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Nuclear Magenetic Resonance Evidence for the Role of the Flexible Regions of the E1 Component of the Pyruvate Dehydrogenase Complex from Gram Negative Bacteria
Author(s) -
Song Jaeyoung,
Nemeria Natalia S.,
Kale Sachin,
Kakalis Lazaros
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.465.3
Subject(s) - pyruvate dehydrogenase complex , heteronuclear single quantum coherence spectroscopy , dehydrogenase , protein subunit , stereochemistry , crystallography , chemistry , dihydrolipoyl transacetylase , nuclear magnetic resonance spectroscopy , biophysics , enzyme , biochemistry , biology , pyruvate dehydrogenase kinase , gene
Sequence‐specific NMR assignments were made for six residues in the N‐terminal 1–55 region, and one in each of the inner (401–413) and outer (541–557) active center loops of the E1 component of E. coli pyruvate dehydrogenase complex (E1ec, 2x100 kDa), flexible regions not seen in the x‐ray structure. The 2D HSQC spectrum revealed 103 resonances of the 109 expected in the three regions. Site‐specific substitutions and appropriate labeling patterns assisted the assignments. Resonances assigned to the N‐terminal region diminished in size/were absent in the spectrum in the presence of E2ec1‐190 comprising the lipoyl domain, peripheral subunit binding domain and hinge regions, indicating that the N‐terminal region became organized. Assignment of backbone NH resonances for residues G402 and G542 revealed dynamic features of the loops. These resonances, but not those assigned to the N‐terminal region, were no longer seen on saturation of the enzyme with a stable pre‐decarboxylation analogue C2alpha‐phosphonolactylThDP (PLThDP), indicating that the dynamic loop regions became ordered. The results provide the first structural support to the notion that the N‐terminal region of the E1ec of this entire class of bacterial pyruvate dehydrogenase complexes is responsible for binding the E2 component specifically, and dynamic motions of loops of E1ec are restricted by PLThDP. Supported by NIH‐GM‐050380.