title: Structure and function of the catalytic domain of the dihydrolipoyl acetyltransferase component in Escherichia coli pyruvate dehydrogenase complex.
abstract: The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s(-1), comparable with the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4'-aminopyrimidine tautomer of bound thiamin diphosphate (AP).
authors.affiliation: From the Department of Chemistry, Rutgers University, Newark, New Jersey 07102.
authors.forename: Junjie
authors.initials: J
authors.lastname: Wang
authors.affiliation: From the Department of Chemistry, Rutgers University, Newark, New Jersey 07102.
authors.forename: Natalia S
authors.initials: NS
authors.lastname: Nemeria
authors.affiliation: the Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
authors.forename: Krishnamoorthy
authors.initials: K
authors.lastname: Chandrasekhar
authors.affiliation: From the Department of Chemistry, Rutgers University, Newark, New Jersey 07102.
authors.forename: Sowmini
authors.initials: S
authors.lastname: Kumaran
authors.affiliation: the Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
authors.forename: Palaniappa
authors.initials: P
authors.lastname: Arjunan
authors.affiliation: the Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
authors.forename: Shelley
authors.initials: S
authors.lastname: Reynolds
authors.affiliation: the Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Calero
authors.affiliation: From the Department of Chemistry, Rutgers University, Newark, New Jersey 07102.
authors.forename: Roman
authors.initials: R
authors.lastname: Brukh
authors.affiliation: From the Department of Chemistry, Rutgers University, Newark, New Jersey 07102.
authors.forename: Lazaros
authors.initials: L
authors.lastname: Kakalis
authors.affiliation: the Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, the Veterans Affairs Medical Center, Pittsburgh, Pennsylvania 15240, and fureyw@pitt.edu.
authors.forename: William
authors.initials: W
authors.lastname: Furey
authors.affiliation: From the Department of Chemistry, Rutgers University, Newark, New Jersey 07102, frjordan@rutgers.edu.
authors.forename: Frank
authors.initials: F
authors.lastname: Jordan
mesh_terms: D000105:Acetyl Coenzyme A / Q000096:biosynthesis; D000107:Acetylation; D000595:Amino Acid Sequence; D050260:Carbohydrate Metabolism / Q000502:physiology; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D041961:Deuterium Exchange Measurement; D051047:Dihydrolipoyllysine-Residue Acetyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D011768:Pyruvate Dehydrogenase Complex; D000105:Acetyl Coenzyme A; D019289:Pyruvic Acid; D051047:Dihydrolipoyllysine-Residue Acetyltransferase
keywords: Carbohydrate Metabolism; Enzyme Catalysis; HD Exchange; Mass Spectrometry (MS); Nuclear Magnetic Resonance (NMR); Pyruvate Dehydrogenase Complex (PDC); X-ray Crystallography
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