Pyruvate formate-lyase activating enzyme: elucidation of a novel mechanism for glycyl radical formation

Pyruvate formate lyase activating enzyme is a member of a novel superfamily of enzymes that utilize S-adenosylmethionine to initiate radical catalysis. This enzyme has been isolated with several different iron-sulfur clusters, but single turnover monitored by EPR has identified the [4Fe-4S](1+) cluster as the catalytically active cluster; this cluster is believed to be oxidized to the [4Fe-4S](2+) state during turnover. The [4Fe-4S] cluster is coordinated by a three-cysteine motif common to the radical/S-adenosylmethionine superfamily, suggesting the presence of a unique iron in the cluster. The unique iron site has been confirmed by Mossbauer and ENDOR spectroscopy experiments, which also provided the first evidence for direct coordination of S-adenosylmethionine to an iron-sulfur cluster, in this case the unique iron of the [4Fe-4S] cluster. Coordination to the unique iron anchors the S-adenosylmethionine in the active site, and allows for a close association between the sulfonium of S-adenosylmethionine and the cluster as observed by ENDOR spectroscopy. The evidence to date leads to a mechanistic proposal involving inner-sphere electron transfer from the cluster to the sulfonium of S-adenosylmethionine, followed by or concomitant with C-S bond homolysis to produce a 5'-deoxyadenosyl radical; this transient radical abstracts a hydrogen atom from G734 to activate pyruvate formate lyase.     

GABA shunt deficiencies and accumulation of reactive oxygen intermediates: insight from Arabidopsis mutants

In plants, succinic semialdehyde dehydrogenase (SSADH)-deficiency results in the accumulation of reactive oxygen intermediates (ROI), necrotic lesions, dwarfism, and hypersensitivity to environmental stresses. We report that Arabidopsis ssadh knockout mutants contain five times the normal level of gamma-hydroxybutyrate (GHB), which in SSADH-deficient mammals accounts for phenotypic abnormalities. Moreover, the level of GHB in Arabidopsis is light dependent. Treatment with gamma-vinyl-gamma-aminobutyrate, a specific gamma-aminobutyrate (GABA)-transaminase inhibitor, prevents the accumulation of ROI and GHB in ssadh mutants, inhibits cell death, and improves growth. These results provide novel evidence for the relationship between the GABA shunt and ROI, which may, in part, explain the phenotype of SSADH-deficient plants and animals.     

Monocarboxylate transporters in subsarcolemmal and intermyofibrillar mitochondria

Whether subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria contain monocarboxylate transporters (MCTs) is controversial. We have examined the presence of MCT1, 2, and 4 in highly purified SS and IMF mitochondria. These mitochondria were not contaminated with plasma membrane, sarcoplasmic reticulum or endosomal compartments, as the marker proteins for these sub-cellular compartments (Na+-K+-ATPase, Ca2+-ATPase, and the transferrin receptor) were not present in SS or IMF mitochondria. MCT1, MCT2, and MCT4 were all present at the plasma membrane. However, MCT1 and MCT4 were associated with SS mitochondria. In contrast, the IMF mitochondria were completely devoid of MCT1 and MCT4. However, MCT2 was associated with both SS and IMF mitochondria. These observations suggest that SS and IMF mitochondria have different capacities for metabolizing monocarboxylates. Thus, the controversy as to whether mitochondria can take up and oxidize lactate will need to take account of the different distribution of MCTs between SS and IMF mitochondria.     

Determinants of substrate specificity in KdcA, a thiamin diphosphate-dependent decarboxylase

Thiamin diphosphate-dependent decarboxylases catalyze the non-oxidative decarboxylation of 2-keto carboxylic acids. Although they display relatively low sequence similarity, and broadly different range of substrates, these enzymes show a common homotetrameric structure. Here we describe a kinetic characterization of the substrate spectrum of a recently identified member of this class, the branched chain 2-keto acid decarboxylase (KdcA) from Lactococcus lactis. In order to understand the structural basis for KdcA substrate recognition we developed a homology model of its structure. Ser286, Phe381, Val461 and Met358 were identified as residues that appeared to shape the substrate binding pocket. Subsequently, site-directed mutagenesis was carried out on these residues with a view to converting KdcA into a pyruvate decarboxylase. The results show that the mutations all lowered the Km value for pyruvate and both the S286Y and F381W variants also had greatly increased values of k(cat) with pyruvate as a substrate.     

Anabolic five subunit-type pyruvate:ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6

The thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. A gene cluster, porEDABG, encoding pyruvate:ferredoxin oxidoreductase (POR), which plays a key role in this cycle, was cloned and sequenced. The nucleotide sequence and the gene organization were similar to those of the five subunit-type 2-oxoglutarate:ferredoxin oxidoreductase from this strain, although the anabolic POR had been previously reported to consist of four subunits. A small protein (8 kDa) encoded by porE, which had not been detected in the previous work, was identified in the purified recombinant POR expressed in Escherichia coli, indicating that the enzyme is also a five-subunit type. Incorporation of PorE in the wild-type POR enzyme was confirmed by immunological analysis. PorA, PorB, PorG, and PorE were similar to the alpha, beta, gamma, and delta subunits of the four subunit-type 2-oxoacid oxidoreductases, respectively, and had conserved specific motifs. PorD had no specific motifs but was essential for the expression of the active enzyme.     

Assay of the pyruvate dehydrogenase complex by coupling with recombinant chicken liver arylamine N-acetyltransferase

The activity of the pyruvate dehydrogenase complex has long been determined in some laboratories by coupling the production of acetyl-coenzyme A (acetyl-CoA) to the acetylation of 4-aminoazobenzene-4'-sulfonic acid by arylamine N-acetyltransferase. The assay has some advantages, but its use has been limited by the need for large amounts of arylamine N-acetyltransferase. Here we report production of recombinant chicken liver arylamine N-acetyltransferase and optimization of its use in miniaturized assays for the pyruvate dehydrogenase complex and its kinase.     

Identification of a novel duplication CFTRdup2 and functional impact of large rearrangements identified in the CFTR gene

The aim of this study was to investigate associations of two candidate gene SNPs of the endocannabinoid receptor type 1 gene (CNR1) with overweight, obesity and obesity-related traits in Chinese retired women. The study subjects were a subsample of the Taizhou Retiree Women Cohort, consisting of 2812 retired women aged 50-64 years recruited from Taizhou, Jiangsu, China. Neither rs2023239 nor rs806381 polymorphism was significantly associated with body mass index-defined overweight and obesity or waist-to-hip-ratio-defined obesity. For obesity-related traits, rs2023239 was significantly associated with glutamate pyruvate transaminase (GPT) (median, 18.00 vs 17.00 for TT and TC genotypes, respectively, P=0.043). The rs806381 also showed significant association with triglyceride (TG) (mean±SD, 1.46±0.20 vs 1.53±0.20 for GA and GG+AA genotypes, respectively, P=0.013) under the dominant genetic model. In conclusion, the rs2023239 and rs806381 polymorphisms of CNR1 were not associated with increased overweight and obesity risk. But the rs2023239 polymorphism was significantly associated with GPT, and the rs806381 polymorphism was significantly associated with TG.     

Inactivation of Pyruvate Dehydrogenase Kinase 2 by Mitochondrial Reactive Oxygen Species

Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H₂O₂) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H₂O₂ derives from superoxide (O₂^˙̄), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O₂^˙̄ production, such as may occur under nutrient excess and low ATP demand, the increase in O₂^˙̄ and H₂O₂ may provide feedback signals to modulate mitochondrial metabolism.     

Plasma membrane potential oscillations in insulin secreting Ins-1 832/13 cells do not require glycolysis and are not initiated by fluctuations in mitochondrial bioenergetics

Oscillations in plasma membrane potential play a central role in glucose-induced insulin secretion from pancreatic β-cells and related insulinoma cell lines. We have employed a novel fluorescent plasma membrane potential (Δψ(p)) indicator in combination with indicators of cytoplasmic free Ca(2+) ([Ca(2+)](c)), mitochondrial membrane potential (Δψ(m)), matrix ATP concentration, and NAD(P)H fluorescence to investigate the role of mitochondria in the generation of plasma membrane potential oscillations in clonal INS-1 832/13 β-cells. Elevated glucose caused oscillations in plasma membrane potential and cytoplasmic free Ca(2+) concentration over the same concentration range required for insulin release, although considerable cell-to-cell heterogeneity was observed. Exogenous pyruvate was as effective as glucose in inducing oscillations, both in the presence and absence of 2.8 mM glucose. Increased glucose and pyruvate each produced a concentration-dependent mitochondrial hyperpolarization. The causal relationships between pairs of parameters (Δψ(p) and [Ca(2+)](c), Δψ(p) and NAD(P)H, matrix ATP and [Ca(2+)](c), and Δψ(m) and [Ca(2+)](c)) were investigated at single cell level. It is concluded that, in these β-cells, depolarizing oscillations in Δψ(p) are not initiated by mitochondrial bioenergetic changes. Instead, regardless of substrate, it appears that the mitochondria may simply be required to exceed a critical bioenergetic threshold to allow release of insulin. Once this threshold is exceeded, an autonomous Δψ(p) oscillatory mechanism is initiated.