Selective proteolysis of the protein X subunit of the bovine heart pyruvate dehydrogenase complex. Effects on dihydrolipoamide dehydrogenase (E3) affinity and enzymic properties of the complex

Neagle, J.; Lindsay, J. Gordon

doi:10.1042/bj2780423

Cited by 34 publications

(14 citation statements)

References 18 publications

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“…PDHc comprises three catalytic subunits, E1, E2, and E3, two regulatory subunits, E1 kinase and E1 phosphatase, and a further component, E3 binding protein (E3BP) [Neagle and Lindsay, 1991]. Despite its lack of catalytic activity, E3BP performs an essential structural role in the formation and cohesion of the PDHc.…”

Section: Introductionmentioning

confidence: 99%

A large genomic deletion in thePDHX gene caused by the retrotranspositional insertion of a full-length LINE-1 element

Miné¹,

Chen

Brivet

et al. 2007

Hum. Mutat.

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The long interspersed element-1 (LINE-1 or L1) retrotransposition has altered the human genome in many ways. In particular, recent in vitro studies have demonstrated that the retrotranspositional insertion of L1 elements has resulted in significant genomic deletions. Here we provide evidence for its operation in the human genome by identifying a approximately 46-kb pathological genomic deletion in the PDHX gene directly linked to the insertion of a full-length L1 element, in a patient with pyruvate dehydrogenase complex (PDHc) deficiency. Both the deduced bottom and top strand cleavage sites in the PDHX gene coincide with the consensus L1 endonuclease (EN) target sequence 5'-TTTT/A-3', while the full-length L1 element is followed by a 67-bp poly(A) tail. Interestingly, two hairpin structures, potentially formed by the inverted repeats present immediately 5' to the top strand nick site and 3' to the bottom strand nick site, may have facilitated the accessibility of L1 EN to the target sequences and also brought the two otherwise distantly located sequences into close proximity. Since the L1 element inserted in the PDHX gene is full-length, we favor the model of the template jumping as opposed to that of the microhomology-mediated end-joining for linking the 5' end of the nascent L1 copy to its genomic target. Our finding not only serves as an important complement to the in vitro approaches to studying L1 retrotransposition, but also reveals a novel mechanism causing human genetic disease.

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Section: Introductionmentioning

confidence: 99%

A large genomic deletion in thePDHX gene caused by the retrotranspositional insertion of a full-length LINE-1 element

Miné¹,

Chen

Brivet

et al. 2007

Hum. Mutat.

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“…Along with E3BP, the human E2 component forms the structural core of PDC and provides the binding site for E1 (2). E3BP (previously known as protein X) provides primarily the binding site for E3 (3,4). In the absence of E3BP, PDC catalysis is supported at a rate of 4% only (5,6).…”

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confidence: 99%

How Dihydrolipoamide Dehydrogenase-binding Protein Binds Dihydrolipoamide Dehydrogenase in the Human Pyruvate Dehydrogenase Complex

Ciszak

Makal

Hong

et al. 2006

Journal of Biological Chemistry

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The dihydrolipoamide dehydrogenase-binding protein (E3BP) and the dihydrolipoamide acetyltransferase (E2) component enzyme form the structural core of the human pyruvate dehydrogenase complex by providing the binding sites for two other component proteins, dihydrolipoamide dehydrogenase (E3) and pyruvate dehydrogenase (E1), as well as pyruvate dehydrogenase kinases and phosphatases. Despite a high similarity between the primary structures of E3BP and E2, the E3-binding domain of human E3BP is highly specific to human E3, whereas the E1-binding domain of human E2 is highly specific to human E1. In this study, we characterized binding of human E3 to the E3-binding domain of E3BP by x-ray crystallography at 2.6-Å resolution, and we used this structural information to interpret the specificity for selective binding. Two subunits of E3 form a single recognition site for the E3-binding domain of E3BP through their hydrophobic interface. The hydrophobic residues Pro 133 , Pro 154 , and Ile 157 in the E3-binding domain of E3BP insert themselves into the surface of both E3 polypeptide chains. Numerous ionic and hydrogen bonds between the residues of three interacting polypeptide chains adjacent to the central hydrophobic patch add to the stability of the subcomplex. The specificity of pairing for human E3BP with E3 is interpreted from its subcomplex structure to be most likely due to conformational rigidity of the binding fragment of the E3-binding domain of E3BP and its exquisite amino acid match with the E3 target interface. The human pyruvate dehydrogenase complex (PDC)2 with an approximate molecular mass of 8 ϫ 10 6 Da consists of multiple copies of three catalytic enzymes known as pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3) as well as the dedicated E3-binding protein (E3BP) (1). In addition, pyruvate dehydrogenase kinase and phosphatase interacting with the complex are responsible for regulation of PDC activity by a reversible phosphorylation/dephosphorylation mechanism that involves covalent modification of E1. PDC plays a key role in regulation of the flux of two-carbon acetyl units from pyruvate through acetyl-CoA into the Krebs cycle, yielding CO 2 , NADH, and H ϩ . The E1 component catalyzes the decarboxylation of pyruvate and the reductive acetylation of the lipoyl moieties of E2. The E2 component transfers the acetyl group to CoA. The E3 component oxidizes the reduced lipoyl moieties through reduction of NAD ϩ to NADH, thus preparing the lipoyl moiety for another cycle of catalytic reaction. Along with E3BP, the human E2 component forms the structural core of PDC and provides the binding site for E1 (2). E3BP (previously known as protein X) provides primarily the binding site for E3 (3, 4). In the absence of E3BP, PDC catalysis is supported at a rate of 4% only (5, 6). Both E2 and E3BP share considerable sequence identity (37%) as revealed in pairwise sequence alignment (7). The principal differences are that mammalian E3BP has a single lipoyl ...

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“…These include the pyruvate dehydrogenase (E 1 ) and dihydrolipoamide dehydrogenase (E 3 ). E 3 requires a binding protein (BP) to anchor it to the core of the yeast (12) and mammalian PDCs (13)(14)(15) though, in E. coli and Bacillus stearothermophilus PDCs, BP is not required (1)(2)(3)(4).…”

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On the Unique Structural Organization of the Saccharomyces cerevisiae Pyruvate Dehydrogenase Complex

Stoops

Cheng

Yazdi

et al. 1997

Journal of Biological Chemistry

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Dihydrolipoamide acyltransferase (E 2 ), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of ␣-keto acids, forms the central core to which the other components attach. We have determined the structures of the truncated 60-mer core dihydrolipoamide acetyltransferase (tE 2 ) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex and complexes of the tE 2 core associated with a truncated binding protein (tBP), intact binding protein (BP), and the BP associated with its dihydrolipoamide dehydrogenase (BP⅐E 3 ). The tE 2 core is a pentagonal dodecahedron consisting of 20 cone-shaped trimers interconnected by 30 bridges. Previous studies have given rise to the generally accepted belief that the other components are bound on the outside of the E 2 scaffold. However, this investigation shows that the 12 large openings in the tE 2 core permit the entrance of tBP, BP, and BP⅐E 3 into a large central cavity where the BP component apparently binds near the tip of the tE 2 trimer. The bone-shaped E 3 molecule is anchored inside the central cavity through its interaction with BP. One end of E 3 has its catalytic site within the surface of the scaffold for interaction with other external catalytic domains. Though tE 2 has 60 potential binding sites, it binds only about 30 copies of tBP, 15 of BP, and 12 of BP⅐E 3 . Thus, E 2 is unusual in that the stoichiometry and arrangement of the tBP, BP, and E 3 ⅐BP components are determined by the geometric constraints of the underlying scaffold.Pyruvate dehydrogenase complexes (PDCs) 1 are among the largest (M r ϳ10 6 -10 7 ) and most complex multienzyme structures known. They consist of a central core that has both functional and structural roles in organizing the complex, the dihydrolipoamide acetyltransferase (E 2 ) subunits associate to form the core complex that also serves as a scaffold to which the other components are attached (1-4). Electron microscopy (5-8) and x-ray crystallography (9 -11) studies have revealed two fundamental morphologies of the E 2 cores. The cubic E 2 core of the Escherichia coli PDC has 24 subunits arranged with octahedral symmetry, whereas the pentagonal dodecahedral E 2 core of the PDC complexes from eukaryotes and some Grampositive bacteria has 60 subunits arranged with icosahedral symmetry. The subunits form cone-shaped trimers at each of the 8 and 20 vertices of the cubic and dodecahedral structures, respectively. These trimers are interconnected by bridges to form a cage-like complex (8 -11).The E 2 subunit is a multidomain structure to which the other constituents of the functional PDC (1-4) bind (see Fig. 1). These include the pyruvate dehydrogenase (E 1 ) and dihydrolipoamide dehydrogenase (E 3 ). E 3 requires a binding protein (BP) to anchor it to the core of the yeast (12) and mammalian PDCs (13-15) though, in E. coli and Bacillus stearothermophilus PDCs, BP is not required (1-4).It is widely held that the constituent proteins are bound to the out...

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Selective proteolysis of the protein X subunit of the bovine heart pyruvate dehydrogenase complex. Effects on dihydrolipoamide dehydrogenase (E3) affinity and enzymic properties of the complex

Cited by 34 publications

References 18 publications

A large genomic deletion in thePDHX gene caused by the retrotranspositional insertion of a full-length LINE-1 element

A large genomic deletion in thePDHX gene caused by the retrotranspositional insertion of a full-length LINE-1 element

How Dihydrolipoamide Dehydrogenase-binding Protein Binds Dihydrolipoamide Dehydrogenase in the Human Pyruvate Dehydrogenase Complex

On the Unique Structural Organization of the Saccharomyces cerevisiae Pyruvate Dehydrogenase Complex

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