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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 alpha(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 loops, (b) an E2 component construct consisting of a lipoyl domain and peripheral subunit binding domain, and (c) a peptide corresponding to the amino acid sequence of the E2 peripheral subunit binding domain. Deductions from the NMR studies are in excellent agreement with our functional finding, providing a clear indication that the N-terminal region of the E1 interacts with the E2 peripheral subunit binding domain and that this interaction precedes reductive acetylation. The results provide the first structural support to the notion that the N-terminal region of the E1 component of this entire class of bacterial pyruvate dehydrogenase complexes is responsible for binding the E2 component.

SUBMITTER: Song J

PROVIDER: S-EPMC2836073 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Nuclear magnetic resonance evidence for the role of the flexible regions of the E1 component of the pyruvate dehydrogenase complex from gram-negative bacteria.

Song Jaeyoung J Park Yun-Hee YH Nemeria Natalia S NS Kale Sachin S Kakalis Lazaros L Jordan Frank F

The Journal of biological chemistry 20091207 7

Most bacterial pyruvate dehydrogenase complexes from either gram-positive or gram-negative bacteria have E1 components with an alpha(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 E ...[more]

PMID: 19996100

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Project description:Hyperthyroidism increases heart rate, contractility, cardiac output, and metabolic rate. It is also accompanied by alterations in the regulation of cardiac substrate use. Specifically, hyperthyroidism increases the ex vivo activity of pyruvate dehydrogenase kinase, thereby inhibiting glucose oxidation via pyruvate dehydrogenase. Cardiac hypertrophy is another effect of hyperthyroidism, with an increase in the abundance of mitochondria. Although the hypertrophy is initially beneficial, it can eventually lead to heart failure. The aim of this study was to use hyperpolarized magnetic resonance spectroscopy to investigate the rate and regulation of in vivo pyruvate dehydrogenase flux in the hyperthyroid heart and to establish whether modulation of flux through pyruvate dehydrogenase would alter cardiac hypertrophy.Hyperthyroidism was induced in 18 male Wistar rats with 7 daily intraperitoneal injections of freshly prepared triiodothyronine (0.2 mg x kg(-1) x d(-1)). In vivo pyruvate dehydrogenase flux, assessed with hyperpolarized magnetic resonance spectroscopy, was reduced by 59% in hyperthyroid animals (0.0022 ± 0.0002 versus 0.0055 ± 0.0005 second(-1); P=0.0003), and this reduction was completely reversed by both short- and long-term delivery of dichloroacetic acid, a pyruvate dehydrogenase kinase inhibitor. Hyperpolarized [2-(13)C]pyruvate was also used to evaluate Krebs cycle metabolism and demonstrated a unique marker of anaplerosis, the level of which was significantly increased in the hyperthyroid heart. Cine magnetic resonance imaging showed that long-term dichloroacetic acid treatment significantly reduced the hypertrophy observed in hyperthyroid animals (100 ± 20 versus 200 ± 30 mg; P=0.04) despite no change in the increase observed in cardiac output.This work has demonstrated that inhibition of glucose oxidation in the hyperthyroid heart in vivo is mediated by pyruvate dehydrogenase kinase. Relieving this inhibition can increase the metabolic flexibility of the hyperthyroid heart and reduce the level of hypertrophy that develops while maintaining the increased cardiac output required to meet the higher systemic metabolic demand.

Project description:The region encompassing residues 401-413 on the E1 component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli comprises a loop (the inner loop) which was not seen in the X-ray structure in the presence of thiamin diphosphate, the required cofactor for the enzyme. This loop is seen in the presence of a stable analogue of the pre-decarboxylation intermediate, the covalent adduct between the substrate analogue methyl acetylphosphonate and thiamin diphosphate, C2α-phosphonolactylthiamin diphosphate. It has been shown that the residue H407 and several other residues on this loop are required to reduce the mobility of the loop so electron density corresponding to it can be seen once the pre-decarboxylation intermediate is formed. Concomitantly, the loop encompassing residues 541-557 (the outer loop) appears to work in tandem with the inner loop and there is a hydrogen bond between the two loops ensuring their correlated motion. The inner loop was shown to: a) sequester the active center from carboligase side reactions; b) assist the interaction between the E1 and the E2 components, thereby affecting the overall reaction rate of the entire multienzyme complex; c) control substrate access to the active center. Using viscosity effects on kinetics it was shown that formation of the pre-decarboxylation intermediate is specifically affected by loop movement. A cysteine-less variant was created for the E1 component, onto which cysteines were substituted at selected loop positions. Introducing an electron spin resonance spin label and an (19)F NMR label onto these engineered cysteines, the loop mobility was examined: a) both methods suggested that in the absence of ligand, the loop exists in two conformations; b) line-shape analysis of the NMR signal at different temperatures, enabled estimation of the rate constant for loop movement, and this rate constant was found to be of the same order of magnitude as the turnover number for the enzyme under the same conditions. Furthermore, this analysis gave important insights into rate-limiting thermal loop dynamics. Overall, the results suggest that the dynamic properties correlate with catalytic events on the E1 component of the pyruvate dehydrogenase complex.

Dataset Information

Nuclear magnetic resonance evidence for the role of the flexible regions of the E1 component of the pyruvate dehydrogenase complex from gram-negative bacteria.

Publications

Nuclear magnetic resonance evidence for the role of the flexible regions of the E1 component of the pyruvate dehydrogenase complex from gram-negative bacteria.

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