Thiamine diphosphate (ThDP) is used as a cofactor in many key metabolic enzymes. We present evidence that the ThDPs in the two active sites of the E1 (EC 1.2.4.1) component of the pyruvate dehydrogenase complex communicate over a distance of 20 angstroms by reversibly shuttling a proton through an acidic tunnel in the protein. This "proton wire" permits the co-factors to serve reciprocally as general acid/base in catalysis and to switch the conformation of crucial active-site peptide loops. This synchronizes the progression of chemical events and can account for the oligomeric organization, conformational asymmetry, and "ping-pong" kinetic properties of E1 and other thiamine-dependent enzymes.

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RNA turnover is an essential element of cellular homeostasis and response to environmental change. Whether the ribonucleases that mediate RNA turnover can respond to cellular metabolic status is an unresolved question. Here we present evidence that the Krebs cycle metabolite citrate affects the activity of Escherichia coli polynucleotide phosphorylase (PNPase) and, conversely, that cellular metabolism is affected widely by PNPase activity. An E. coli strain that requires PNPase for viability has suppressed growth in the presence of increased citrate concentration. Transcriptome analysis reveals a PNPase-mediated response to citrate, and PNPase deletion broadly impacts on the metabolome. In vitro, citrate directly binds and modulates PNPase activity, as predicted by crystallographic data. Binding of metal-chelated citrate in the active site at physiological concentrations appears to inhibit enzyme activity. However, metal-free citrate is bound at a vestigial active site, where it stimulates PNPase activity. Mutagenesis data confirmed a potential role of this vestigial site as an allosteric binding pocket that recognizes metal-free citrate. Collectively, these findings suggest that RNA degradative pathways communicate with central metabolism. This communication appears to be part of a feedback network that may contribute to global regulation of metabolism and cellular energy efficiency.

SummaryThe pyruvate dehydrogenase multienzyme assembly (PDH) generates acetyl coenzyme A and reducing equivalents from pyruvate in a multiple-step process that is a nexus of central metabolism. We report crystal structures of the Geobacillus stearothermophilus PDH E1p subunit with ligands that mimic the prereaction complex and the postdecarboxylation product. The structures implicate residues that help to orient substrates, nurture intermediates, and organize surface loops so that they can engage a mobile lipoyl domain that receives the acetyl group and shuttles it to the next active site. The structural and enzymatic data suggest that H128β performs a dual role: first, as electrostatic catalyst of the reaction of pyruvate with the thiamine cofactor; and second, as a proton donor in the second reaction of acetyl group with the lipoate. We also identify I206α as a key residue in mediating the conformation of active-site loops. We propose that a simple conformational flip of the H271α side chain assists transfer of the acetyl group from thiamine cofactor to lipoyl domain in synchrony with reduction of the dithiolane ring.

Previous studies have shown that a combination of weight loss and fish oil supplementation reduce cardiovascular disease and diabetes risks by increasing adiponectin and reducing triacylglyceride concentrations, while weight loss alone significantly improves insulin sensitivity and reduces inflammation. Here, a metabolomic approach, using a combination of 1H-Nuclear Magnetic Resonance spectroscopy, and gas and liquid chromatography and mass spectrometry, was employed to elucidate the metabolic changes in blood plasma following weight loss and fish oil supplementation. The intervention study was conducted over 24 weeks, with 93 female subjects randomised to one of three groups. Two groups followed a 12-week weight loss program, followed by a 12-week weight maintenance period and were randomised to fish or placebo oil capsules; a control group did not follow the weight loss program and were given placebo oil capsules. Lipid profiles changed dramatically upon fish oil intake and subtly across the two weight loss groups. While the fish oil supplementation increased the proportion of various phospholipid species, previously reported reductions in total triacylglycerides (TAGs) upon fish oil intake were shown to be driven by a reduction in a specific subset of the measured TAGs. This remodelling of triglycerides may represent further beneficial effects of fish oil supplementation.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-009-0161-7) contains supplementary material, which is available to authorized users.