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William Furey - One of the best experts on this subject based on the ideXlab platform.
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structure and function of the catalytic domain of the dihydrolipoyl acetyltransferase component in escherichia coli pyruvate dehydrogenase complex
Journal of Biological Chemistry, 2014Co-Authors: Junjie Wang, Natalia S. Nemeria, Palaniappa Arjunan, Krishnamoorthy Chandrasekhar, Sowmini Kumaran, Shelley Reynolds, Guillermo Calero, Roman Brukh, Lazaros Kakalis, William FureyAbstract:The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s−1, comparable with the rate of NADH production in the PDHc reaction. Hence, neither Reductive Acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4′-aminopyrimidine tautomer of bound thiamin diphosphate (AP).
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insight to the interaction of the dihydrolipoamide acetyltransferase e2 core with the peripheral components in the escherichia coli pyruvate dehydrogenase complex via multifaceted structural approaches
Journal of Biological Chemistry, 2013Co-Authors: Krishnamoorthy Chandrasekhar, Natalia S. Nemeria, Frank Jordan, Palaniappa Arjunan, Jaeyoung Song, Junjie Wang, M Sax, Yunhee Park, Sowmini Kumaran, William FureyAbstract:Multifaceted structural approaches were undertaken to investigate interaction of the E2 component with E3 and E1 components from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), as a representative of the PDHc from Gram-negative bacteria. The crystal structure of E3 at 2.5 A resolution reveals similarity to other E3 structures and was an important starting point for understanding interaction surfaces between E3 and E2. Biochemical studies revealed that R129E-E2 and R150E-E2 substitutions in the peripheral subunit-binding domain (PSBD) of E2 greatly diminished PDHc activity, affected interactions with E3 and E1 components, and affected Reductive Acetylation of E2. Because crystal structures are unavailable for any complete E2-containing complexes, peptide-specific hydrogen/deuterium exchange mass spectrometry was used to identify loci of interactions between 3-lipoyl E2 and E3. Two peptides from the PSBD, including Arg-129, and three peptides from E3 displayed statistically significant reductions in deuterium uptake resulting from interaction between E3 and E2. Of the peptides identified on E3, two were from the catalytic site, and the third was from the interface domain, which for all known E3 structures is believed to interact with the PSBD. NMR clearly demonstrates that there is no change in the lipoyl domain structure on complexation with E3. This is the first instance where the entire wild-type E2 component was employed to understand interactions with E3. A model for PSBD-E3 binding was independently constructed and found to be consistent with the importance of Arg-129, as well as revealing other electrostatic interactions likely stabilizing this complex.
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a dynamic loop at the active center of the escherichia coli pyruvate dehydrogenase complex e1 component modulates substrate utilization and chemical communication with the e2 component
Journal of Biological Chemistry, 2007Co-Authors: Sachin Kale, William Furey, Palaniappa Arjunan, Frank JordanAbstract:Abstract Our crystallographic studies have shown that two active center loops (an inner loop formed by residues 401-413 and outer loop formed by residues 541-557) of the E1 component of the Escherichia coli pyruvate dehydrogenase complex become organized only on binding a substrate analog that is capable of forming a stable thiamin diphosphate-bound covalent intermediate. We showed that residue His-407 on the inner loop has a key role in the mechanism, especially in the Reductive Acetylation of the E. coli dihydrolipoamide transacetylase component, whereas crystallographic results showed a role of this residue in a disorder-order transformation of these two loops, and the ordered conformation gives rise to numerous new contacts between the inner loop and the active center. We present mapping of the conserved residues on the inner loop. Kinetic, spectroscopic, and crystallographic studies on some inner loop variants led us to conclude that charged residues flanking His-407 are important for stabilization/ordering of the inner loop thereby facilitating completion of the active site. The results further suggest that a disorder to order transition of the dynamic inner loop is essential for substrate entry to the active site, for sequestering active site chemistry from undesirable side reactions, as well as for communication between the E1 and E2 components of the E. coli pyruvate dehydrogenase multienzyme complex.
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histidine 407 a phantom residue in the e1 subunit of the escherichia coli pyruvate dehydrogenase complex activates Reductive Acetylation of lipoamide on the e2 subunit an explanation for conservation of active sites between the e1 subunit and transketolase
Biochemistry, 2002Co-Authors: Natalia S. Nemeria, Wen Wei, Frank Jordan, Palaniappa Arjunan, Andrew Brunskill, Farzad Sheibani, Yan Yan, Sheng Zhang, William FureyAbstract:Least squares alignment of the E. coli pyruvate dehydrogenase multienzyme complex E1 subunit and yeast transketolase crystal structures indicates a general structural similarity between the two enzymes and provides a plausible location for a short-loop region in the E1 structure that was unobserved due to disorder. The residue H407, located in this region, is shown to be able to penetrate the active site. Suggested by this comparison, the H407A E1 variant was created, and H407 was shown to participate in the Reductive Acetylation of both an independently expressed lipoyl domain and the intact 1-lipoyl E2 subunit. While the H407A substitution only modestly affected the reaction through pyruvate decarboxylation (ca. 14% activity compared to parental E1), the overall complex has a much impaired activity, at most 0.15% compared to parental E1. Isothermal titration calorimetry measurements show that the binding of the lipoyl domain to the H407A E1 variant is much weaker than that to parental E1. At the same ti...
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inhibition of the escherichia coli pyruvate dehydrogenase complex e1 subunit and its tyrosine 177 variants by thiamin 2 thiazolone and thiamin 2 thiothiazolone diphosphates evidence for reversible tight binding inhibition
Journal of Biological Chemistry, 2001Co-Authors: Natalia S. Nemeria, Palaniappa Arjunan, Yan Yan, William Furey, Zhen Zhang, Angela Brown, John R Guest, Frank JordanAbstract:Abstract Variants of the pyruvate dehydrogenase subunit (E1; EC 1.2.4.1) of the Escherichia coli pyruvate dehydrogenase multienzyme complex with Y177A and Y177F substitutions were created. Both variants displayed pyruvate dehydrogenase multienzyme complex activity at levels of 11% (Y177A E1) and 7% (Y177F E1) of the parental enzyme. TheK m values for thiamin diphosphate (ThDP) were 1.58 μm (parental E1) and 6.65 μm (Y177A E1), whereas the Y177F E1 variant was not saturated at 200 μm. According to fluorescence studies, binding of ThDP was unaffected by the Tyr177 substitutions. The ThDP analogs thiamin 2-thiazolone diphosphate (ThTDP) and thiamin 2-thiothiazolone diphosphate (ThTTDP) behaved as tight-binding inhibitors of parental E1 (K i = 0.003 μm for ThTDP andK i = 0.064 μm for ThTTDP) and the Y177A and Y177F variants. This analysis revealed that ThTDP and ThTTDP bound to parental E1 via a two-step mechanism, but that ThTDP bound to the Y177A variant via a one-step mechanism. Binding of ThTDP was affected and that of ThTTDP was unaffected by substitutions at Tyr177. Addition of ThDP or ThTDP to parental E1 resulted in similar CD spectral changes in the near-UV region. In contrast, binding of ThTTDP to either parental E1 or the Y177A and Y177F variants was accompanied by the appearance of a positive band at 330 nm, indicating that ThTTDP was bound in a chiral environment. In combination with x-ray structural evidence on the location of Tyr177, the kinetic and spectroscopic data suggest that Tyr177 has a role in stabilization of some transition state(s) in the reaction pathway, starting with the free enzyme and culminating with the first irreversible step (decarboxylation), as well as in Reductive Acetylation of the dihydrolipoamide acetyltransferase component.
Frank Jordan - One of the best experts on this subject based on the ideXlab platform.
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Inter-chain Acetyl Transfer in the E2 Component of Bacterial Pyruvate Dehydrogenase Suggests a Model with Different Roles for Each Chain in a Trimer of the Homooligomeric Component
2015Co-Authors: Jaeyoung Song, Frank JordanAbstract:The bacterial pyruvate dehydrogenase complex carries out conversion of pyruvate to acetyl-Coenzyme A with the assistance of thiamin diphosphate (ThDP), several other cofactors and three principal protein components E1, E2 and E3, each present in multiple copies. The E2 component forms the core of the complexes, each copy consisting of variable numbers of lipoyl domains (LD, lipoic acid covalently amidated onto a lysine residue), peripheral subunit binding domain (PSBD), and catalytic (or core) domain (CD). The reaction starts with a ThDP-dependent decarboxylation on E1 to an enamine/C2α carbanion, followed by oxidation and acetyl transfer to form S-acetyldihydrolipoamide E2, then transfer of this acetyl group from LD to coenzyme A on CD. The dihydrolipoamide E2 is finally re-oxidized by the E3 component. This report investigates whether the acetyl group is passed from the LD to the CD in an intra- or inter-chain reaction. Using an E. coli E2 component having a single LD, two types of constructs were prepared: one has a Lys to Ala substitution on LD at the Lys carrying the lipoic acid, making E2 incompetent towards posttranslational ligation of lipoic acid, hence, towards Reductive Acetylation; the other in which the His believed to catalyze the transthiolAcetylation in the CD is substituted to A or C, th
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insight to the interaction of the dihydrolipoamide acetyltransferase e2 core with the peripheral components in the escherichia coli pyruvate dehydrogenase complex via multifaceted structural approaches
Journal of Biological Chemistry, 2013Co-Authors: Krishnamoorthy Chandrasekhar, Natalia S. Nemeria, Frank Jordan, Palaniappa Arjunan, Jaeyoung Song, Junjie Wang, M Sax, Yunhee Park, Sowmini Kumaran, William FureyAbstract:Multifaceted structural approaches were undertaken to investigate interaction of the E2 component with E3 and E1 components from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), as a representative of the PDHc from Gram-negative bacteria. The crystal structure of E3 at 2.5 A resolution reveals similarity to other E3 structures and was an important starting point for understanding interaction surfaces between E3 and E2. Biochemical studies revealed that R129E-E2 and R150E-E2 substitutions in the peripheral subunit-binding domain (PSBD) of E2 greatly diminished PDHc activity, affected interactions with E3 and E1 components, and affected Reductive Acetylation of E2. Because crystal structures are unavailable for any complete E2-containing complexes, peptide-specific hydrogen/deuterium exchange mass spectrometry was used to identify loci of interactions between 3-lipoyl E2 and E3. Two peptides from the PSBD, including Arg-129, and three peptides from E3 displayed statistically significant reductions in deuterium uptake resulting from interaction between E3 and E2. Of the peptides identified on E3, two were from the catalytic site, and the third was from the interface domain, which for all known E3 structures is believed to interact with the PSBD. NMR clearly demonstrates that there is no change in the lipoyl domain structure on complexation with E3. This is the first instance where the entire wild-type E2 component was employed to understand interactions with E3. A model for PSBD-E3 binding was independently constructed and found to be consistent with the importance of Arg-129, as well as revealing other electrostatic interactions likely stabilizing this complex.
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Interchain Acetyl Transfer in the E2 Component of Bacterial Pyruvate Dehydrogenase Suggests a Model with Different Roles for Each Chain in a Trimer of the Homooligomeric Component
2012Co-Authors: Jaeyoung Song, Frank JordanAbstract:The bacterial pyruvate dehydrogenase complex carries out conversion of pyruvate to acetyl-coenzyme A with the assistance of thiamin diphosphate (ThDP), several other cofactors, and three principal protein components, E1–E3, each present in multiple copies. The E2 component forms the core of the complexes, each copy consisting of variable numbers of lipoyl domains (LDs, lipoic acid covalently amidated at a lysine residue), peripheral subunit binding domains (PSBDs), and catalytic (or core) domains (CDs). The reaction starts with a ThDP-dependent decarboxylation on E1 to an enamine/C2α̃ carbanion, followed by oxidation and acetyl transfer to form S-acetyldihydrolipoamide E2, and then transfer of this acetyl group from the LD to coenzyme A on the CD. The dihydrolipoamide E2 is finally reoxidized by the E3 component. This report investigates whether the acetyl group is passed from the LD to the CD in an intra- or interchain reaction. Using an Escherichia coli E2 component having a single LD, two types of constructs were prepared: one with a Lys to Ala substitution in the LD at the Lys carrying the lipoic acid, making E2 incompetent toward post-translational ligation of lipoic acid and, hence, toward Reductive Acetylation, and the other in which the His believed to catalyze the transthiolAcetylation in the CD is substituted with A or C, the absence of His rendering it incompetent toward acetyl-CoA formation. Both kinetic evidence and mass spectrometric evidence support interchain transfer of the acetyl groups, providing a novel model for the presence of multiples of three chains in all E2 components, and their assembly in bacterial enzymes
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a dynamic loop at the active center of the escherichia coli pyruvate dehydrogenase complex e1 component modulates substrate utilization and chemical communication with the e2 component
Journal of Biological Chemistry, 2007Co-Authors: Sachin Kale, William Furey, Palaniappa Arjunan, Frank JordanAbstract:Abstract Our crystallographic studies have shown that two active center loops (an inner loop formed by residues 401-413 and outer loop formed by residues 541-557) of the E1 component of the Escherichia coli pyruvate dehydrogenase complex become organized only on binding a substrate analog that is capable of forming a stable thiamin diphosphate-bound covalent intermediate. We showed that residue His-407 on the inner loop has a key role in the mechanism, especially in the Reductive Acetylation of the E. coli dihydrolipoamide transacetylase component, whereas crystallographic results showed a role of this residue in a disorder-order transformation of these two loops, and the ordered conformation gives rise to numerous new contacts between the inner loop and the active center. We present mapping of the conserved residues on the inner loop. Kinetic, spectroscopic, and crystallographic studies on some inner loop variants led us to conclude that charged residues flanking His-407 are important for stabilization/ordering of the inner loop thereby facilitating completion of the active site. The results further suggest that a disorder to order transition of the dynamic inner loop is essential for substrate entry to the active site, for sequestering active site chemistry from undesirable side reactions, as well as for communication between the E1 and E2 components of the E. coli pyruvate dehydrogenase multienzyme complex.
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Expression and purification of the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase subunits of the Escherichia coli pyruvate dehydrogenase multienzyme complex: a mass spectrometric assay for Reductive Acetylation of dihydrolipoa
Protein Expression and Purification, 2003Co-Authors: Wen Wei, Natalia S. Nemeria, Frank JordanAbstract:Plasmids were constructed for overexpression of the Escherichia coli dihydrolipoamide acetyltransferase (1-lip E2, with a single hybrid lipoyl domain per subunit) and dihydrolipoamide dehydrogenase (E3). A purification protocol is presented that yields homogeneous recombinant 1-lip E2 and E3 proteins. The hybrid lipoyl domain was also expressed independently. Masses of 45,953+/-73Da (1-lip E2), 50,528+/-5.5Da (apo-E3), 51,266+/-48Da (E3 including FAD), and 8982+/-4.0 (lipoyl domain) were determined by MALDI-TOF mass spectrometry. The purified 1-lip E2 and E3 proteins were functionally active according to the overall PDHc activity measurement. The lipoyl domain was fully acetylated after just 30 s of incubation with E1 and pyruvate. The mass of the acetylated lipoyl domain is 9019+/-2Da according to MALDI-TOF mass spectrometry. Treatment of the 1-lip E2 subunit with trypsin resulted in the appearance of the lipoyl domain with a mass of 10,112+/-3Da. When preincubated with E1 and pyruvate, this tryptic fragment was acetylated according to the mass increase. MALDI-TOF mass spectrometry was thus demonstrated to be a fast and precise method for studying the Reductive Acetylation of the recombinant 1-lip E2 subunit by E1 and pyruvate.
Richard N. Perham - One of the best experts on this subject based on the ideXlab platform.
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reaction mechanism of the heterotetrameric α2β2 e1 component of 2 oxo acid dehydrogenase multienzyme complexes
Biochemistry, 2003Co-Authors: Markus Fries, Hyo Il Jung, Richard N. PerhamAbstract:Pyruvate decarboxylase (E1) catalyzes the first two reactions of the four involved in oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase (PDH) multienzyme complex. It requires thiamin diphosphate to bring about the decarboxylation of pyruvate, which is followed by the Reductive Acetylation of a lipoyl group covalently bound to the N(6) amino group of a lysine residue in the second catalytic component, a dihydrolipoyl acetyltransferase (E2). Replacement of two histidine residues in the E1alpha and E1beta chains of the heterotetrameric E1 (alpha(2)beta(2)) component of the PDH complex of Bacillus stearothermophilus, considered possible proton donors at the active site, was carried out. Subsequent characterization of the mutants permitted different roles to be assigned to these two particular residues in the reaction catalyzed by E1: E1alpha His271 to stabilize the dianion formed during decarboxylation of the 2-oxo acid and E1beta His128 to provide the proton required to protonate the incoming dithiolane ring in the subsequent Reductive Acetylation of the lipoyl goup. On the basis of these and other results from a separate investigation into the roles of individual residues in a loop region in the E1alpha chain close to the active site of E1 [Fries, M., Chauhan, H. J., Domingo, G. J., Jung, H., and Perham, R. N. (2002) Eur. J. Biochem. 270, 861-870] together with work from other laboratories, a detailed mechanism for the E1 reaction can be formulated.
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Recognition of the Lipoyl Domain is the Ultimate Determinant of Substrate Channelling in the Pyruvate Dehydrogenase Multienzyme Complex
2001Co-Authors: Jones D. Dafydd, Katherine M Stott, Pedro A Reche, Richard N. PerhamAbstract:Reductive Acetylation of the lipoyl domain (E2plip) of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli is catalysed speci®cally by its partner pyruvate decarboxylase (E1p), and no productive interaction occurs with the analogous 2-oxoglutarate decarboxylase (E1o) of the 2-oxoglutarate dehydrogenase complex. Residues in the lipoyl-lysine b-turn region of the unlipoylated E2plip domain (E2plip apo ) undergo signi®cant changes in both chemical shift and transverse relaxation time (T 2 ) in the presence of E1p but not E1o. Residue Gly11, in a prominent surface loop between b-strands 1 and 2 in the E2plip domain, was also observed to undergo a signi®cant change in chemical shift. Addition of pyruvate to the mixture of E2plip apo and E1p caused larger changes in chemical shift and the appearance of multiple cross-peaks for certain residues, suggesting that the domain was experiencing more than one type of interaction. Residues in both b-strands 4 and 5, together with those in the prominent surface loop and the following b-strand 2, appeared to be interacting with E1p, as did a small patch of residues centred around Glu31. The values of T 2 across the polypeptide chain backbone were also lower than in the presence of E1p alone, suggesting that E2plip apo binds more tightly after the addition of pyruvate. The lipoylated domain (E2plip holo ) also exhibited signi®cant changes in chemical shift and decreases in the overall T 2 relaxation times in the presence of E1p, the residues principally affected being restricted to the half of the domain that contains the lipoyl-lysine (Lys41) residue. In addition, small chemical shift changes and a general drop in T 2 times in the presence of E1o were observed, indicating that E2plip holo can interact, weakly but non-productively, with E1o. It is evident that recognition of the protein domain is the ultimate determinant of whether Reductive Acetylation of the lipoyl group occurs, and that this is ensured by a mosaic of interactions with the Elp
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Expression in Escherichia coli of a sub-gene encoding the lipoyl and peripheral subunit-binding domains of the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus.
Biochemical Journal, 1992Co-Authors: D S Hipps, Richard N. PerhamAbstract:A sub-gene encoding the N-terminal 170 residues of the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was over-expressed in Escherichia coli. The expressed polypeptide consists of the lipoyl domain, inter-domain linker and peripheral subunit-binding domain; these were found to have folded into their native functional conformations as judged by Reductive Acetylation of the lipoyl domain, limited proteolysis of the linker region and ability to bind the dihydrolipoamide dehydrogenase dimer. The di-domain was largely (80%) unlipoylated; a small proportion (4%) was correctly modified with lipoic acid and the remainder (16%) was aberrantly modified with octanoic acid. A polyclonal antiserum was raised that recognized both the di-domain and the individual component domains. The 400 MHz 1H-n.m.r. spectrum of the di-domain showed resonances corresponding to those seen in spectra of the lipoyl domain, plus others characteristic of amino acid residues in the flexible linker region. Further, as yet unidentified, resonances are likely to be derived from the peripheral subunit-binding domain. The existence and independent folding of the peripheral subunit-binding domain is thus confirmed and its purification in large-scale amounts for detailed structural analysis is now possible.
Palaniappa Arjunan - One of the best experts on this subject based on the ideXlab platform.
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structure and function of the catalytic domain of the dihydrolipoyl acetyltransferase component in escherichia coli pyruvate dehydrogenase complex
Journal of Biological Chemistry, 2014Co-Authors: Junjie Wang, Natalia S. Nemeria, Palaniappa Arjunan, Krishnamoorthy Chandrasekhar, Sowmini Kumaran, Shelley Reynolds, Guillermo Calero, Roman Brukh, Lazaros Kakalis, William FureyAbstract:The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s−1, comparable with the rate of NADH production in the PDHc reaction. Hence, neither Reductive Acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4′-aminopyrimidine tautomer of bound thiamin diphosphate (AP).
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insight to the interaction of the dihydrolipoamide acetyltransferase e2 core with the peripheral components in the escherichia coli pyruvate dehydrogenase complex via multifaceted structural approaches
Journal of Biological Chemistry, 2013Co-Authors: Krishnamoorthy Chandrasekhar, Natalia S. Nemeria, Frank Jordan, Palaniappa Arjunan, Jaeyoung Song, Junjie Wang, M Sax, Yunhee Park, Sowmini Kumaran, William FureyAbstract:Multifaceted structural approaches were undertaken to investigate interaction of the E2 component with E3 and E1 components from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), as a representative of the PDHc from Gram-negative bacteria. The crystal structure of E3 at 2.5 A resolution reveals similarity to other E3 structures and was an important starting point for understanding interaction surfaces between E3 and E2. Biochemical studies revealed that R129E-E2 and R150E-E2 substitutions in the peripheral subunit-binding domain (PSBD) of E2 greatly diminished PDHc activity, affected interactions with E3 and E1 components, and affected Reductive Acetylation of E2. Because crystal structures are unavailable for any complete E2-containing complexes, peptide-specific hydrogen/deuterium exchange mass spectrometry was used to identify loci of interactions between 3-lipoyl E2 and E3. Two peptides from the PSBD, including Arg-129, and three peptides from E3 displayed statistically significant reductions in deuterium uptake resulting from interaction between E3 and E2. Of the peptides identified on E3, two were from the catalytic site, and the third was from the interface domain, which for all known E3 structures is believed to interact with the PSBD. NMR clearly demonstrates that there is no change in the lipoyl domain structure on complexation with E3. This is the first instance where the entire wild-type E2 component was employed to understand interactions with E3. A model for PSBD-E3 binding was independently constructed and found to be consistent with the importance of Arg-129, as well as revealing other electrostatic interactions likely stabilizing this complex.
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a dynamic loop at the active center of the escherichia coli pyruvate dehydrogenase complex e1 component modulates substrate utilization and chemical communication with the e2 component
Journal of Biological Chemistry, 2007Co-Authors: Sachin Kale, William Furey, Palaniappa Arjunan, Frank JordanAbstract:Abstract Our crystallographic studies have shown that two active center loops (an inner loop formed by residues 401-413 and outer loop formed by residues 541-557) of the E1 component of the Escherichia coli pyruvate dehydrogenase complex become organized only on binding a substrate analog that is capable of forming a stable thiamin diphosphate-bound covalent intermediate. We showed that residue His-407 on the inner loop has a key role in the mechanism, especially in the Reductive Acetylation of the E. coli dihydrolipoamide transacetylase component, whereas crystallographic results showed a role of this residue in a disorder-order transformation of these two loops, and the ordered conformation gives rise to numerous new contacts between the inner loop and the active center. We present mapping of the conserved residues on the inner loop. Kinetic, spectroscopic, and crystallographic studies on some inner loop variants led us to conclude that charged residues flanking His-407 are important for stabilization/ordering of the inner loop thereby facilitating completion of the active site. The results further suggest that a disorder to order transition of the dynamic inner loop is essential for substrate entry to the active site, for sequestering active site chemistry from undesirable side reactions, as well as for communication between the E1 and E2 components of the E. coli pyruvate dehydrogenase multienzyme complex.
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histidine 407 a phantom residue in the e1 subunit of the escherichia coli pyruvate dehydrogenase complex activates Reductive Acetylation of lipoamide on the e2 subunit an explanation for conservation of active sites between the e1 subunit and transketolase
Biochemistry, 2002Co-Authors: Natalia S. Nemeria, Wen Wei, Frank Jordan, Palaniappa Arjunan, Andrew Brunskill, Farzad Sheibani, Yan Yan, Sheng Zhang, William FureyAbstract:Least squares alignment of the E. coli pyruvate dehydrogenase multienzyme complex E1 subunit and yeast transketolase crystal structures indicates a general structural similarity between the two enzymes and provides a plausible location for a short-loop region in the E1 structure that was unobserved due to disorder. The residue H407, located in this region, is shown to be able to penetrate the active site. Suggested by this comparison, the H407A E1 variant was created, and H407 was shown to participate in the Reductive Acetylation of both an independently expressed lipoyl domain and the intact 1-lipoyl E2 subunit. While the H407A substitution only modestly affected the reaction through pyruvate decarboxylation (ca. 14% activity compared to parental E1), the overall complex has a much impaired activity, at most 0.15% compared to parental E1. Isothermal titration calorimetry measurements show that the binding of the lipoyl domain to the H407A E1 variant is much weaker than that to parental E1. At the same ti...
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inhibition of the escherichia coli pyruvate dehydrogenase complex e1 subunit and its tyrosine 177 variants by thiamin 2 thiazolone and thiamin 2 thiothiazolone diphosphates evidence for reversible tight binding inhibition
Journal of Biological Chemistry, 2001Co-Authors: Natalia S. Nemeria, Palaniappa Arjunan, Yan Yan, William Furey, Zhen Zhang, Angela Brown, John R Guest, Frank JordanAbstract:Abstract Variants of the pyruvate dehydrogenase subunit (E1; EC 1.2.4.1) of the Escherichia coli pyruvate dehydrogenase multienzyme complex with Y177A and Y177F substitutions were created. Both variants displayed pyruvate dehydrogenase multienzyme complex activity at levels of 11% (Y177A E1) and 7% (Y177F E1) of the parental enzyme. TheK m values for thiamin diphosphate (ThDP) were 1.58 μm (parental E1) and 6.65 μm (Y177A E1), whereas the Y177F E1 variant was not saturated at 200 μm. According to fluorescence studies, binding of ThDP was unaffected by the Tyr177 substitutions. The ThDP analogs thiamin 2-thiazolone diphosphate (ThTDP) and thiamin 2-thiothiazolone diphosphate (ThTTDP) behaved as tight-binding inhibitors of parental E1 (K i = 0.003 μm for ThTDP andK i = 0.064 μm for ThTTDP) and the Y177A and Y177F variants. This analysis revealed that ThTDP and ThTTDP bound to parental E1 via a two-step mechanism, but that ThTDP bound to the Y177A variant via a one-step mechanism. Binding of ThTDP was affected and that of ThTTDP was unaffected by substitutions at Tyr177. Addition of ThDP or ThTDP to parental E1 resulted in similar CD spectral changes in the near-UV region. In contrast, binding of ThTTDP to either parental E1 or the Y177A and Y177F variants was accompanied by the appearance of a positive band at 330 nm, indicating that ThTTDP was bound in a chiral environment. In combination with x-ray structural evidence on the location of Tyr177, the kinetic and spectroscopic data suggest that Tyr177 has a role in stabilization of some transition state(s) in the reaction pathway, starting with the free enzyme and culminating with the first irreversible step (decarboxylation), as well as in Reductive Acetylation of the dihydrolipoamide acetyltransferase component.
Natalia S. Nemeria - One of the best experts on this subject based on the ideXlab platform.
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structure and function of the catalytic domain of the dihydrolipoyl acetyltransferase component in escherichia coli pyruvate dehydrogenase complex
Journal of Biological Chemistry, 2014Co-Authors: Junjie Wang, Natalia S. Nemeria, Palaniappa Arjunan, Krishnamoorthy Chandrasekhar, Sowmini Kumaran, Shelley Reynolds, Guillermo Calero, Roman Brukh, Lazaros Kakalis, William FureyAbstract:The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s−1, comparable with the rate of NADH production in the PDHc reaction. Hence, neither Reductive Acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4′-aminopyrimidine tautomer of bound thiamin diphosphate (AP).
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insight to the interaction of the dihydrolipoamide acetyltransferase e2 core with the peripheral components in the escherichia coli pyruvate dehydrogenase complex via multifaceted structural approaches
Journal of Biological Chemistry, 2013Co-Authors: Krishnamoorthy Chandrasekhar, Natalia S. Nemeria, Frank Jordan, Palaniappa Arjunan, Jaeyoung Song, Junjie Wang, M Sax, Yunhee Park, Sowmini Kumaran, William FureyAbstract:Multifaceted structural approaches were undertaken to investigate interaction of the E2 component with E3 and E1 components from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), as a representative of the PDHc from Gram-negative bacteria. The crystal structure of E3 at 2.5 A resolution reveals similarity to other E3 structures and was an important starting point for understanding interaction surfaces between E3 and E2. Biochemical studies revealed that R129E-E2 and R150E-E2 substitutions in the peripheral subunit-binding domain (PSBD) of E2 greatly diminished PDHc activity, affected interactions with E3 and E1 components, and affected Reductive Acetylation of E2. Because crystal structures are unavailable for any complete E2-containing complexes, peptide-specific hydrogen/deuterium exchange mass spectrometry was used to identify loci of interactions between 3-lipoyl E2 and E3. Two peptides from the PSBD, including Arg-129, and three peptides from E3 displayed statistically significant reductions in deuterium uptake resulting from interaction between E3 and E2. Of the peptides identified on E3, two were from the catalytic site, and the third was from the interface domain, which for all known E3 structures is believed to interact with the PSBD. NMR clearly demonstrates that there is no change in the lipoyl domain structure on complexation with E3. This is the first instance where the entire wild-type E2 component was employed to understand interactions with E3. A model for PSBD-E3 binding was independently constructed and found to be consistent with the importance of Arg-129, as well as revealing other electrostatic interactions likely stabilizing this complex.
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Expression and purification of the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase subunits of the Escherichia coli pyruvate dehydrogenase multienzyme complex: a mass spectrometric assay for Reductive Acetylation of dihydrolipoa
Protein Expression and Purification, 2003Co-Authors: Wen Wei, Natalia S. Nemeria, Frank JordanAbstract:Plasmids were constructed for overexpression of the Escherichia coli dihydrolipoamide acetyltransferase (1-lip E2, with a single hybrid lipoyl domain per subunit) and dihydrolipoamide dehydrogenase (E3). A purification protocol is presented that yields homogeneous recombinant 1-lip E2 and E3 proteins. The hybrid lipoyl domain was also expressed independently. Masses of 45,953+/-73Da (1-lip E2), 50,528+/-5.5Da (apo-E3), 51,266+/-48Da (E3 including FAD), and 8982+/-4.0 (lipoyl domain) were determined by MALDI-TOF mass spectrometry. The purified 1-lip E2 and E3 proteins were functionally active according to the overall PDHc activity measurement. The lipoyl domain was fully acetylated after just 30 s of incubation with E1 and pyruvate. The mass of the acetylated lipoyl domain is 9019+/-2Da according to MALDI-TOF mass spectrometry. Treatment of the 1-lip E2 subunit with trypsin resulted in the appearance of the lipoyl domain with a mass of 10,112+/-3Da. When preincubated with E1 and pyruvate, this tryptic fragment was acetylated according to the mass increase. MALDI-TOF mass spectrometry was thus demonstrated to be a fast and precise method for studying the Reductive Acetylation of the recombinant 1-lip E2 subunit by E1 and pyruvate.
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histidine 407 a phantom residue in the e1 subunit of the escherichia coli pyruvate dehydrogenase complex activates Reductive Acetylation of lipoamide on the e2 subunit an explanation for conservation of active sites between the e1 subunit and transketolase
Biochemistry, 2002Co-Authors: Natalia S. Nemeria, Wen Wei, Frank Jordan, Palaniappa Arjunan, Andrew Brunskill, Farzad Sheibani, Yan Yan, Sheng Zhang, William FureyAbstract:Least squares alignment of the E. coli pyruvate dehydrogenase multienzyme complex E1 subunit and yeast transketolase crystal structures indicates a general structural similarity between the two enzymes and provides a plausible location for a short-loop region in the E1 structure that was unobserved due to disorder. The residue H407, located in this region, is shown to be able to penetrate the active site. Suggested by this comparison, the H407A E1 variant was created, and H407 was shown to participate in the Reductive Acetylation of both an independently expressed lipoyl domain and the intact 1-lipoyl E2 subunit. While the H407A substitution only modestly affected the reaction through pyruvate decarboxylation (ca. 14% activity compared to parental E1), the overall complex has a much impaired activity, at most 0.15% compared to parental E1. Isothermal titration calorimetry measurements show that the binding of the lipoyl domain to the H407A E1 variant is much weaker than that to parental E1. At the same ti...
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inhibition of the escherichia coli pyruvate dehydrogenase complex e1 subunit and its tyrosine 177 variants by thiamin 2 thiazolone and thiamin 2 thiothiazolone diphosphates evidence for reversible tight binding inhibition
Journal of Biological Chemistry, 2001Co-Authors: Natalia S. Nemeria, Palaniappa Arjunan, Yan Yan, William Furey, Zhen Zhang, Angela Brown, John R Guest, Frank JordanAbstract:Abstract Variants of the pyruvate dehydrogenase subunit (E1; EC 1.2.4.1) of the Escherichia coli pyruvate dehydrogenase multienzyme complex with Y177A and Y177F substitutions were created. Both variants displayed pyruvate dehydrogenase multienzyme complex activity at levels of 11% (Y177A E1) and 7% (Y177F E1) of the parental enzyme. TheK m values for thiamin diphosphate (ThDP) were 1.58 μm (parental E1) and 6.65 μm (Y177A E1), whereas the Y177F E1 variant was not saturated at 200 μm. According to fluorescence studies, binding of ThDP was unaffected by the Tyr177 substitutions. The ThDP analogs thiamin 2-thiazolone diphosphate (ThTDP) and thiamin 2-thiothiazolone diphosphate (ThTTDP) behaved as tight-binding inhibitors of parental E1 (K i = 0.003 μm for ThTDP andK i = 0.064 μm for ThTTDP) and the Y177A and Y177F variants. This analysis revealed that ThTDP and ThTTDP bound to parental E1 via a two-step mechanism, but that ThTDP bound to the Y177A variant via a one-step mechanism. Binding of ThTDP was affected and that of ThTTDP was unaffected by substitutions at Tyr177. Addition of ThDP or ThTDP to parental E1 resulted in similar CD spectral changes in the near-UV region. In contrast, binding of ThTTDP to either parental E1 or the Y177A and Y177F variants was accompanied by the appearance of a positive band at 330 nm, indicating that ThTTDP was bound in a chiral environment. In combination with x-ray structural evidence on the location of Tyr177, the kinetic and spectroscopic data suggest that Tyr177 has a role in stabilization of some transition state(s) in the reaction pathway, starting with the free enzyme and culminating with the first irreversible step (decarboxylation), as well as in Reductive Acetylation of the dihydrolipoamide acetyltransferase component.
