The Experts below are selected from a list of 273 Experts worldwide ranked by ideXlab platform
Joan B Broderick - One of the best experts on this subject based on the ideXlab platform.
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mechanistic studies of radical sam enzymes Pyruvate Formate Lyase activating enzyme and lysine 2 3 aminomutase case studies
Methods in Enzymology, 2018Co-Authors: Amanda S Byer, William E Broderick, Elizabeth C Mcdaniel, Stella Impano, Joan B BroderickAbstract:Abstract The radical SAM enzyme superfamily is large and diverse, with ever-increasing numbers of examples of characterized reactions. This chapter focuses on the methodology we have developed over the last 25 years for working with these enzymes, with the specific examples discussed being the Pyruvate Formate-Lyase activating enzyme (PFL-AE) and lysine 2,3-aminomutase (LAM). Both enzymes are purified from overexpressing Escherichia coli, but differ in that PFL-AE is expressed without an affinity tag and does not require iron–sulfur cluster reconstitution, while LAM purification is carried out through use of a His6 affinity tag and the enzyme benefits from cluster reconstitution. Because of radical SAM enzymes’ catalytic need for a [4Fe–4S] cluster, we present methods for characterization and incorporation of a full [4Fe–4S] cluster in addition to enzyme activity assay protocols. Synthesis of SAM (S-adenosyl- l -methionine) and its analogs have played an important role in our mechanistic studies of radical SAM enzymes, and their synthetic methods are also presented in detail.
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spectroscopic investigation of Pyruvate Formate Lyase activating enzyme a look into epr endor and mossabuer spectroscopy
Research Journal of Applied Sciences Engineering and Technology, 2014Co-Authors: Danilo Ortillo, Joan B BroderickAbstract:Electron Paramagnetic Resonance (EPR) and Electron Nuclear Double Resonance (ENDOR) spectroscopies are extremely powerful and versatile methods for the characterization of paramagnetic systems in biology, chemistry and physics. For iron centers in the radical SAM enzymes however, Mossbauer spectroscopy has proven to be both powerful and useful as a complementary spectroscopic technique in determining not just the oxidation states but also the type of iron species present in the catalytic center. The cluster content of the radical SAM protein, Pyruvate Formate-Lyase-Activating Enzyme (PFL-AE), was characterized using EPR and Mossbauer techniques while additional ENDOR analysis helped determine the novel interaction of the co-substrate, SAdenosylmethionine (SAM or AdoMet) with the Fe-S cluster of PFL-AE. The anchoring role of the Fe-S cluster to the co-substrate derived from the spectroscopic data supports the mechanism where a SAM-based radical species is involved during catalysis.
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Pyruvate Formate Lyase and its activation by Pyruvate Formate Lyase activating enzyme
Journal of Biological Chemistry, 2014Co-Authors: Adam V Crain, Joan B BroderickAbstract:Abstract The activation of Pyruvate Formate-Lyase (PFL) by Pyruvate Formate-Lyase activating enzyme (PFL-AE) involves formation of a specific glycyl radical on PFL by the PFL-AE in a reaction requiring S-adenosylmethionine (SAM). Surface plasmon resonance (SPR) experiments were performed under anaerobic conditions on the oxygen sensitive PFL-AE to determine the kinetics and equilibrium constant for its interaction with PFL. These experiments show that the interaction is very slow and rate-limited by large conformational changes. A novel SAM binding assay was used to accurately determine the equilibrium constants for SAM binding to PFL-AE alone and in complex with PFL. The PFL-AE bound SAM with the same affinity (~ 6 μM) regardless of the presence or absence of PFL. Activation of PFL in the presence of its substrate Pyruvate or the analogue oxamate resulted in stoichiometric conversion of the [4Fe-4S]1+ cluster to the glycyl radical on PFL, however 3.7-fold less activation was achieved in the absence of these small molecules, demonstrating that Pyruvate or oxamate are required for optimal activation. Finally, in vivo concentrations of the entire PFL system were calculated to estimate the amount of bound protein in the cell. PFL, PFL-AE, and SAM are essentially fully bound in vivo, whereas electron donor proteins are partially bound.
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Pyruvate Formate Lyase evidence for an open conformation favored in the presence of its activating enzyme
Journal of Biological Chemistry, 2010Co-Authors: Yi Peng, Susan E Veneziano, Gregory D Gillispie, Joan B BroderickAbstract:Pyruvate Formate-Lyase-activating enzyme (PFL-AE) activates Pyruvate Formate-Lyase (PFL) by generating a catalytically essential radical on Gly-734 of PFL. Crystal structures of unactivated PFL reveal that Gly-734 is buried 8 Å from the surface of the protein in what we refer to here as the closed conformation of PFL. We provide here the first experimental evidence for an alternate open conformation of PFL in which: (i) the glycyl radical is significantly less stable; (ii) the activated enzyme exhibits lower catalytic activity; (iii) the glycyl radical undergoes less H/D exchange with solvent; and (iv) the Tm of the protein is decreased. The evidence suggests that in the open conformation of PFL, the Gly-734 residue is located not in its buried position in the enzyme active site but rather in a more solvent-exposed location. Further, we find that the presence of the PFL-AE increases the proportion of PFL in the open conformation; this observation supports the idea that PFL-AE accesses Gly-734 for direct hydrogen atom abstraction by binding to the Gly-734 loop in the open conformation, thereby shifting the closed ↔ open equilibrium of PFL to the right. Together, our results lead to a model in which PFL can exist in either a closed conformation, with Gly-734 buried in the active site of PFL and harboring a stable glycyl radical, or an open conformation, with Gly-734 more solvent-exposed and accessible to the PFL-AE active site. The equilibrium between these two conformations of PFL is modulated by the interaction with PFL-AE.
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The Iron−Sulfur Cluster of Pyruvate Formate-Lyase Activating Enzyme in Whole Cells: Cluster Interconversion and a Valence-Localized [4Fe-4S]2+ State
Biochemistry, 2009Co-Authors: Jian Yang, Meng Li, William E Broderick, Boi Hanh Huynh, Sunil G Naik, Danilo Ortillo, Ricardo Garcia-serres, Joan B BroderickAbstract:Pyruvate Formate-Lyase activating enzyme (PFL-AE) catalyzes the generation of a catalytically essential glycyl radical on Pyruvate Formate-Lyase (PFL). Purified PFL-AE contains an oxygen-sensitive, labile [4Fe-4S] cluster that undergoes cluster interconversions in vitro, with only the [4Fe-4S]+ cluster state being catalytically active. Such cluster interconversions could play a role in regulating the activity of PFL-AE, and thus of PFL, in response to oxygen levels in vivo. Here we report a Mossbauer investigation on whole cells overexpressing PFL-AE following incubation under aerobic and/or anaerobic conditions and provide evidence that PFL-AE undergoes cluster interconversions in vivo. After 2 h aerobic induction of PFL-AE expression, approximately 44% of the total iron is present in [4Fe-4S]2+ clusters, 6% in [2Fe-2S]2+ clusters, and the remainder as noncluster FeIII (29%) and FeII (21%) species. Subsequent anaerobic incubation of the culture results in approximately 75% of the total iron being present...
William E Broderick - One of the best experts on this subject based on the ideXlab platform.
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mechanistic studies of radical sam enzymes Pyruvate Formate Lyase activating enzyme and lysine 2 3 aminomutase case studies
Methods in Enzymology, 2018Co-Authors: Amanda S Byer, William E Broderick, Elizabeth C Mcdaniel, Stella Impano, Joan B BroderickAbstract:Abstract The radical SAM enzyme superfamily is large and diverse, with ever-increasing numbers of examples of characterized reactions. This chapter focuses on the methodology we have developed over the last 25 years for working with these enzymes, with the specific examples discussed being the Pyruvate Formate-Lyase activating enzyme (PFL-AE) and lysine 2,3-aminomutase (LAM). Both enzymes are purified from overexpressing Escherichia coli, but differ in that PFL-AE is expressed without an affinity tag and does not require iron–sulfur cluster reconstitution, while LAM purification is carried out through use of a His6 affinity tag and the enzyme benefits from cluster reconstitution. Because of radical SAM enzymes’ catalytic need for a [4Fe–4S] cluster, we present methods for characterization and incorporation of a full [4Fe–4S] cluster in addition to enzyme activity assay protocols. Synthesis of SAM (S-adenosyl- l -methionine) and its analogs have played an important role in our mechanistic studies of radical SAM enzymes, and their synthetic methods are also presented in detail.
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the iron sulfur cluster of Pyruvate Formate Lyase activating enzyme in whole cells cluster interconversion and a valence localized 4fe 4s 2 state
Biochemistry, 2009Co-Authors: Jian Yang, Meng Li, William E Broderick, Boi Hanh Huynh, Sunil G Naik, Danilo Ortillo, Ricardo Garciaserres, Joan B BroderickAbstract:Pyruvate Formate-Lyase activating enzyme (PFL-AE) catalyzes the generation of a catalytically essential glycyl radical on Pyruvate Formate-Lyase (PFL). Purified PFL-AE contains an oxygen-sensitive, labile [4Fe-4S] cluster that undergoes cluster interconversions in vitro, with only the [4Fe-4S]+ cluster state being catalytically active. Such cluster interconversions could play a role in regulating the activity of PFL-AE, and thus of PFL, in response to oxygen levels in vivo. Here we report a Mossbauer investigation on whole cells overexpressing PFL-AE following incubation under aerobic and/or anaerobic conditions and provide evidence that PFL-AE undergoes cluster interconversions in vivo. After 2 h aerobic induction of PFL-AE expression, approximately 44% of the total iron is present in [4Fe-4S]2+ clusters, 6% in [2Fe-2S]2+ clusters, and the remainder as noncluster FeIII (29%) and FeII (21%) species. Subsequent anaerobic incubation of the culture results in approximately 75% of the total iron being present...
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The Iron−Sulfur Cluster of Pyruvate Formate-Lyase Activating Enzyme in Whole Cells: Cluster Interconversion and a Valence-Localized [4Fe-4S]2+ State
Biochemistry, 2009Co-Authors: Jian Yang, Meng Li, William E Broderick, Boi Hanh Huynh, Sunil G Naik, Danilo Ortillo, Ricardo Garcia-serres, Joan B BroderickAbstract:Pyruvate Formate-Lyase activating enzyme (PFL-AE) catalyzes the generation of a catalytically essential glycyl radical on Pyruvate Formate-Lyase (PFL). Purified PFL-AE contains an oxygen-sensitive, labile [4Fe-4S] cluster that undergoes cluster interconversions in vitro, with only the [4Fe-4S]+ cluster state being catalytically active. Such cluster interconversions could play a role in regulating the activity of PFL-AE, and thus of PFL, in response to oxygen levels in vivo. Here we report a Mossbauer investigation on whole cells overexpressing PFL-AE following incubation under aerobic and/or anaerobic conditions and provide evidence that PFL-AE undergoes cluster interconversions in vivo. After 2 h aerobic induction of PFL-AE expression, approximately 44% of the total iron is present in [4Fe-4S]2+ clusters, 6% in [2Fe-2S]2+ clusters, and the remainder as noncluster FeIII (29%) and FeII (21%) species. Subsequent anaerobic incubation of the culture results in approximately 75% of the total iron being present...
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structural basis for glycyl radical formation by Pyruvate Formate Lyase activating enzyme
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Jian Yang, Meng Li, William E Broderick, Joan B Broderick, Catherine L DrennanAbstract:Pyruvate Formate-Lyase activating enzyme generates a stable and catalytically essential glycyl radical on G734 of Pyruvate Formate-Lyase via the direct, stereospecific abstraction of a hydrogen atom from Pyruvate Formate-Lyase. The activase performs this remarkable feat by using an iron-sulfur cluster and S-adenosylmethionine (AdoMet), thus placing it among the AdoMet radical superfamily of enzymes. We report here structures of the substrate-free and substrate-bound forms of Pyruvate Formate-Lyase-activating enzyme, the first structures of an AdoMet radical activase. To obtain the substrate-bound structure, we have used a peptide substrate, the 7-mer RVSGYAV, which contains the sequence surrounding G734. Our structures provide fundamental insights into the interactions between the activase and the G734 loop of Pyruvate Formate-Lyase and provide a structural basis for direct and stereospecific H atom abstraction from the buried G734 of Pyruvate Formate-Lyase.
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an anchoring role for fes clusters chelation of the amino acid moiety of s adenosylmethionine to the unique iron site of the 4fe 4s cluster of Pyruvate Formate Lyase activating enzyme
Journal of the American Chemical Society, 2002Co-Authors: Charles J Walsby, William E Broderick, Joan B Broderick, Danilo Ortillo, Brian M HoffmanAbstract:Pyruvate Formate-Lyase activating enzyme (PFL-AE) generates the catalytically essential glycyl radical on Pyruvate Formate-Lyase via the interaction of the catalytically active [4Fe−4S]+ cluster with S-adenosylmethionine (AdoMet). Like other members of the Fe−S/AdoMet family of enzymes, PFL-AE is thought to function via generation of an AdoMet-derived 5‘-deoxyadenosyl radical intermediate; however, the mechanistic steps by which this radical is generated remain to be elucidated. While all of the members of the Fe−S/AdoMet family of enzymes appear to have a unique iron site in the [4Fe−4S] cluster, based on the presence of a conserved three-cysteine cluster binding motif, the role of this unique site has been elusive. Here we utilize 35-GHz pulsed electron nuclear double resonance (ENDOR) studies of the [4Fe−4S]+ cluster of PFL-AE in complex with isotopically labeled AdoMet (denoted [1+/AdoMet]) to show that the unique iron serves to anchor the AdoMet for catalysis. AdoMet labeled with 17O at the carboxyla...
Joachim Knappe - One of the best experts on this subject based on the ideXlab platform.
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stable glycyl radical from Pyruvate Formate Lyase and ribonucleotide reductase iii
Advances in Protein Chemistry, 2001Co-Authors: Joachim Knappe, A Volker F WagnerAbstract:Publisher Summary This chapter reviews the protein chemical and functional similarities between the glycyl radicals of Pyruvate Formate-Lyase (PFL) and ribonucleotide reductase (RNR) III. In catalyzing quite different chemical reactions, the two enzymes illustrate the broad utilization potential that nature has apparently exploited for this cofactor. The investigations of the two enzyme systems have revealed the principles of how the glycyl radical cofactor is generated, stabilized, and utilized to promote the transformation of organic molecules along the radical-chemical reaction coordinates. The selection of the simple glycine residue as a radical storage element for the chemical purpose of transferring hydrogen atoms must be an ancient stratagem in the evolution of life. While glycyl radical enzymes are distributed today among many classes of organisms, but restricted to anaerobes a “ur-ribonucleotide reductase” probably of the class III type is presently proposed as a prerequisite for the first appearance of DNA. The sequences of extant PFL and anaerobic RNR enzymes suggest divergent evolution of these proteins.
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modification of cys 418 of Pyruvate Formate Lyase by methacrylic acid based on its radical mechanism
FEBS Letters, 2000Co-Authors: Wulf Plaga, Gabriele Vielhaber, Jochen Wallach, Joachim KnappeAbstract:Abstract The recently determined crystal structure of Pyruvate Formate-Lyase (PFL) suggested a new view of the mechanism of this glycyl radical enzyme, namely that intermediary thiyl radicals of Cys-418 and Cys-419 participate in different ways [Becker, A. et al. (1999) Nat. Struct. Biol. 6, 969–975]. We report here a suicide reaction of PFL that occurs with the substrate-analog methacrylate with retention of the protein radical (KI=0.42 mM, ki=0.14 min−1). Using [1-14C]methacrylate (synthesized via acetone cyanhydrin), the reaction end-product was identified by peptide mapping and cocrystallization experiments as S-(2-carboxy-(2S)-propyl) substituted Cys-418. The stereoselectivity of the observed Michael addition reaction is compatible with a radical mechanism that involves Cys-418 thiyl as nucleophile and Cys-419 as H-atom donor, thus supporting the functional assignments of these catalytic amino acid residues derived from the protein structure.
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structure and mechanism of the glycyl radical enzyme Pyruvate Formate Lyase
Nature Structural & Molecular Biology, 1999Co-Authors: A Becker, Joachim Knappe, Karin Fritzwolf, Wolfgang Kabsch, Sabine Schultz, A Volker F WagnerAbstract:Pyruvate Formate-Lyase (PFL) from Escherichia coli uses a radical mechanism to reversibly cleave the C1-C2 bond of Pyruvate using the Gly 734 radical and two cysteine residues (Cys 418, Cys 419). We have determined by X-ray crystallography the structures of PFL (non-radical form), its complex with the substrate analog oxamate, and the C418A,C419A double mutant. The atomic model (a dimer of 759-residue monomers) comprises a 10-stranded β/α barrel assembled in an antiparallel manner from two parallel five-stranded β-sheets; this architecture resembles that of ribonucleotide reductases. Gly 734 and Cys 419, positioned at the tips of opposing hairpin loops, meet in the apolar barrel center (Cα–Sγ = 3.7 A). Oxamate fits into a compact pocket where C2 is juxtaposed with Cys 418Sγ (3.3 A), which in turn is close to Cys 419Sγ (3.7 A). Our model of the active site is suggestive of a snapshot of the catalytic cycle, when the Pyruvate-carbonyl awaits attack by the Cys 418 thiyl radical. We propose a homolytic radical mechanismfor PFL that involves Cys 418 and Cys 419 both as thiyl radicals, with distinct chemical functions.
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a dehydroalanyl residue can capture the 5 deoxyadenosyl radical generated froms adenosylmethionine by Pyruvate Formate Lyase activating enzyme
Biochemical and Biophysical Research Communications, 1999Co-Authors: A Volker F Wagner, Jens Demand, Gerhard Schilling, Thomas Pils, Joachim KnappeAbstract:Abstract The glycyl radical (Gly-734) contained in the active form of Pyruvate Formate-Lyase (PFL) of Escherichia coli is produced post-translationally by Pyruvate Formate-Lyase-activating enzyme (PFL activase), employing adenosylmethionine (AdoMet) and dihydroflavodoxin as co-substrates. Previous 2 H-labelings found incorporation of the pro- S hydrogen of Gly-734 into the 5′-deoxyadenosine co-product, indicating that a deoxyadenosyl radical intermediate, generated by reductive cleavage of AdoMet, serves as the actual H atom abstracting species in this system. We have now examined an octapeptide (Suc-Arg-Val-Pro-ΔAla-Tyr-Ala-Val-Arg-NH 2 ) that is analogous to the Gly-734 site of the PFL polypeptide but contains a dehydroalanyl residue (ΔAla) in the glycyl position. Applied to the PFL activase reaction, this peptide becomes C -adenosylated at the olefinic β carbon of ΔAla. The modified peptide was isolated in μmol-quantities and characterized, after chymotryptic truncation, by MS and 2D NMR. PFL activase functions catalytically (k cat ≥ 1 min −1 ) in the peptide modification reaction, which occurs with stoichiometric consumption of AdoMet. The mechanism appears to involve addition of the nucleophilic deoxyadenosyl radical to the electrophilic CC double bond of ΔAla, followed by quenching of the peptide backbone-centered adduct radical by the buffer medium. The trapping-property of the ΔAla residue should be exploitable in investigating of how the Fe 4 S 4 protein PFL activase generates the highly reactive deoxyadenosyl radical.
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A Dehydroalanyl Residue Can Capture the 5′-Deoxyadenosyl Radical Generated fromS-Adenosylmethionine by Pyruvate Formate-Lyase-Activating Enzyme
Biochemical and Biophysical Research Communications, 1999Co-Authors: A.f.volker Wagner, Gerhard Schilling, Jens Demand, Thomas Pils, Joachim KnappeAbstract:Abstract The glycyl radical (Gly-734) contained in the active form of Pyruvate Formate-Lyase (PFL) of Escherichia coli is produced post-translationally by Pyruvate Formate-Lyase-activating enzyme (PFL activase), employing adenosylmethionine (AdoMet) and dihydroflavodoxin as co-substrates. Previous 2 H-labelings found incorporation of the pro- S hydrogen of Gly-734 into the 5′-deoxyadenosine co-product, indicating that a deoxyadenosyl radical intermediate, generated by reductive cleavage of AdoMet, serves as the actual H atom abstracting species in this system. We have now examined an octapeptide (Suc-Arg-Val-Pro-ΔAla-Tyr-Ala-Val-Arg-NH 2 ) that is analogous to the Gly-734 site of the PFL polypeptide but contains a dehydroalanyl residue (ΔAla) in the glycyl position. Applied to the PFL activase reaction, this peptide becomes C -adenosylated at the olefinic β carbon of ΔAla. The modified peptide was isolated in μmol-quantities and characterized, after chymotryptic truncation, by MS and 2D NMR. PFL activase functions catalytically (k cat ≥ 1 min −1 ) in the peptide modification reaction, which occurs with stoichiometric consumption of AdoMet. The mechanism appears to involve addition of the nucleophilic deoxyadenosyl radical to the electrophilic CC double bond of ΔAla, followed by quenching of the peptide backbone-centered adduct radical by the buffer medium. The trapping-property of the ΔAla residue should be exploitable in investigating of how the Fe 4 S 4 protein PFL activase generates the highly reactive deoxyadenosyl radical.
Jian Yang - One of the best experts on this subject based on the ideXlab platform.
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monovalent cation activation of the radical sam enzyme Pyruvate Formate Lyase activating enzyme
Journal of the American Chemical Society, 2017Co-Authors: Krista A Shisler, Adam V Crain, Rachel U Hutcheson, Masaki Horitani, Kaitlin S Duschene, Amanda S Byer, Eric M Shepard, Ashley Rasmussen, Jian YangAbstract: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 positionin...
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the iron sulfur cluster of Pyruvate Formate Lyase activating enzyme in whole cells cluster interconversion and a valence localized 4fe 4s 2 state
Biochemistry, 2009Co-Authors: Jian Yang, Meng Li, William E Broderick, Boi Hanh Huynh, Sunil G Naik, Danilo Ortillo, Ricardo Garciaserres, Joan B BroderickAbstract:Pyruvate Formate-Lyase activating enzyme (PFL-AE) catalyzes the generation of a catalytically essential glycyl radical on Pyruvate Formate-Lyase (PFL). Purified PFL-AE contains an oxygen-sensitive, labile [4Fe-4S] cluster that undergoes cluster interconversions in vitro, with only the [4Fe-4S]+ cluster state being catalytically active. Such cluster interconversions could play a role in regulating the activity of PFL-AE, and thus of PFL, in response to oxygen levels in vivo. Here we report a Mossbauer investigation on whole cells overexpressing PFL-AE following incubation under aerobic and/or anaerobic conditions and provide evidence that PFL-AE undergoes cluster interconversions in vivo. After 2 h aerobic induction of PFL-AE expression, approximately 44% of the total iron is present in [4Fe-4S]2+ clusters, 6% in [2Fe-2S]2+ clusters, and the remainder as noncluster FeIII (29%) and FeII (21%) species. Subsequent anaerobic incubation of the culture results in approximately 75% of the total iron being present...
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The Iron−Sulfur Cluster of Pyruvate Formate-Lyase Activating Enzyme in Whole Cells: Cluster Interconversion and a Valence-Localized [4Fe-4S]2+ State
Biochemistry, 2009Co-Authors: Jian Yang, Meng Li, William E Broderick, Boi Hanh Huynh, Sunil G Naik, Danilo Ortillo, Ricardo Garcia-serres, Joan B BroderickAbstract:Pyruvate Formate-Lyase activating enzyme (PFL-AE) catalyzes the generation of a catalytically essential glycyl radical on Pyruvate Formate-Lyase (PFL). Purified PFL-AE contains an oxygen-sensitive, labile [4Fe-4S] cluster that undergoes cluster interconversions in vitro, with only the [4Fe-4S]+ cluster state being catalytically active. Such cluster interconversions could play a role in regulating the activity of PFL-AE, and thus of PFL, in response to oxygen levels in vivo. Here we report a Mossbauer investigation on whole cells overexpressing PFL-AE following incubation under aerobic and/or anaerobic conditions and provide evidence that PFL-AE undergoes cluster interconversions in vivo. After 2 h aerobic induction of PFL-AE expression, approximately 44% of the total iron is present in [4Fe-4S]2+ clusters, 6% in [2Fe-2S]2+ clusters, and the remainder as noncluster FeIII (29%) and FeII (21%) species. Subsequent anaerobic incubation of the culture results in approximately 75% of the total iron being present...
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structural basis for glycyl radical formation by Pyruvate Formate Lyase activating enzyme
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Jian Yang, Meng Li, William E Broderick, Joan B Broderick, Catherine L DrennanAbstract:Pyruvate Formate-Lyase activating enzyme generates a stable and catalytically essential glycyl radical on G734 of Pyruvate Formate-Lyase via the direct, stereospecific abstraction of a hydrogen atom from Pyruvate Formate-Lyase. The activase performs this remarkable feat by using an iron-sulfur cluster and S-adenosylmethionine (AdoMet), thus placing it among the AdoMet radical superfamily of enzymes. We report here structures of the substrate-free and substrate-bound forms of Pyruvate Formate-Lyase-activating enzyme, the first structures of an AdoMet radical activase. To obtain the substrate-bound structure, we have used a peptide substrate, the 7-mer RVSGYAV, which contains the sequence surrounding G734. Our structures provide fundamental insights into the interactions between the activase and the G734 loop of Pyruvate Formate-Lyase and provide a structural basis for direct and stereospecific H atom abstraction from the buried G734 of Pyruvate Formate-Lyase.
Danilo Ortillo - One of the best experts on this subject based on the ideXlab platform.
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spectroscopic investigation of Pyruvate Formate Lyase activating enzyme a look into epr endor and mossabuer spectroscopy
Research Journal of Applied Sciences Engineering and Technology, 2014Co-Authors: Danilo Ortillo, Joan B BroderickAbstract:Electron Paramagnetic Resonance (EPR) and Electron Nuclear Double Resonance (ENDOR) spectroscopies are extremely powerful and versatile methods for the characterization of paramagnetic systems in biology, chemistry and physics. For iron centers in the radical SAM enzymes however, Mossbauer spectroscopy has proven to be both powerful and useful as a complementary spectroscopic technique in determining not just the oxidation states but also the type of iron species present in the catalytic center. The cluster content of the radical SAM protein, Pyruvate Formate-Lyase-Activating Enzyme (PFL-AE), was characterized using EPR and Mossbauer techniques while additional ENDOR analysis helped determine the novel interaction of the co-substrate, SAdenosylmethionine (SAM or AdoMet) with the Fe-S cluster of PFL-AE. The anchoring role of the Fe-S cluster to the co-substrate derived from the spectroscopic data supports the mechanism where a SAM-based radical species is involved during catalysis.
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the iron sulfur cluster of Pyruvate Formate Lyase activating enzyme in whole cells cluster interconversion and a valence localized 4fe 4s 2 state
Biochemistry, 2009Co-Authors: Jian Yang, Meng Li, William E Broderick, Boi Hanh Huynh, Sunil G Naik, Danilo Ortillo, Ricardo Garciaserres, Joan B BroderickAbstract:Pyruvate Formate-Lyase activating enzyme (PFL-AE) catalyzes the generation of a catalytically essential glycyl radical on Pyruvate Formate-Lyase (PFL). Purified PFL-AE contains an oxygen-sensitive, labile [4Fe-4S] cluster that undergoes cluster interconversions in vitro, with only the [4Fe-4S]+ cluster state being catalytically active. Such cluster interconversions could play a role in regulating the activity of PFL-AE, and thus of PFL, in response to oxygen levels in vivo. Here we report a Mossbauer investigation on whole cells overexpressing PFL-AE following incubation under aerobic and/or anaerobic conditions and provide evidence that PFL-AE undergoes cluster interconversions in vivo. After 2 h aerobic induction of PFL-AE expression, approximately 44% of the total iron is present in [4Fe-4S]2+ clusters, 6% in [2Fe-2S]2+ clusters, and the remainder as noncluster FeIII (29%) and FeII (21%) species. Subsequent anaerobic incubation of the culture results in approximately 75% of the total iron being present...
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The Iron−Sulfur Cluster of Pyruvate Formate-Lyase Activating Enzyme in Whole Cells: Cluster Interconversion and a Valence-Localized [4Fe-4S]2+ State
Biochemistry, 2009Co-Authors: Jian Yang, Meng Li, William E Broderick, Boi Hanh Huynh, Sunil G Naik, Danilo Ortillo, Ricardo Garcia-serres, Joan B BroderickAbstract:Pyruvate Formate-Lyase activating enzyme (PFL-AE) catalyzes the generation of a catalytically essential glycyl radical on Pyruvate Formate-Lyase (PFL). Purified PFL-AE contains an oxygen-sensitive, labile [4Fe-4S] cluster that undergoes cluster interconversions in vitro, with only the [4Fe-4S]+ cluster state being catalytically active. Such cluster interconversions could play a role in regulating the activity of PFL-AE, and thus of PFL, in response to oxygen levels in vivo. Here we report a Mossbauer investigation on whole cells overexpressing PFL-AE following incubation under aerobic and/or anaerobic conditions and provide evidence that PFL-AE undergoes cluster interconversions in vivo. After 2 h aerobic induction of PFL-AE expression, approximately 44% of the total iron is present in [4Fe-4S]2+ clusters, 6% in [2Fe-2S]2+ clusters, and the remainder as noncluster FeIII (29%) and FeII (21%) species. Subsequent anaerobic incubation of the culture results in approximately 75% of the total iron being present...
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an anchoring role for fes clusters chelation of the amino acid moiety of s adenosylmethionine to the unique iron site of the 4fe 4s cluster of Pyruvate Formate Lyase activating enzyme
Journal of the American Chemical Society, 2002Co-Authors: Charles J Walsby, William E Broderick, Joan B Broderick, Danilo Ortillo, Brian M HoffmanAbstract:Pyruvate Formate-Lyase activating enzyme (PFL-AE) generates the catalytically essential glycyl radical on Pyruvate Formate-Lyase via the interaction of the catalytically active [4Fe−4S]+ cluster with S-adenosylmethionine (AdoMet). Like other members of the Fe−S/AdoMet family of enzymes, PFL-AE is thought to function via generation of an AdoMet-derived 5‘-deoxyadenosyl radical intermediate; however, the mechanistic steps by which this radical is generated remain to be elucidated. While all of the members of the Fe−S/AdoMet family of enzymes appear to have a unique iron site in the [4Fe−4S] cluster, based on the presence of a conserved three-cysteine cluster binding motif, the role of this unique site has been elusive. Here we utilize 35-GHz pulsed electron nuclear double resonance (ENDOR) studies of the [4Fe−4S]+ cluster of PFL-AE in complex with isotopically labeled AdoMet (denoted [1+/AdoMet]) to show that the unique iron serves to anchor the AdoMet for catalysis. AdoMet labeled with 17O at the carboxyla...
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electron nuclear double resonance spectroscopic evidence that s adenosylmethionine binds in contact with the catalytically active 4fe 4s cluster of Pyruvate Formate Lyase activating enzyme
Journal of the American Chemical Society, 2002Co-Authors: Charles J Walsby, William E Broderick, Joan B Broderick, Jennifer Cheek, Danilo Ortillo, Wei Hong, Brian M HoffmanAbstract:Pyruvate Formate-Lyase activating enzyme (PFL-AE) is a representative member of an emerging family of enzymes that utilize iron−sulfur clusters and S-adenosylmethionine (AdoMet) to initiate radical catalysis. Although these enzymes have diverse functions, evidence is emerging that they operate by a common mechanism in which a [4Fe−4S]+ interacts with AdoMet to generate a 5‘-deoxyadenosyl radical intermediate. To date, however, it has been unclear whether the iron−sulfur cluster is a simple electron-transfer center or whether it participates directly in the radical generation chemistry. Here we utilize electron paramagnetic resonance (EPR) and pulsed 35 GHz electron-nuclear double resonance (ENDOR) spectroscopy to address this question. EPR spectroscopy reveals a dramatic effect of AdoMet on the EPR spectrum of the [4Fe−4S]+ of PFL-AE, changing it from rhombic (g = 2.02, 1.94, 1.88) to nearly axial (g = 2.01, 1.88, 1.87). 2H and 13C ENDOR spectroscopy was performed on [4Fe−4S]+-PFL-AE (S = 1/2) in the pres...
