Nuclear Magnetic Resonance Evidence for the Role of the Flexible Regions of the E1 Component of the Pyruvate Dehydrogenase Complex from Gram-negative Bacteria

Abstract

Most bacterial pyruvate dehydrogenase complexes from either Gram-positive or Gram-negative bacteria have E1 components with an ␣ 2 homodimeric quaternary structure. In a sequel to our previous publications, we present the first NMR study on the flexible regions of the E1 component from Escherichia coli and its biological relevance. We report sequence-specific NMR assignments for 6 residues in the N-terminal 1-55 region and for a glycine in each of the two mobile active center loops of the E1 component, a 200-kDa homodimer. This was accomplished by using site-specific substitutions and appropriate labeling patterns along with a peptide with the sequence corresponding to the N-terminal 1-35 amino acids of the E1 component. To study the functions of these mobile regions, we also examined the spectra in the presence of (a) a reaction intermediate analog known to affect the mobility of the active center The pyruvate dehydrogenase complex (PDHc) 3 is a molecular machine consisting of multiple copies of three distinct proteins in bacteria (5 MDa) (1, 2). In most bacteria (both Gramnegative and Gram-positive) the complex has octahedral symmetry. The PDHc catalyzes the oxidative decarboxylation of pyruvate according to Equation 1.The components of the Escherichia coli PDHc have the following roles (Equations 2-6) (Refs. 3-5). The thiamin diphosphate (ThDP)-dependent E1 component (E1ec, pyruvate dehydrogenase) is an ␣ 2 homodimer and carries out consecutively pyruvate decarboxylation and reductive acetylation of the E2 component (Equation 3). E2 (E2ec, dihydrolipoamide acetyltransferase) has a dual function. Its covalently attached lipoamide is reductively acetylated by the E1 component and pyruvate, and subsequently, the acetyl group is transferred to coenzyme A (CoA), and the principal metabolic product acetyl-CoA is released (Equation 4). E3 (E3ec, dihydrolipoamide dehydrogenase) has tightly bound FAD and NAD ϩ ; it regenerates the lipoamide from the reduced dihydrolipoamide form (Equation 5) (Scheme 1).Our laboratory has been studying the PDHc from E. coli (PDHc-ec) as a representative of this large class of enzymes (6 -10). In the x-ray structure of E1ec with ThDP, there were identified three disordered regions corresponding to the N-terminal 1-55 residues, to amino acids spanning residues 401-413 (inner active center loop) and 541-557 (outer active center loop) (7). We have now shown that all three of these regions have important function. To study the function of the N-terminal region of E1ec, we followed up on earlier work of de Kok and

Structure and Function of the Catalytic Domain of the Dihydrolipoyl Acetyltransferase Component in Escherichia coli Pyruvate Dehydrogenase Complex

“…Sodium pyruvate and tris(2-carboxyethyl)phosphine (TCEP) were from Sigma-Aldrich. (27), 1-lip E2p (28), and E3 (27) followed reported protocols.…”

“…The LD h is composed of residues 1-33 from LD1 and residues 238 -289 from LD3 of the E. coli 3-lip E2p (29). The C-terminally truncated E2p proteins were expressed in BL21 (DE3) cells at 37°C, similarly to that reported for the E2p(1-190) didomain (28). The culture was grown in LB medium supplemented with 50 g/ml ampicillin and 0.30 mM lipoic acid, and protein expression was induced by 0.5 mM isopropyl 1-thio-␤-D-galactopyranoside for 5-6 h at 37°C.…”

“…Proteins-Construction of plasmids for expression of C-terminally truncated E2p proteins was as reported for E2p didomain (28). Briefly, the pET15b-E2p plasmid encoding His 6 tag 1-lip E2p from the N-terminal end (containing a single hybrid lipoyl domain, LD h , in place of three LDs in the wild type 3-lip E2p) was used as a template, and the amplification primers and their complements were used for site-directed mutagenesis to introduce a TAA stop codon in place of Lys 97 , Lys 165 , or Lys 191 , leading to C-terminally truncated E2p proteins: E2p(1-96) (LD h , comprising LD h and part of the linker) and E2p(1-164) and E2p didomains, both comprising LD h , PSBD, and linkers of different lengths (see Fig.…”

“…The C-terminally truncated proteins were purified using a nickelSepharose Fast Flow column (GE Healthcare). All proteins were additionally purified using G3000SW TSK size exclusion chromatography with a high performance liquid chromatography system (28).…”

Human 2-Oxoglutarate Dehydrogenase Complex E1 Component Forms a Thiamin-derived Radical by Aerobic Oxidation of the Enamine Intermediate

“…The E1o-h was dialyzed against 50 mM KH 2 PO 4 (pH 7.5) containing 0.30 M NaCl, 0.50 mM ThDP, 2.0 mM MgCl 2 , and 1.0 mM benzamidine⅐HCl at 4°C for 15 h. The protein was concentrated, and buffer was exchanged to 50 mM HEPES (pH 7.5) containing 0.15 M NaCl, 0.50 mM ThDP, 1.0 mM MgCl 2 , and 1 mM benzamidine⅐HCl and was stored at Ϫ20°C. The His 6 -tagged E2o-h was purified in a manner similar to that reported for E2p-ec (34).…”