Recognition of the lipoyl domain is the ultimate determinant of substrate channelling in the pyruvate dehydrogenase multienzyme complex

Jones, D. Dafydd; Stott, Katherine; Reche, Pedro A.; Perham, Richard N.

doi:10.1006/jmbi.2000.4257

Cited by 23 publications

(23 citation statements)

References 33 publications

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“…With this ordering, a tunnel that can accommodate the lipoyl-lysine side chain bound to an E2 component in PDHc is completely formed, and a new surface is created at the mouth of the funnel, which, based on the distance from the active site and the known length of the lipoyl-lysine side chain, is likely to bind to the corresponding E2 lipoyl domain. These results are consistent with a previous report indicating that E1-E2 lipoyl domain binding stability is increased in the presence of the substrate pyruvate (31). Given the strong structural similarity between substrate and analogue, we suggest that the ordering observed in the E1-PLThDP complex would also be present when the intermediate from the natural substrate pyruvate is formed, and the increased E1-E2 stability may be explained by E2-lipoyl domain interactions with the resulting newly ordered E1 surface residues.…”

Section: Discussionsupporting

confidence: 94%

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: Discussionsupporting

confidence: 94%

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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“…Whether and how this hydrophobic patch and the surrounding charged groups interact with the cognate E1, E2, and E3 components remain to be elucidated. Nevertheless, the above observation is in accord with the notion that recognition of the lipoyl domain by its cognate E1 components is complex and cannot be attributed to any particular determinant on the domain (21,66). It is important to point out that this region is also the least defined region (Fig.…”

Section: Discussionsupporting

confidence: 79%

Solution Structure and Dynamics of the Lipoic Acid-bearing Domain of Human Mitochondrial Branched-chain α-Keto Acid Dehydrogenase Complex

Chang

Chou

Chuang

et al. 2002

Journal of Biological Chemistry

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The lipoyl-bearing domain (LBD) of the transacylase (E2) subunit of the branched-chain ␣-keto acid dehydrogenase complex plays a central role in substrate channeling in this mitochondrial multienzyme complex. We have employed multidimensional heteronuclear NMR techniques to determine the structure and dynamics of the LBD of the human branched-chain ␣-keto acid dehydrogenase complex (hbLBD). Similar to LBD from other members of the ␣-keto acid dehydrogenase family, the solution structure of hbLBD is a flattened ␤-barrel formed by two four-stranded antiparallel ␤-sheets. The lipoyl Lys 44 residue resides at the tip of a ␤-hairpin comprising a sharp type I ␤-turn and the two connecting ␤-strands 4 and 5. A prominent V-shaped groove formed by a surface loop, L1, connecting ␤1-and ␤2-strands and the lipoyl lysine ␤-hairpin constitutes the functional pocket. We further applied reduced spectral density functions formalism to extract dynamic information of hbLBD from 15 N-T 1 , 15 N-T 2 , and ( 1 H-15 N) nuclear Overhauser effect data obtained at 600 MHz. The results showed that residues surrounding the lipoyl lysine region comprising the L1 loop and the Lys 44 ␤-turn are highly flexible, whereas ␤-sheet S1 appears to display a slow conformational exchange process.The mammalian mitochondrial branched-chain ␣-keto acid dehydrogenase (BCKD) 1 complex catalyzes the oxidative decarboxylation of branched-chain ␣-keto acids derived from leucine, isoleucine, and valine to give rise to branched-chain acyl-CoAs (1). The reaction products are indirectly channeled into the Krebs cycle or linked to lipid and cholesterol biosynthesis. In patients with inherited maple syrup urine disease, the activity of the BCKD complex is deficient, which results in the accumulation of branched-chain ␣-keto acids. This metabolic block has severe clinical consequences including often fatal ketoacidosis, neurological derangement, and mental retardation in survivors (2).The mammalian BCKD complex is a member of the highly conserved ␣-keto acid dehydrogenase complexes consisting of the pyruvate dehydrogenase complex (PDC), the ␣-ketoglutarate dehydrogenase complex (KGDC), and the BCKD complex with similar structure and function (3). The mammalian BCKD complex consists of three catalytic components as follows: a heterotetrameric (␣ 2 ␤ 2 ) branched-chain ␣-keto acid decarboxylase or E1, a homo-24-meric dihydrolipoyltransacylase or E2, and a homodimeric dihydrolipoamide dehydrogenase or E3. E1 and E2 components are specific for the BCKD complex, whereas the E3 component is common among the three ␣-keto acid dehydrogenase complexes. In addition, the mammalian BCKD complex contains two regulatory enzymes, the BCKD kinase and the BCKD phosphatase that regulate activity of the BCKD complex through phosphorylation (inactivation)/dephosphorylation (activation) cycles (4). The BCKD complex is organized around the cubic E2 core, to which 12 copies of E1, and unspecified copies of E3, the BCKD kinase, and the BCKD phosphatase are attached through ionic interact...

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“…One of the first features noticed in this overlay is that the active sites of different thiamine enzymes show a high degree of variation in shape and composition. This is not so surprising given the significant differences in substrate specificity, particularly in the second half-reaction, where the substrate may be as small as a proton, as in the case of pyruvate decarboxylase (PDC, EC 4.1.1.1), or as large as an entire protein domain, as seen in pyruvate dehydrogenase [27]. Interestingly, the site occupied by the Review Article 897 phosphate-proximal His is a potential proton donor in PDH-E1 and TK exclusively.…”

Section: Conserved Aspects Of the Catalytic Mechanismmentioning

confidence: 91%

Structure, mechanism and catalytic duality of thiamine-dependent enzymes

Frank

Leeper

Luisi

2007

Cell. Mol. Life Sci.

229

223

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Thiamine is an essential cofactor that is required for processes of general metabolism amongst all organisms, and it is likely to have played a role in the earliest stages of the evolution of life. Here, we review from a structural perspective the enzymatic mechanisms that involve this cofactor. We explore asymmetry within homodimeric thiamine diphosphate (ThDP)-dependent enzyme structures and discuss how this may be correlated with the kinetic properties of half-of-the-sites reactivity, and negative cooperativity. It is likely these structural and kinetic hallmarks may arise through reciprocal coupling of active sites. This mode of communication between distant active sites is not unique to ThDP-dependent enzymes, but is widespread in other classes of oligomeric enzyme. Thus, it appears likely to be a general phenomenon reflecting a powerful mechanism of accelerating the rate of a chemical pathway. Finally, we speculate on the early evolutionary history of the cofactor and its ancient association with protein and RNA.

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Recognition of the lipoyl domain is the ultimate determinant of substrate channelling in the pyruvate dehydrogenase multienzyme complex

Cited by 23 publications

References 33 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

Solution Structure and Dynamics of the Lipoic Acid-bearing Domain of Human Mitochondrial Branched-chain α-Keto Acid Dehydrogenase Complex

Structure, mechanism and catalytic duality of thiamine-dependent enzymes

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