title: Amino-terminal residues 1-45 of the Escherichia coli pyruvate dehydrogenase complex E1 subunit interact with the E2 subunit and are required for activity of the complex but not for reductive acetylation of the E2 subunit.
abstract: While N-terminal amino acids 1-55 are not seen in the structure of the Escherichia coli pyruvate dehydrogenase complex E1 subunit (PDHc-E1), mass spectrometric analysis indicated that this amino-terminal region of PDHc-E1 was protected by PDHc-E2. Hence, five deletion constructs of PDHc-E1 were created, Delta6-15, Delta16-25, Delta26-35, Delta36-45, and Delta46-55, along with single-site substitutions at Asp7, Asp9, Pro10, Ile11, Glu12, Thr13, Arg14, and Asp15. The decarboxylation of pyruvate and the ability of PDHc-E1 to dimerize are not affected by any of the deletions or substitutions. While Delta46-55 and the Pro10Ala, Ile11Ala, and Thr13Ala variants could form a complex with PDHc-E2, and produced NADH in the overall assay, Delta16-25, Delta26-35, and Delta36-45 and the Asp7Ala, Asp9Ala, Glu12Gln, Glu12Asp, Arg14Ala, and Asp15Ala variants failed in both respects. Remarkably, all constructs of PDHc-E1 from E. coli, as well as PDHc-E1 from Mycobacterium tuberculosis, could carry out reductive acetylation of the E. coli lipoyl domain, but only constructs of the E. coli PDHc-E1 could reductively acetylate E. coli PDHc-E2. It was concluded that there are at least two loci of interaction between the PDHc-E1 and PDHc-E2 subunits: (1) the thiamin diphosphate-bound substrate on PDHc-E1 and the lipoylamide of PDHc-E2, as reflected by the ability to reductively acetylate the latter; and (2) amino terminal residues 1-45 of PDHc-E1 with regions of PDHc-E2 (so far undefined for the E. coli complex), as reflected by the overall activity of the entire complex. These studies add important information regarding recognition within this multienzyme complex class with an alpha(2) E1 assembly.
authors.affiliation: Department of Chemistry, Rutgers, the State University of New Jersey, Newark, New Jersey 07102, USA.
authors.forename: Yun-Hee
authors.initials: YH
authors.lastname: Park
authors.forename: Wen
authors.initials: W
authors.lastname: Wei
authors.forename: Leon
authors.initials: L
authors.lastname: Zhou
authors.forename: Natalia
authors.initials: N
authors.lastname: Nemeria
authors.forename: Frank
authors.initials: F
authors.lastname: Jordan
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D019281:Dimerization; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014644:Genetic Variation; D006358:Hot Temperature; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D030481:Pyruvate Dehydrogenase (Lipoamide) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000378:metabolism*; D017384:Sequence Deletion; D008063:Thioctic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D014357:Trypsin
chemical_list: D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; D011768:Pyruvate Dehydrogenase Complex; D008063:Thioctic Acid; C013091:lipoamide; D030481:Pyruvate Dehydrogenase (Lipoamide); D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D014357:Trypsin