The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms

Pyruvate is a keystone molecule critical for numerous aspects of eukaryotic and human metabolism. Pyruvate is the end-product of glycolysis, is derived from additional sources in the cellular cytoplasm, and is ultimately destined for transport into mitochondria as a master fuel input undergirding citric acid cycle carbon flux. In mitochondria, pyruvate drives ATP production by oxidative phosphorylation and multiple biosynthetic pathways intersecting the citric acid cycle. Mitochondrial pyruvate metabolism is regulated by many enzymes, including the recently discovered mitochondria pyruvate carrier, pyruvate dehydrogenase, and pyruvate carboxylase, to modulate overall pyruvate carbon flux. Mutations in any of the genes encoding for proteins regulating pyruvate metabolism may lead to disease. Numerous cases have been described. Aberrant pyruvate metabolism plays an especially prominent role in cancer, heart failure, and neurodegeneration. Because most major diseases involve aberrant metabolism, understanding and exploiting pyruvate carbon flux may yield novel treatments that enhance human health.

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Regulation of pyruvate metabolism and human disease.

In Saccharomyces cerevisiae, reduction of NAD+ to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD+. At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial ‘internal’ NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol–acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate–oxaloacetate shuttle, a malate–aspartate shuttle and a malate–pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate–citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.

Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

Pyruvate carboxylase (PC; EC 6.4.1.1), a member of the biotin-dependent enzyme family, catalyses the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC has been found in a wide variety of prokaryotes and eukaryotes. In mammals, PC plays a crucial role in gluconeogenesis and lipogenesis, in the biosynthesis of neurotransmitter substances, and in glucose-induced insulin secretion by pancreatic islets. The reaction catalysed by PC and the physical properties of the enzyme have been studied extensively. Although no high-resolution three-dimensional structure has yet been determined by X-ray crystallography, structural studies of PC have been conducted by electron microscopy, by limited proteolysis, and by cloning and sequencing of genes and cDNA encoding the enzyme. Most well characterized forms of active PC consist of four identical subunits arranged in a tetrahedron-like structure. Each subunit contains three functional domains: the biotin carboxylation domain, the transcarboxylation domain and the biotin carboxyl carrier domain. Different physiological conditions, including diabetes, hyperthyroidism, genetic obesity and postnatal development, increase the level of PC expression through transcriptional and translational mechanisms, whereas insulin inhibits PC expression. Glucocorticoids, glucagon and catecholamines cause an increase in PC activity or in the rate of pyruvate carboxylation in the short term. Molecular defects of PC in humans have recently been associated with four point mutations within the structural region of the PC gene, namely Val145-->Ala, Arg451-->Cys, Ala610-->Thr and Met743-->Thr.

/pdf/structure-function-and-regulation-of-pyruvate-carboxylase-2p3v3g1k4u.pdf

Structure, function and regulation of pyruvate carboxylase

The contents of citric acid cycle compounds were measured in rat hind limb muscle in situ during rest, exercise, and recovery from exercise. The following changes in citric acid cycle intermediates were observed during exercise for 15 min. The contents of fumarate and malate increased fourfold over resting values. The contents of citrate, isocitrate, and succinate rose 68%, 48% and 87% respectively, but the increase in these intermediates was delayed relative to the increase in fumarate and malate. The content of 2-oxoglutarate did not change significantly. The content of oxaloacetate increased from less than 1 nmol/g dry weight in resting muscle to 8 nmol/g dry weight during exercise. The orthophosphate content doubled. The sum of the contents of citric acid cycle intermediates doubled. The ratios of the contents of malate/fumarate and citrate/isocitrate remained constant. The constancy of the citrate/isocitrate ratio indicates that the concentration of intra-cellular free Mg2+ is not affected by exercise. All metabolite levels returned to resting values during recovery. Comparison of these data with the rate of operation of the purine nucleotide cycle leads us to conclude that the increase in citric acid cycle intermediates can be accounted for by the operation of the purine nucleotide cycle.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1980.tb04877.x

The Purine‐Nucleotide Cycle

The overall activity and distribution of pyruvate carboxylase and of phosphoenolpyruvate carboxykinase in rat liver was studied by applying an improved technique for the fractional extraction of hepatic enzymes. In the rat the mean activity of pyruvate carboxylase amounted to 10 units/g fresh liver (1 unit = 1 μmole pyruvate carboxylated/min at 30°). In the human liver the corresponding value was about 1 unit/g fresh wt. For phosphoenolpyruvate carboxy-kinase 2.5 and 10 units/g fresh wt. were measured in rat and human liver, respectively. During fractional extraction only negligible quantities of pyruvate carboxylase appeared in the soluble cytoplasmatic fraction. It is concluded that the bulk of enzyme activity is located within the mitochondria. By digitonin treatment of isolated rat liver mitochondria pyruvate carboxylase could be localized within the matrix space. Phosphoenolpyruvate carboxykinase was found to be located mainly in the soluble cytoplasmatic fraction. Our results strongly support the view that in rat liver gluconeogenesis pyruvate has to enter the mitochondria prior to its carboxy-lation to oxaloacetate.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1969.tb00503.x

I. Böttger

Papers

Intracellular Localization of Pyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase in Rat Liver