Nucleotide sequence of a cDNA for the dihydrolipoamide acetyltransferase component of human pyruvate dehydrogenase complex

Thekkumkara, Thomas J.; Ho, Lap; Wexler, Isaiah D.; Pons, Gabriel; Liu, Te-Chung; Patel, Mulchand S.

doi:10.1016/0014-5793(88)80337-5

Cited by 96 publications

(58 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Both bands appeared to represent the same protein by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) protein identification analysis. The sequences of two peptides were determined, and both showed high homology to the mitochondrial protein dihydrolipoamide acetyltransferase (Thekkumkara et al, 1988;Matuda et al, 1992), which is the E2 component of the pyruvate dehydrogenas complex (PDC-E2). These results suggest that Vp67 may be Xenopus PDC-E2.…”

Section: Purification Of Vp67mentioning

confidence: 99%

“…Additional primers were based on available vertebrate PDC-E2 cDNA sequences (Thekkumkara et al, 1988;Matuda et al, 1992). Two overlapping PCR products (ϳ200 bp and 400 bp) were successfully amplified from Xenopus total oocyte cDNA.…”

Section: Cloning Of Vp67 Cdnamentioning

confidence: 99%

“…As shown in Figure 5A, Xenopus Vp67 is highly related to human PDC-E2 (Thekkumkara et al, 1988), with overall amino acid identity of ϳ82% for the mature protein. The two peptide sequences obtained from the purified Vp67 are present in the translated cDNA sequence (boxed, Fig.…”

Section: Vp67 Is a Xenopus Homolog Of Pdc-e2mentioning

confidence: 99%

See 2 more Smart Citations

Apparent mitochondrial asymmetry in Xenopus eggs

Володина

Denegre

Mowry

2003

Developmental Dynamics

View full text Add to dashboard Cite

Section: Purification Of Vp67mentioning

confidence: 99%

Section: Cloning Of Vp67 Cdnamentioning

confidence: 99%

See 1 more Smart Citation

Apparent mitochondrial asymmetry in Xenopus eggs

Володина

Denegre

Mowry

2003

Developmental Dynamics

View full text Add to dashboard Cite

“…An alignment of the amino acid sequences of the E2 chains from B. stearothermophilus, S. cerevisiae [21,381 and human liver [39] PDH complexes is shown in Fig. 4.…”

Section: Comparison Of the B Stearothermophilus E2 Chain With Other mentioning

confidence: 99%

Cloning and sequence analysis of the genes encoding the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

Borges

Hawkins

Packman

et al. 1990

European Journal of Biochemistry

View full text Add to dashboard Cite

A 2641-bp EcoRI fragment of DNA that encodes the C-terminal part of the dihydrolipoyl acetyltransferase (E2) component and the dihydrolipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. A 705-bp truncated open reading frame was located at the 5' end of the insert which, together with the 588-bp truncated open reading frame at the 3' end of another EcoRI fragment of B. stearothermophilus DNA previously cloned and sequenced [Hawkins, C. F., Borges, A. & Perham, R. N. (1990) Eur. J. Biochem. 191,, was identified as the gene, pdhC, encoding the E2 polypeptide chain. Direct sequence analysis of the purified E2 chain confirmed that the two EcoRI fragments are adjoining in the B. stearothermophilus genome. The E3 gene, pdhD, begins just 4 bp downstream from the stop codon of the pdhC gene. The amino acid sequences deduced from the pdhC and pdhD genes correspond to proteins of 427 amino acids (E2, M , 46265) and 469 amino acids (E3, M , 49193), respectively. Both genes are preceded by potential ribosome-binding sites and the E3 gene is followed by a stemloop structure characteristic of rho-independent transcription terminators. The B. stearothermophilus E2 and E3 chains exhibit substantial sequence similarity with the corresponding subunits of other 2-0x0-acid dehydrogenase multienzyme complexes. The cloning and sequence analysis described here complete the description of the gene cluster (pdhA, B, C and D ) which encodes the B. stearothermophilus pyruvate dehydrogenase multienzyme complex.The oxidative decarboxylation of 2-0x0-acids is accomplished in most organisms by the 2-0x0-acid dehydrogenase multienzyme complexes, producing the corresponding acyl-CoA and NADH. For the pyruvate dehydrogenase (PDH) complex, the constituent enzymes that function successively in the mechanism are pyruvate dehydrogenase (El), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3) (for recent reviews see [I, 21).The PDH complex from the thermophilic, Gram-positive bacterium Bacillus stearothermophilus is assembled round a core of the E2 component of 60 copies of the E2 polypeptide chain organized with icosahedral symmetry [3]. Multiple cop- ies of the El (comprising two polypeptide subunits, Elcl and Elp) and E3 components are attached to this core tightly but non-covalently. The M, of the individual components as well as the morphology of the E2 core [3, 41 closely resemble those of the PDH complexes of eukaryotes. Its thermostability makes the B. stearothermophilus PDH complex a good system for studying the structure of a 2-0x0-acid dehydrogenase complex based on icosahedral symmetry, which differs from the PDH and 2-oxoglutarate dehydrogenase complexes of Escherichia coli in that the latter have octahedral E2 cores [5, 61. The sequence of the first 21 1 amino acid residues from the N-terminus of the B. stearothermophilus E2 chain has been determined [7], showing that this ace...

show abstract

“…The core structure of PDC is formed by association of 60 dihydrolipoyl acetyltransferase (E2) subunits. E2 is a segmented protein with four domains connected by mobile linker regions (25,26). Twenty trimers of the C-terminal inner domain of E2 assemble at the vertices of dodecahedron; these trimers catalyze the transacetylation reaction (27,28).…”

mentioning

confidence: 99%

Marked Differences between Two Isoforms of Human Pyruvate Dehydrogenase Kinase

Baker

Yan

Peng

et al. 2000

Journal of Biological Chemistry

110

192

View full text Add to dashboard Cite

Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co-expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 M L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in k cat along with about a 2-fold decrease in the K m of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g. using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2-60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated >3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors.

show abstract

Nucleotide sequence of a cDNA for the dihydrolipoamide acetyltransferase component of human pyruvate dehydrogenase complex

Cited by 96 publications

References 15 publications

Apparent mitochondrial asymmetry in Xenopus eggs

Apparent mitochondrial asymmetry in Xenopus eggs

Cloning and sequence analysis of the genes encoding the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

Marked Differences between Two Isoforms of Human Pyruvate Dehydrogenase Kinase

Contact Info

Product

Resources

About