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Michael J. Danson - One of the best experts on this subject based on the ideXlab platform.
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Discovery of a putative acetoin dehydrogenase Complex in the hyperthermophilic archaeon Sulfolobus solfataricus.
FEBS Letters, 2010Co-Authors: Karl A. P. Payne, Michael J. DansonAbstract:Like many other aerobic archaea, the hyperthermophile Sulfolobus solfataricus possesses a gene cluster encoding components of a putative 2-oxoacid dehydrogenase Complex. In the current paper, we have cloned and expressed the first two genes of this cluster and demonstrate that the protein products form an α2β2 hetero-tetramer possessing the catalytic activity characteristic of the first component enzyme of an acetoin dehydrogenase Multienzyme Complex. This represents the first report of an acetoin Multienzyme Complex in archaea, and contrasts with the branched-chain 2-oxoacid dehydrogenase Complex activities characterised in two other archaea, Thermoplasma acidophilum and Haloferax volcanii.
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discovery of the catalytic function of a putative 2 oxoacid dehydrogenase Multienzyme Complex in the thermophilic archaeon thermoplasma acidophilum
FEBS Letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase Multienzyme Complex. However, no catalytic activity of any such Complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an α2β2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase Multienzyme Complex. This represents the first report of the catalytic function of these putative archaeal Multienzyme Complexes.
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discovery of the catalytic function of a putative 2 oxoacid dehydrogenase Multienzyme Complex in the thermophilic archaeon thermoplasma acidophilum
FEBS Letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase Multienzyme Complex. However, no catalytic activity of any such Complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an α2β2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase Multienzyme Complex. This represents the first report of the catalytic function of these putative archaeal Multienzyme Complexes.
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Discovery of the catalytic function of a putative 2-oxoacid dehydrogenase Multienzyme Complex in the thermophilic archaeon Thermoplasma acidophilum.
FEBS letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase Multienzyme Complex. However, no catalytic activity of any such Complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an alpha2beta2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase Multienzyme Complex. This represents the first report of the catalytic function of these putative archaeal Multienzyme Complexes.
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2-Oxoacid dehydrogenase Multienzyme Complexes in the halophilic Archaea? Gene sequences and protein structural predictions
Microbiology, 2000Co-Authors: Keith A. Jolley, Deborah G. Maddocks, Shan L. Gyles, Zoë Mullan, Sen-lin Tang, Michael L. Dyall-smith, Michael J. DansonAbstract:All Archaea catalyse the conversion of pyruvate to acetyl-CoA via a simple pyruvate oxidoreductase. This is in contrast to the Eukarya and most aerobic bacteria, which use the pyruvate dehydrogenase Multienzyme Complex [PDHC], consisting of multiple copies of three component enzymes: E1 (pyruvate decarboxylase), E2 (lipoate acetyl-transferase) and E3 (dihydrolipoamide dehydrogenase, DHLipDH). Until now no PDHC activity has been found in the Archaea, although DHLipDH has been discovered in the extremely halophilic Archaea and its gene sequence has been determined. In this paper, the discovery and sequencing of an operon containing the DHLipDH gene in the halophilic archaeon Haloferax volcanii are reported. Upstream of the DHLipDH gene are 3 ORFs which show highest sequence identities with the E1α, E1β and E2 genes of the PDHC from Gram-positive organisms. Structural predictions of the proposed protein product of the E2 gene show a domain structure characteristic of the E2 component in PDHCs, and catalytically important residues, including the lysine to which the lipoic acid cofactor is covalently bound, are conserved. Northern analyses indicate the transcription of the whole operon, but no PDHC enzymic activity could be detected in cell extracts. The presence in the E2 gene of an insertion (equivalent to approximately 100 aa) not found in bacterial or eukaryal E2 proteins, might be predicted to prevent Multienzyme Complex assembly. This is the first detailed report of the genes for a putative 2-oxoacid dehydrogenase Complex in the Archaea, and the evolutionary and metabolic consequences of these findings are discussed.
Richard N. Perham - One of the best experts on this subject based on the ideXlab platform.
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extended polypeptide linkers establish the spatial architecture of a pyruvate dehydrogenase Multienzyme Complex
Structure, 2008Co-Authors: Jeffrey S Lengyel, Richard N. Perham, Katherine Stott, Bernard R Brooks, Andrea Balbo, Peter Schuck, Sriram Subramaniam, Jacqueline L S MilneAbstract:Summary Icosahedral pyruvate dehydrogenase (PDH) enzyme Complexes are molecular machines consisting of a central E2 core decorated by a shell of peripheral enzymes (E1 and E3) found localized at a distance of ∼75–90 A from the core. Using a combination of biochemical, biophysical, and cryo-electron microscopic techniques, we show here that the gap between the E2 core and the shell of peripheral enzymes is maintained by the flexible but extended conformation adopted by 60 linker polypeptides that radiate outwards from the inner E2 core, irrespective of the E1 or E3 occupancy. The constancy of the gap is thus not due to protein-protein interactions in the outer protein shell. The extended nature of the E2 inner-linker regions thereby creates the restricted annular space in which the lipoyl domains of E2 that carry catalytic intermediates shuttle between E1, E2, and E3 active sites, while their conformational flexibility facilitates productive encounters.
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crystal structure of the e1 component of the escherichia coli 2 oxoglutarate dehydrogenase Multienzyme Complex
Journal of Molecular Biology, 2007Co-Authors: Rene A W Frank, Fred Northrop, Richard N. Perham, Amanda J Price, Ben F. LuisiAbstract:Abstract The thiamine-dependent E1o component (EC 1.2.4.2) of the 2-oxoglutarate dehydrogenase Complex catalyses a rate-limiting step of the tricarboxylic acid cycle (TCA) of aerobically respiring organisms. We describe the crystal structure of Escherichia coli E1o in its apo and holo forms at 2.6 A and 3.5 A resolution, respectively. The structures reveal the characteristic fold that binds thiamine diphosphate and resemble closely the α 2 β 2 hetero-tetrameric E1 components of other 2-oxo acid dehydrogenase Complexes, except that in E1o, the α and β subunits are fused as a single polypeptide. The extended segment that links the α-like and β-like domains forms a pocket occupied by AMP, which is recognised specifically. Also distinctive to E1o are N-terminal extensions to the core fold, and which may mediate interactions with other components of the 2-oxoglutarate dehydrogenase Multienzyme Complex. The active site pocket contains a group of three histidine residues and one serine that appear to confer substrate specificity and the capacity to accommodate the TCA metabolite oxaloacetate. Oxaloacetate inhibits E1o activity at physiological concentrations, and we suggest that the inhibition may allow coordinated activity within the TCA cycle. We discuss the implications for metabolic control in facultative anaerobes, and for energy homeostasis of the mammalian brain.
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thermodynamic analysis of the binding of component enzymes in the assembly of the pyruvate dehydrogenase Multienzyme Complex of bacillus stearothermophilus
Protein Science, 2002Co-Authors: Hyo Il Jung, Simon J Bowden, Alan Cooper, Richard N. PerhamAbstract:The peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2, EC 2.3.1.12) binds tightly but mutually exclusively to dihydrolipoyl dehydrogenase (E3, EC 1.8.1.4) and pyruvate decarboxylase (E1, EC 1.2.4.1) in the pyruvate dehydrogenase Multienzyme Complex of Bacillus stearothermophilus. Isothermal titration calorimetry (ITC) experiments demonstrated that the enthalpies of binding (ΔH°) of both E3 and E1 with the PSBD varied with salt concentration, temperature, pH, and buffer composition. There is little significant difference in the free energies of binding (ΔG° = −12.6 kcal/mol for E3 and = −12.9 kcal/mol for E1 at pH 7.4 and 25°C). However, the association with E3 was characterized by a small, unfavorable enthalpy change (ΔH° = +2.2 kcal/mol) and a large, positive entropy change (TΔS° = +14.8 kcal/mol), whereas that with E1 was accompanied by a favorable enthalpy change (ΔH° = −8.4 kcal/mol) and a less positive entropy change (TΔS° = +4.5 kcal/mol). Values of ΔCp of −316 cal/molK and −470 cal/molK were obtained for the binding of E3 and E1, respectively. The value for E3 was not compatible with the ΔCp calculated from the nonpolar surface area buried in the crystal structure of the E3-PSBD Complex. In this instance, a large negative ΔCp is not indicative of a classical hydrophobic interaction. In differential scanning calorimetry experiments, the midpoint melting temperature (Tm) of E3 increased from 91°C to 97.1°C when it was bound to PSBD, and that of E1 increased from 65.2°C to 70.0°C. These high Tm values eliminate unfolding as a major source of the anomalous ΔCp effects at the temperatures (10–37°C) used for the ITC experiments.
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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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protein protein interactions in the pyruvate dehydrogenase Multienzyme Complex dihydrolipoamide dehydrogenase Complexed with the binding domain of dihydrolipoamide acetyltransferase
Structure, 1996Co-Authors: Sharmila S Mande, Richard N. Perham, Steve Sarfaty, Mark D Allen, Wim G J HolAbstract:Abstract Background: The ubiquitous pyruvate dehydrogenase Multienzyme Complex is built around an octahedral or icosahedral core of dihydrolipoamide acetyltransferase (E2) chains, to which multiple copies of pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) bind tightly but non-covalently. E2 is a flexible multidomain protein that mediates interactions with E1 and E3 through a remarkably small binding domain (E2BD). Results In the Bacillus stearothermophilus Complex, the E2 core is an icosahedral assembly of 60 E2 chains. The crystal structure of the E3 dimer (101 kDa) Complexed with E2BD (4 kDa) has been solved to 2.6 a resolution. Interactions between E3 and E2BD are dominated by an electrostatic zipper formed by Arg135 and Arg139 in the N-terminal helix of E2BD and Asp344 and Glu431 of one of the monomers of E3. E2BD interacts with both E3 monomers, but the binding site is located close to the twofold axis. Thus, in agreement with earlier biochemical results, it is impossible for two molecules of E2BD to bind simultaneously to one E3 dimer. Conclusion Combining this new structure for the E3–E2BD Complex with previously determined structures of the E2 catalytic domain and the E2 lipoyl domain creates a model of the E2 core showing how the lipoyl domain can move between the active sites of E2 and E3 in the Multienzyme Complex.
William Furey - One of the best experts on this subject based on the ideXlab platform.
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novel binding motif and new flexibility revealed by structural analyses of a pyruvate dehydrogenase dihydrolipoyl acetyltransferase subComplex from the escherichia coli pyruvate dehydrogenase Multienzyme Complex
Journal of Biological Chemistry, 2014Co-Authors: Palaniappa Arjunan, Krishnamoorthy Chandrasekhar, Natalia S. Nemeria, Frank Jordan, Junjie Wang, Shelley Reynolds, Ian S Brown, Guillermo Calero, William FureyAbstract:Abstract The Escherichia coli pyruvate dehydrogenase Multienzyme Complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large Complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subComplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subComplex, both of which probably have consequences in the overall Multienzyme Complex assembly. This represents the first structure of an E1p-E2p didomain subComplex involving a homodimeric E1p, and the results may be applicable to a large range of Complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subComplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly.
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efficient coupling of catalysis and dynamics in the e1 component of escherichia coli pyruvate dehydrogenase Multienzyme Complex
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Sachin Kale, William Furey, Gozde Ulas, Jaeyoung Song, Gary W Brudvig, Frank JordanAbstract:Protein motions are ubiquitous and are intrinsically coupled to catalysis. Their specific roles, however, remain largely elusive. Dynamic loops at the active center of the E1 component of Escherichia coli pyruvate dehydrogenase Multienzyme Complex are essential for several catalytic functions starting from a predecarboxylation event and culminating in transfer of the acetyl moiety to the E2 component. Monitoring the kinetics of E1 and its loop variants at various solution viscosities, we show that the rate of a chemical step is modulated by loop dynamics. A cysteine-free E1 construct was site-specifically labeled on the inner loop (residues 401-413), and the EPR nitroxide label revealed ligand-induced conformational dynamics of the loop and a slow "open close" conformational equilibrium in the unliganded state. An (19)F NMR label placed at the same residue revealed motion on the millisecond-second time scale and suggested a quantitative correlation of E1 catalysis and loop dynamics for the 200,000-Da protein. Thermodynamic studies revealed that these motions may promote covalent addition of substrate to the enzyme-bound thiamin diphosphate by reducing the free energy of activation. Furthermore, the global dynamics of E1 presumably regulate and streamline the catalytic steps of the overall Complex by inducing an entirely entropic (nonmechanical) negative cooperativity with respect to substrate binding at higher temperatures. Our results are consistent with, and reinforce the hypothesis of, coupling of catalysis and regulation with enzyme dynamics and suggest the mechanism by which it is achieved in a key branchpoint enzyme in sugar metabolism.
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Active-site changes in the pyruvate dehydrogenase Multienzyme Complex E1 apoenzyme component from Escherichia coli observed at 2.32 A resolution
Acta Crystallographica Section D Biological Crystallography, 2006Co-Authors: Krishnamoorthy Chandrasekhar, Palaniappa Arjunan, Martin Sax, Natalia S. Nemeria, Frank Jordan, William FureyAbstract:The first enzymatic component, E1 (EC 1.2.4.1), of the pyruvate dehydrogenase Multienzyme Complex (PDHc) utilizes thiamine diphosphate (ThDP) and Mg(2+) as cofactors. The structure of a branched-chain-specific E1 apoenzyme from the heterotetrameric alpha(2)beta(2) E1 family was recently reported and showed that disorder-to-order transformations in two active-site loops take place upon cofactor binding. To ascertain what effect the absence of cofactor may have in the homodimeric alpha(2) Escherichia coli PDHc E1, the corresponding apoenzyme has been prepared and its three-dimensional structure determined and analyzed at 2.32 A by crystallographic methods. This represents the first reported apoenzyme structure for any E1 component from the homodimeric alpha(2) family. Electron-density features occurring in the region where the cofactor pyrimidine ring would normally be expected to bind are of size, shape and location compatible with water molecules that form a hydrogen-bonded linkage between residues Glu571 and Val192, which normally make conserved interactions with the ThDP cofactor. A histidine side chain that normally forms hydrogen bonds to ThDP is disordered in its absence and partially occupies two sites. Unlike in the reported heterotetrameric branched-chain apo-E1, no disorder/order loop transformations are evident in apo-PDHc E1 relative to the holo-E1 enzyme (PDHc E1-ThDP-Mg(2+)). Differences in the extent of hydrogen-bonding networks found in the apo-E1 enzyme, the holo-E1 enzyme and in an inhibitor Complex with bound thiamine 2-thiazolone diphosphate (ThTDP), PDHc E1-ThTDP-Mg(2+), are described.
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Structure of the pyruvate dehydrogenase Multienzyme Complex E1 component from Escherichia coli at 1.85 A resolution.
Biochemistry, 2002Co-Authors: Palaniappa Arjunan, Krishnamoorthy Chandrasekhar, Martin Sax, Natalia S. Nemeria, Frank Jordan, Yan Yan, John R Guest, Andrew Brunskill, William FureyAbstract:The crystal structure of the recombinant thiamin diphosphate-dependent E1 component from the Escherichia coli pyruvate dehydrogenase Multienzyme Complex (PDHc) has been determined at a resolution of 1.85 A. The E. coli PDHc E1 component E1p is a homodimeric enzyme and crystallizes with an intact dimer in an asymmetric unit. Each E1p subunit consists of three domains: N-terminal, middle, and C-terminal, with all having α/β folds. The functional dimer contains two catalytic centers located at the interface between subunits. The ThDP cofactors are bound in the “V” conformation in clefts between the two subunits (binding involves the N-terminal and middle domains), and there is a common ThDP binding fold. The cofactors are completely buried, as only the C2 atoms are accessible from solution through the active site clefts. Significant structural differences are observed between individual domains of E1p relative to heterotetrameric Multienzyme Complex E1 components operating on branched chain substrates. Thes...
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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, William Furey, Yan Yan, 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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novel binding motif and new flexibility revealed by structural analyses of a pyruvate dehydrogenase dihydrolipoyl acetyltransferase subComplex from the escherichia coli pyruvate dehydrogenase Multienzyme Complex
Journal of Biological Chemistry, 2014Co-Authors: Palaniappa Arjunan, Krishnamoorthy Chandrasekhar, Natalia S. Nemeria, Frank Jordan, Junjie Wang, Shelley Reynolds, Ian S Brown, Guillermo Calero, William FureyAbstract:Abstract The Escherichia coli pyruvate dehydrogenase Multienzyme Complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large Complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subComplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subComplex, both of which probably have consequences in the overall Multienzyme Complex assembly. This represents the first structure of an E1p-E2p didomain subComplex involving a homodimeric E1p, and the results may be applicable to a large range of Complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subComplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly.
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efficient coupling of catalysis and dynamics in the e1 component of escherichia coli pyruvate dehydrogenase Multienzyme Complex
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Sachin Kale, William Furey, Gozde Ulas, Jaeyoung Song, Gary W Brudvig, Frank JordanAbstract:Protein motions are ubiquitous and are intrinsically coupled to catalysis. Their specific roles, however, remain largely elusive. Dynamic loops at the active center of the E1 component of Escherichia coli pyruvate dehydrogenase Multienzyme Complex are essential for several catalytic functions starting from a predecarboxylation event and culminating in transfer of the acetyl moiety to the E2 component. Monitoring the kinetics of E1 and its loop variants at various solution viscosities, we show that the rate of a chemical step is modulated by loop dynamics. A cysteine-free E1 construct was site-specifically labeled on the inner loop (residues 401-413), and the EPR nitroxide label revealed ligand-induced conformational dynamics of the loop and a slow "open close" conformational equilibrium in the unliganded state. An (19)F NMR label placed at the same residue revealed motion on the millisecond-second time scale and suggested a quantitative correlation of E1 catalysis and loop dynamics for the 200,000-Da protein. Thermodynamic studies revealed that these motions may promote covalent addition of substrate to the enzyme-bound thiamin diphosphate by reducing the free energy of activation. Furthermore, the global dynamics of E1 presumably regulate and streamline the catalytic steps of the overall Complex by inducing an entirely entropic (nonmechanical) negative cooperativity with respect to substrate binding at higher temperatures. Our results are consistent with, and reinforce the hypothesis of, coupling of catalysis and regulation with enzyme dynamics and suggest the mechanism by which it is achieved in a key branchpoint enzyme in sugar metabolism.
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Active-site changes in the pyruvate dehydrogenase Multienzyme Complex E1 apoenzyme component from Escherichia coli observed at 2.32 A resolution
Acta Crystallographica Section D Biological Crystallography, 2006Co-Authors: Krishnamoorthy Chandrasekhar, Palaniappa Arjunan, Martin Sax, Natalia S. Nemeria, Frank Jordan, William FureyAbstract:The first enzymatic component, E1 (EC 1.2.4.1), of the pyruvate dehydrogenase Multienzyme Complex (PDHc) utilizes thiamine diphosphate (ThDP) and Mg(2+) as cofactors. The structure of a branched-chain-specific E1 apoenzyme from the heterotetrameric alpha(2)beta(2) E1 family was recently reported and showed that disorder-to-order transformations in two active-site loops take place upon cofactor binding. To ascertain what effect the absence of cofactor may have in the homodimeric alpha(2) Escherichia coli PDHc E1, the corresponding apoenzyme has been prepared and its three-dimensional structure determined and analyzed at 2.32 A by crystallographic methods. This represents the first reported apoenzyme structure for any E1 component from the homodimeric alpha(2) family. Electron-density features occurring in the region where the cofactor pyrimidine ring would normally be expected to bind are of size, shape and location compatible with water molecules that form a hydrogen-bonded linkage between residues Glu571 and Val192, which normally make conserved interactions with the ThDP cofactor. A histidine side chain that normally forms hydrogen bonds to ThDP is disordered in its absence and partially occupies two sites. Unlike in the reported heterotetrameric branched-chain apo-E1, no disorder/order loop transformations are evident in apo-PDHc E1 relative to the holo-E1 enzyme (PDHc E1-ThDP-Mg(2+)). Differences in the extent of hydrogen-bonding networks found in the apo-E1 enzyme, the holo-E1 enzyme and in an inhibitor Complex with bound thiamine 2-thiazolone diphosphate (ThTDP), PDHc E1-ThTDP-Mg(2+), are described.
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Structure of the pyruvate dehydrogenase Multienzyme Complex E1 component from Escherichia coli at 1.85 A resolution.
Biochemistry, 2002Co-Authors: Palaniappa Arjunan, Krishnamoorthy Chandrasekhar, Martin Sax, Natalia S. Nemeria, Frank Jordan, Yan Yan, John R Guest, Andrew Brunskill, William FureyAbstract:The crystal structure of the recombinant thiamin diphosphate-dependent E1 component from the Escherichia coli pyruvate dehydrogenase Multienzyme Complex (PDHc) has been determined at a resolution of 1.85 A. The E. coli PDHc E1 component E1p is a homodimeric enzyme and crystallizes with an intact dimer in an asymmetric unit. Each E1p subunit consists of three domains: N-terminal, middle, and C-terminal, with all having α/β folds. The functional dimer contains two catalytic centers located at the interface between subunits. The ThDP cofactors are bound in the “V” conformation in clefts between the two subunits (binding involves the N-terminal and middle domains), and there is a common ThDP binding fold. The cofactors are completely buried, as only the C2 atoms are accessible from solution through the active site clefts. Significant structural differences are observed between individual domains of E1p relative to heterotetrameric Multienzyme Complex E1 components operating on branched chain substrates. Thes...
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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, William Furey, Yan Yan, 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.
Caroline Heath - One of the best experts on this subject based on the ideXlab platform.
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discovery of the catalytic function of a putative 2 oxoacid dehydrogenase Multienzyme Complex in the thermophilic archaeon thermoplasma acidophilum
FEBS Letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase Multienzyme Complex. However, no catalytic activity of any such Complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an α2β2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase Multienzyme Complex. This represents the first report of the catalytic function of these putative archaeal Multienzyme Complexes.
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discovery of the catalytic function of a putative 2 oxoacid dehydrogenase Multienzyme Complex in the thermophilic archaeon thermoplasma acidophilum
FEBS Letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase Multienzyme Complex. However, no catalytic activity of any such Complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an α2β2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase Multienzyme Complex. This represents the first report of the catalytic function of these putative archaeal Multienzyme Complexes.
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Discovery of the catalytic function of a putative 2-oxoacid dehydrogenase Multienzyme Complex in the thermophilic archaeon Thermoplasma acidophilum.
FEBS letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase Multienzyme Complex. However, no catalytic activity of any such Complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an alpha2beta2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase Multienzyme Complex. This represents the first report of the catalytic function of these putative archaeal Multienzyme Complexes.