Ligand-induced Conformational Changes and a Reaction Intermediate in Branched-chain 2-Oxo Acid Dehydrogenase (E1) from Thermus thermophilus HB8, as Revealed by X-ray Crystallography

Nakai, Tadashi; Nakagawa, Noriko; Maoka, N.; Masui, Ryoji; Kuramitsu, Seiki; Kamiya, Nobuo

doi:10.1016/j.jmb.2004.02.011

Cited by 45 publications

(75 citation statements)

References 49 publications

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“…It is noteworthy that the order-disorder transformation in this ␣2 E1 dimer differs considerably from those that have been observed in certain ␣2␤2 E1 heterotetramers (14,32). In the latter cases, the addition of the cofactor alone to the disordered apoenzyme produced an ordered holoenzyme.…”

Section: Discussionmentioning

confidence: 58%

“…In the ␣2 homodimeric E1 protein, disorder occurs in both the apoenzymes and holoenzymes, and it is only the formation of the pre-decarboxylation reaction intermediate that triggers ordering. In addition, the loops in ␣2 E1 that exhibit transformation are not the same as those reported to undergo transformation in the ␣2␤2 cases (14,32,33).…”

Section: Discussionmentioning

confidence: 72%

“…However, unlike the C2␣-CO 2 bond normally cleaved in the reaction with pyruvate, the C2␣-PO 3 Me bond remains intact. The reaction is therefore trapped in a pre-CO 2 release-like state, and the structure represents a covalently bound, pre-decarboxylation reaction intermediate analogue.There have been three covalently bound reaction intermediate structures reported for ThDP-dependent enzymes (11,12,14), but they all represented the planar enamine intermediate (Fig. 1, top right object) that exists only after decarboxylation.…”

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confidence: 99%

“…There have been three covalently bound reaction intermediate structures reported for ThDP-dependent enzymes (11,12,14), but they all represented the planar enamine intermediate (Fig. 1, top right object) that exists only after decarboxylation.…”

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confidence: 99%

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A Thiamin-bound, Pre-decarboxylation Reaction Intermediate Analogue in the Pyruvate Dehydrogenase E1 Subunit Induces Large Scale Disorder-to-Order Transformations in the Enzyme and Reveals Novel Structural Features in the Covalently Bound Adduct

Arjunan

Sax

Brunskill

et al. 2006

Journal of Biological Chemistry

169

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Thiamin-dependent enzymes play key roles in sugar metabolism, typically catalyzing the decarboxylation of ␣-keto acids and the transfer of an aldehyde or an acyl group (1-5). Examples include the E1 components in pyruvate dehydrogenase complexes (PDHc), 2 pyruvate decarboxylase, transketolase, etc. Crystallographic studies (6 -12) have elucidated many of the structural, stereochemical, and biochemical details in the mechanism of action of these enzymes and in the catalytic role of the cofactor ThDP (thiamin diphosphate, vitamin B1 diphosphate, Fig. 1, top left). Despite the enormous contributions made by these and other studies to our understanding of how such enzymes function, important details still remain obscure. There are, for example, no detailed structural data on the first ThDP-bound intermediate in the presence of any enzyme (for example, ␣-lactylthiamin diphosphate (␣-LThDP) in PDHc E1 and pyruvate decarboxylase), which is postulated to form in the currently accepted mechanism of thiamin catalysis (Fig. 1, top, third object from the right). In an effort to obtain structural information pertaining to this key intermediate, we have determined the crystal structure of PDHc E1 from Escherichia coli in complex with ␣-phosphonolactylthiamin diphosphate (PLThDP).PLThDP is the product of the reaction between ThDP and methylacetylphosphonate, with the latter being an analogue of the true substrate pyruvate and a potent inhibitor of PDHc. The complex formed with PLThDP instead of ThDP therefore mimics the structure of the enzyme-bound, reactive tetrahedral intermediate ␣-LThDP (13) in the decarboxylation step of the PDHc E1 reaction. It differs from the complex formed with the true substrate only in the replacement of the carboxylate group by a methyl phosphonate (PO 3 Me) group. However, unlike the C2␣-CO 2 bond normally cleaved in the reaction with pyruvate, the C2␣-PO 3 Me bond remains intact. The reaction is therefore trapped in a pre-CO 2 release-like state, and the structure represents a covalently bound, pre-decarboxylation reaction intermediate analogue.There have been three covalently bound reaction intermediate structures reported for ThDP-dependent enzymes (11,12,14), but they all represented the planar enamine intermediate (Fig. 1, top right object) that exists only after decarboxylation. The E1-PLThDP structure is thus the first structural example of a covalently bound, pre-decarboxylation reaction intermediate analogue in any ThDP-dependent enzyme.* This work was supported by a grant from the Veterans Affairs Merit Review Program and National Institutes of Health Grant GM-61791 (to W. F.) and by National Institutes of Health Grant GM-62330 (to F. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (codes 2G25 and 2G28)

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Section: Discussionmentioning

confidence: 58%

Section: Discussionmentioning

confidence: 72%

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confidence: 99%

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confidence: 99%

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A Thiamin-bound, Pre-decarboxylation Reaction Intermediate Analogue in the Pyruvate Dehydrogenase E1 Subunit Induces Large Scale Disorder-to-Order Transformations in the Enzyme and Reveals Novel Structural Features in the Covalently Bound Adduct

Arjunan

Sax

Brunskill

et al. 2006

Journal of Biological Chemistry

169

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“…More specifically, the existence of a network of hydrogen bonding interactions has been identified as a "proton wire" that mediates the shuttling of a proton between active sites, giving rise to "half-site" reactivity (75). Taken together with findings on other ThDP-dependent enzymes (77,78), such as differential kinetics of co-factor binding to the two sites (79) and co-crystal structures in which substrate analogs are observed to bind in only one of the possible active sites (80,81), this work on pyruvate dehydrogenase raises questions concerning the generality of this molecular mechanism in mediating active site communication. There is no evidence, however, for the existence of such a proton wire in the crystal structure of the OXC homodimer.…”

Section: Resultsmentioning

confidence: 90%

Structural Basis for Activation of the Thiamin Diphosphate-dependent Enzyme Oxalyl-CoA Decarboxylase by Adenosine Diphosphate

Berthold

Moussatche

Richards

et al. 2005

Journal of Biological Chemistry

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Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate-dependent enzyme that plays an important role in the catabolism of the highly toxic compound oxalate. We have determined the crystal structure of the enzyme from Oxalobacter formigenes from a hemihedrally twinned crystal to 1.73 Å resolution and characterized the steady-state kinetic behavior of the decarboxylase. The monomer of the tetrameric enzyme consists of three ␣/␤-type domains, commonly seen in this class of enzymes, and the thiamin diphosphatebinding site is located at the expected subunit-subunit interface between two of the domains with the cofactor bound in the conserved V-conformation. Although oxalyl-CoA decarboxylase is structurally homologous to acetohydroxyacid synthase, a molecule of ADP is bound in a region that is cognate to the FAD-binding site observed in acetohydroxyacid synthase and presumably fulfils a similar role in stabilizing the protein structure. This difference between the two enzymes may have physiological importance since oxalyl-CoA decarboxylation is an essential step in ATP generation in O. formigenes, and the decarboxylase activity is stimulated by exogenous ADP. Despite the significant degree of structural conservation between the two homologous enzymes and the similarity in catalytic mechanism to other thiamin diphosphate-dependent enzymes, the active site residues of oxalyl-CoA decarboxylase are unique. A suggestion for the reaction mechanism of the enzyme is presented.Oxalic acid is one of nature's most highly oxidized organic compounds, and its dianion is a strong chelator of metal cations, especially Ca 2ϩ , causing oxalate to be highly toxic to many organisms (1). In humans, elevated levels of oxalate are associated with several diseases, including the formation of calcium oxalate stones in the kidney (urolithiasis), renal failure, cardiomyopathy, and cardiac conductance disorders (1-3). Relatively large amounts of oxalate are introduced into the body through the diet, although this diacid may also arise as a byproduct of normal cellular metabolism (4). Because humans, in common with other mammals, are not able to degrade oxalate, this compound must be eliminated by excretion in the urine or via the intestine (5). The recent observation that a symbiotic, gut-dwelling bacterium, Oxalobacter formigenes, may regulate oxalate homeostasis in humans, therefore, has important implications for efforts to develop new strategies for treating oxalate-related diseases (6). O. formigenes is an obligate anaerobe bacterium found in the gastrointestinal tracts of vertebrates, including humans, and is unusual in that it employs oxalate as the sole energy source for its survival (7,8). As a result, this bacterium not only degrades free oxalate entering the intestine lumen but also creates a transepithelial gradient favoring oxalate secretion and preventing absorption of oxalic acid in the lower tract of the intestine (9). A direct correlation between the number of recurrent kidney stone episodes and a lack of O. formigenes in the ...

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Single‐Turnover Kinetics Reveal a Distinct Mode of Thiamine Diphosphate‐Dependent Catalysis in Vitamin K Biosynthesis

et al. 2018

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MenD, or (1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase, uses a thiamine diphosphate (ThDP)-dependent tetrahedral Breslow intermediate rather than a canonical enamine for catalysis in the biosynthesis of vitamin K. By real-time monitoring of the cofactor chemical state with circular dichroism spectroscopy, we found that a new post-decarboxylation intermediate was formed from a multistep process that was rate limited by binding of the α-ketoglutarate substrate before it quickly relaxed to the characterized tetrahedral Breslow intermediate. In addition, the chemical steps leading to the reactive post-decarboxylation intermediates were not affected by the electrophilic substrate, isochorismate, whereas release of the product was found to limit the whole catalytic process. Moreover, these intermediates are likely kinetically stabilized owing to the low biological availability of isochorismate under physiological conditions, in contrast to the tight coupling of enamine formation with binding of the electrophilic acceptor in some other ThDP-dependent enzymes. Together with the unusual tetrahedral structure of the intermediates, these findings strongly support a new ThDP-dependent catalytic mode distinct from canonical enamine chemistry.

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Ligand-induced Conformational Changes and a Reaction Intermediate in Branched-chain 2-Oxo Acid Dehydrogenase (E1) from Thermus thermophilus HB8, as Revealed by X-ray Crystallography

Cited by 45 publications

References 49 publications

A Thiamin-bound, Pre-decarboxylation Reaction Intermediate Analogue in the Pyruvate Dehydrogenase E1 Subunit Induces Large Scale Disorder-to-Order Transformations in the Enzyme and Reveals Novel Structural Features in the Covalently Bound Adduct

A Thiamin-bound, Pre-decarboxylation Reaction Intermediate Analogue in the Pyruvate Dehydrogenase E1 Subunit Induces Large Scale Disorder-to-Order Transformations in the Enzyme and Reveals Novel Structural Features in the Covalently Bound Adduct

Structural Basis for Activation of the Thiamin Diphosphate-dependent Enzyme Oxalyl-CoA Decarboxylase by Adenosine Diphosphate

Single‐Turnover Kinetics Reveal a Distinct Mode of Thiamine Diphosphate‐Dependent Catalysis in Vitamin K Biosynthesis

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