbono/química , Catálisis , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia por Spin del Electrón , Electrones , Histidina/análogos & derivados , Histidina/química , Hierro/químicaRESUMEN
Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to cleave SAM to initiate diverse radical reactions. These reactions are thought to involve the 5'-deoxyadenosyl radical intermediate, which has not yet been detected. We used rapid freeze-quenching to trap a catalytically competent intermediate in the reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme. Characterization of the intermediate by electron paramagnetic resonance and (13)C, (57)Fe electron nuclear double-resonance spectroscopies reveals that it contains an organometallic center in which the 5' carbon of a SAM-derived deoxyadenosyl moiety forms a bond with the unique iron site of the [4Fe-4S] cluster. Discovery of this intermediate extends the list of enzymatic bioorganometallic centers to the radical SAM enzymes, the largest enzyme superfamily known, and reveals intriguing parallels to B12 radical enzymes.
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Biocatálisis , Enzimas/química , Proteínas Hierro-Azufre/química , S-Adenosilmetionina/química , Acetiltransferasas , Espectroscopía de Resonancia por Spin del Electrón , Resonancia Magnética Nuclear Biomolecular , Compuestos Organometálicos/química , Vitamina B 12/químicaRESUMEN
In an effort to examine the relative position of a hairpin loop in New Delhi metallo-ß-lactamase, NDM-1, during catalysis, rapid freeze quench double electron electron resonance (RFQ-DEER) spectroscopy was used. A doubly-labeled mutant of NDM-1, which had one spin label on the invariant loop at position 69 and another label at position 235, was prepared and characterized. The reaction of the doubly spin labeled mutant with chromacef was freeze quenched at 500µs and 10ms. DEER results showed that the average distance between labels decreased by 4Å in the 500µs quenched sample and by 2Å in the 10ms quenched sample, as compared to the distance in the unreacted enzyme, although the peaks corresponding to distance distributions were very broad. DEER spectra with the doubly spin labeled enzyme with two inhibitors showed that the distance between the loop residue at position 69 and the spin label at position 235 does not change upon inhibitor binding. This study suggests that the hairpin loop in NDM-1 moves over the metal ion during the catalysis and then moves back to its original position after hydrolysis, which is consistent with a previous hypothesis based on NMR solution studies on a related metallo-ß-lactamase. This study also demonstrates that this loop motion occurs in the millisecond time domain.
Asunto(s)
beta-Lactamasas/metabolismo , Catálisis , Simulación de Dinámica Molecular , Inhibidores de beta-Lactamasas/farmacologíaRESUMEN
Lysine 2,3-aminomutase (LAM) is a radical S-adenosyl-L-methionine (SAM) enzyme and, like other members of this superfamily, LAM utilizes radical-generating machinery comprising SAM anchored to the unique Fe of a [4Fe-4S] cluster via a classical five-membered N,O chelate ring. Catalysis is initiated by reductive cleavage of the SAM S-C5' bond, which creates the highly reactive 5'-deoxyadenosyl radical (5'-dAdoâ¢), the same radical generated by homolytic Co-C bond cleavage in B12 radical enzymes. The SAM surrogate S-3',4'-anhydroadenosyl-L-methionine (anSAM) can replace SAM as a cofactor in the isomerization of L-α-lysine to L-ß-lysine by LAM, via the stable allylic anhydroadenosyl radical (anAdoâ¢). Here electron nuclear double resonance (ENDOR) spectroscopy of the anAdo⢠radical in the presence of (13)C, (2)H, and (15)N-labeled lysine completes the picture of how the active site of LAM from Clostridium subterminale SB4 "tames" the 5'-dAdo⢠radical, preventing it from carrying out harmful side reactions: this "free radical" in LAM is never free. The low steric demands of the radical-generating [4Fe-4S]/SAM construct allow the substrate target to bind adjacent to the S-C5' bond, thereby enabling the 5'-dAdo⢠radical created by cleavage of this bond to react with its partners by undergoing small motions, â¼0.6 Å toward the target and â¼1.5 Å overall, that are controlled by tight van der Waals contact with its partners. We suggest that the accessibility to substrate and ready control of the reactive C5' radical, with "van der Waals control" of small motions throughout the catalytic cycle, is common within the radical SAM enzyme superfamily and is a major reason why these enzymes are the preferred means of initiating radical reactions in nature.