Theglyoxylate cycle, a variation of thetricarboxylic acid cycle, is ananabolic pathway occurring inplants,bacteria,protists, andfungi. Theglyoxylate cycle centers on the conversion ofacetyl-CoA tosuccinate for the synthesis ofcarbohydrates.[1] In microorganisms, the glyoxylate cycle allows cells to use two carbons (C2 compounds), such as acetate, to satisfy cellular carbon requirements when simple sugars such asglucose or fructose are not available.[2] The cycle is generally assumed to be absent in animals, with the exception ofnematodes at the early stages of embryogenesis. In recent years, however, the detection ofmalate synthase (MS) andisocitrate lyase (ICL), key enzymes involved in the glyoxylate cycle, in some animal tissue has raised questions regarding the evolutionary relationship of enzymes inbacteria andanimals and suggests that animals encode alternative enzymes of the cycle that differ in function from known MS and ICL in non-metazoan species.[1][3]
Plants as well as some algae and bacteria can use acetate as the carbon source for the production of carbon compounds. Plants and bacteria employ a modification of the TCA cycle called the glyoxylate cycle to produce four carbon dicarboxylic acid from two carbon acetate units. The glyoxylate cycle bypasses the two oxidative decarboxylation reactions of the TCA cycle and directly converts isocitrate through isocitrate lyase and malate synthase into malate and succinate.
The glyoxylate cycle was discovered in 1957 at theUniversity of Oxford bySir Hans Kornberg and his mentorHans Krebs, resulting in a Nature paperSynthesis of Cell Constituents from C2-Units by a Modified Tricarboxylic Acid Cycle.[4]
The glyoxylate cycle uses five of the eight enzymes associated with thetricarboxylic acid cycle:citrate synthase,aconitase,succinate dehydrogenase,fumarase, andmalate dehydrogenase. The two cycles differ in that in the glyoxylate cycle,isocitrate is converted intoglyoxylate andsuccinate by isocitrate lyase (ICL) instead of into α-ketoglutarate.[1] This bypasses the decarboxylation steps that take place in the citric acid cycle (TCA cycle), allowing simple carbon compounds to be used in the later synthesis of macromolecules, including glucose.[2]Glyoxylate is subsequently combined withacetyl-CoA to producemalate, catalyzed by malate synthase.[1] Malate is also formed in parallel from succinate by the action of succinate dehydrogenase and fumarase.
Fatty acids fromlipids are commonly used as an energy source by vertebrates as fatty acids are degraded throughbeta oxidation into acetate molecules. This acetate, bound to the activethiol group ofcoenzyme A, enters thecitric acid cycle (TCA cycle) where it is fullyoxidized tocarbon dioxide. This pathway thus allowscells to obtainenergy from fat. To use acetate from fat for biosynthesis of carbohydrates, the glyoxylate cycle, whose initial reactions are identical to the TCA cycle, is used.
Cell-wall containing organisms, such asplants,fungi, andbacteria, require very large amounts ofcarbohydrates duringgrowth for the biosynthesis of complex structuralpolysaccharides, such ascellulose,glucans, andchitin. In these organisms, in the absence of available carbohydrates (for example, in certain microbial environments or duringseed germination in plants), the glyoxylate cycle permits the synthesis of glucose from lipids via acetate generated in fatty acid β-oxidation.
The glyoxylate cycle bypasses the steps in the citric acid cycle where carbon is lost in the form of CO2. The two initial steps of the glyoxylate cycle are identical to those in the citric acid cycle:acetate → citrate → isocitrate. In the next step, catalyzed by the first glyoxylate cycle enzyme,isocitrate lyase, isocitrate undergoes cleavage intosuccinate andglyoxylate (the latter gives the cycle its name). Glyoxylate condenses with acetyl-CoA (a step catalyzed bymalate synthase), yieldingmalate. Bothmalate andoxaloacetate can be converted intophosphoenolpyruvate, which is the product ofphosphoenolpyruvate carboxykinase, the first enzyme ingluconeogenesis. The net result of the glyoxylate cycle is therefore the production of glucose from fatty acids. Succinate generated in the first step can enter into the citric acid cycle to eventually form oxaloacetate.[2]
In plants theglyoxylate cycle occurs in specialperoxisomes which are calledglyoxysomes. This cycle allows seeds to use lipids as a source of energy to form the shoot duringgermination. The seed cannot produce biomass using photosynthesis because of lack of an organ to perform this function. The lipid stores of germinating seeds are used for the formation of the carbohydrates that fuel the growth and development of the organism.
The glyoxylate cycle can also provide plants with another aspect of metabolic diversity. This cycle allows plants to take inacetate both as a carbon source and as a source of energy. Acetate is converted to acetyl CoA (similar to the TCA cycle). This acetyl CoA can proceed through the glyoxylate cycle, and some succinate is released during the cycle. The four carbon succinate molecule can be transformed into a variety of carbohydrates through combinations of other metabolic processes; the plant can synthesize molecules using acetate as a source for carbon. The acetyl CoA can also react with glyoxylate to produce some NADPH from NADP+, which is used to drive energy synthesis in the form of ATP later in theelectron transport chain.[5]
The glyoxylate cycle may serve an entirely different purpose in some species of pathogenicfungi. The levels of the main enzymes of the glyoxylate cycle, ICL and MS, are greatly increased upon contact with a human host. Mutants of a particular species of fungi that lacked ICL were also significantly lessvirulent in studies with mice compared to the wild type. The exact link between these two observations is still being explored, but it can be concluded that the glyoxylate cycle is a significant factor in thepathogenesis of thesemicrobes.[6][7]
Vertebrates were once thought to be unable to perform this cycle because there was no evidence of its two keyenzymes, isocitrate lyase and malate synthase. However, some research suggests that this pathway may exist in some, if not all, vertebrates.[8][9] Specifically, some studies show evidence of components of the glyoxylate cycle existing in significant amounts in the liver tissue of chickens. Data such as these support the idea that the cycle could theoretically occur in even the most complex vertebrates.[10] Other experiments have also provided evidence that the cycle is present among certain insect and marine invertebrate species, as well as strong evidence of the cycle's presence in nematode species. However, other experiments refute this claim.[11] Some publications conflict on the presence of the cycle inmammals: for example, one paper has stated that the glyoxylate cycle is active in hibernating bears,[12] but this report was disputed in a later paper.[13] Evidence exists for malate synthase activity in humans due to a dual functional malate/B-methylmalate synthase of mitochondrial origin called CLYBL expressed in brown fat and kidney.[14] Vitamin D may regulate this pathway in vertebrates.[10][15]
Due to the central role of theglyoxylate cycle in the metabolism of pathogenic species including fungi and bacteria, enzymes of the glyoxylate cycle are current inhibition targets for the treatment of diseases. Most reported inhibitors of the glyoxylate cycle target the first enzyme of the cycle (ICL). Inhibitors were reported forCandida albicans for potential use as antifungal agents.[16] The mycobacterial glyoxylate cycle is also being targeted for potential treatments oftuberculosis.[17][18]
The prospect of engineering variousmetabolic pathways intomammals which do not possess them is a topic of great interest for bio-engineers today. The glyoxylate cycle is one of the pathways which engineers have attempted to manipulate into mammalian cells. This is primarily of interest for engineers in order to increase the production of wool in sheep, which is limited by the access to stores of glucose. By introducing the pathway into sheep, the large stores of acetate in cells could be used in order to synthesizeglucose through the cycle, allowing for increased production of wool.[19]Mammals are incapable of executing the pathway due to the lack of two enzymes,isocitrate lyase andmalate synthase, which are needed in order for the cycle to take place. It is believed by some that the genes to produce these enzymes, however, arepseudogenic in mammals, meaning that the gene is not necessarily absent, rather, it is merely "turned off".[1]
In order to engineer the pathway into cells, the genes responsible for coding for the enzymes had to be isolated and sequenced, which was done using the bacteriaE.coli, from which the AceA gene, responsible for encoding forisocitrate lyase, and the AceB gene, responsible for encoding formalate synthase were sequenced.[19] Engineers have been able to successfully incorporate the AceA and AceB genes into mammalian cells in culture, and the cells were successful in translating and transcribing the genes into the appropriate enzymes, proving that the genes could successfully be incorporated into the cell’s DNA without damaging the functionality or health of the cell. However, being able to engineer the pathway into transgenic mice has proven to be difficult for engineers. While the DNA has been expressed in some tissues, including the liver and small intestine in test animals, the level of expression is not high, and not found to be statistically significant. In order to successfully engineer the pathway, engineers would have to fuse the gene with promoters which could be regulated in order to increase the level of expression, and have the expression in the right cells, such asepithelial cells.[20]
Efforts to engineer the pathway into more complex animals, such as sheep, have not been effective. This illustrates that much more research needs to be done on the topic, and suggests it is possible that a high expression of the cycle in animals would not be tolerated by the chemistry of the cell. Incorporating the cycle into mammals will benefit from advances innuclear transfer technology, which will enable engineers to examine and access the pathway for functional integration within the genome before its transfer to animals.[19]
There are possible benefits, however, to the cycle's absence in mammalian cells. The cycle is present inmicroorganisms that cause disease but is absent in mammals, for example humans. There is a strongplausibility of the development of antibiotics that would attack theglyoxylate cycle, which would kill the disease-causing microorganisms that depend on the cycle for their survival, yet would not harm humans where the cycle, and thus the enzymes that the antibiotic would target, are absent.[2]
Metabolism map | ||
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 Majormetabolic pathways inmetro-style map. Click any text (name of pathway or metabolites) to link to the corresponding article. Single lines: pathways common to most lifeforms. Double lines: pathways not in humans (occurs in e.g. plants, fungi, prokaryotes).  Orange nodes:carbohydrate metabolism. Violet nodes:photosynthesis. Red nodes:cellular respiration. Pink nodes:cell signaling. Blue nodes:amino acid metabolism. Grey nodes:vitamin andcofactor metabolism. Brown nodes:nucleotide andprotein metabolism. Green nodes:lipid metabolism. | ||
