Site-directed mutagenesis of a loop at the active site of E1 (α2β2) of the pyruvate dehydrogenase complex

A possible common sequence motif

Markus Fries, Hitesh J. Chauhan, Gonzalo J. Domingo, Hyo Il Jung, Richard N. Perham

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Limited proteolysis of the pyruvate decarboxylase (E1, α2β2) component of the pyruvate dehydrogenase (PDH) multi-enzyme complex of Bacillus stearothermophilus has indicated the importance for catalysis of a site (Tyr281-Arg282) in the E1α subunit (Chauhan, H.J., Domingo, G.J., Jung, H.-I. & Perham, R.N. (2000) Eur. J. Biochem. 267, 7158-7169). This site appears to be conserved in the α-subunit of heterotetrameric E1s and multiple sequence alignments suggest that there are additional conserved amino-acid residues in this region, part of a common pattern with the consensus sequence -YR-H-D-YR-DE-. This region lies about 50 amino acids on the C-terminal side of a 30-residue motif previously recognized as involved in binding thiamin diphosphate (ThDP) in all ThDP-dependent enzymes. The role of individual residues in this set of conserved amino acids in the E1α chain was investigated by means of site-directed mutagenesis. We propose that particular residues are involved in: (a) binding the 2-oxo acid substrate, (b) decarboxylation of the 2-oxo acid and reductive acetylation of the tethered lipoyl domain in the PDH complex, (c) an 'open-close' mechanism of the active site, and (d) phosphorylation by the E1-specific kinase (in eukaryotic PDH and branched chain 2-oxo acid dehydrogenase complexes).

Original languageEnglish
Pages (from-to)861-870
Number of pages10
JournalEuropean Journal of Biochemistry
Volume270
Issue number5
DOIs
Publication statusPublished - 2003 Mar 1

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Pyruvate Dehydrogenase Complex
Mutagenesis
Site-Directed Mutagenesis
Thiamine Pyrophosphate
Keto Acids
Catalytic Domain
Pyruvic Acid
Amino Acids
Pyruvate Dehydrogenase (Lipoamide)
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)
Pyruvate Decarboxylase
Proteolysis
Geobacillus stearothermophilus
Acetylation
Phosphorylation
Decarboxylation
Sequence Alignment
Consensus Sequence
Bacilli
Enzymes

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "Site-directed mutagenesis of a loop at the active site of E1 (α2β2) of the pyruvate dehydrogenase complex: A possible common sequence motif",
abstract = "Limited proteolysis of the pyruvate decarboxylase (E1, α2β2) component of the pyruvate dehydrogenase (PDH) multi-enzyme complex of Bacillus stearothermophilus has indicated the importance for catalysis of a site (Tyr281-Arg282) in the E1α subunit (Chauhan, H.J., Domingo, G.J., Jung, H.-I. & Perham, R.N. (2000) Eur. J. Biochem. 267, 7158-7169). This site appears to be conserved in the α-subunit of heterotetrameric E1s and multiple sequence alignments suggest that there are additional conserved amino-acid residues in this region, part of a common pattern with the consensus sequence -YR-H-D-YR-DE-. This region lies about 50 amino acids on the C-terminal side of a 30-residue motif previously recognized as involved in binding thiamin diphosphate (ThDP) in all ThDP-dependent enzymes. The role of individual residues in this set of conserved amino acids in the E1α chain was investigated by means of site-directed mutagenesis. We propose that particular residues are involved in: (a) binding the 2-oxo acid substrate, (b) decarboxylation of the 2-oxo acid and reductive acetylation of the tethered lipoyl domain in the PDH complex, (c) an 'open-close' mechanism of the active site, and (d) phosphorylation by the E1-specific kinase (in eukaryotic PDH and branched chain 2-oxo acid dehydrogenase complexes).",
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Site-directed mutagenesis of a loop at the active site of E1 (α2β2) of the pyruvate dehydrogenase complex : A possible common sequence motif. / Fries, Markus; Chauhan, Hitesh J.; Domingo, Gonzalo J.; Jung, Hyo Il; Perham, Richard N.

In: European Journal of Biochemistry, Vol. 270, No. 5, 01.03.2003, p. 861-870.

Research output: Contribution to journalArticle

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T2 - A possible common sequence motif

AU - Fries, Markus

AU - Chauhan, Hitesh J.

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