Glyoxylate cycle

Overview of the Glyoxylate Cycle

The glyoxylate cycle, a variation of the Tricarboxylic Acid Cycle, is an anabolic metabolic pathway occurring in plants, bacteria, protists, fungi and several microorganisms, such as E. coli and yeast. The glyoxylate cycle centers on the conversion of acetyl-CoA to succinate for the synthesis of carbohydrates. In microorganisms, the glyoxylate cycle allows cells to utilize simple carbon compounds as a carbon source, when complex complex sources such as glucose are not available. The cycle is generally assumed to be absent in animals, with the exception of nematodes at the early stages of embryogenesis. In recent years, however, the detection of malate synthase (MS) and isocitrate lyase (ICL), key enzymes involved in the gyloxylate cycle, in some animal tissue has raised questions regarding the evolutionary relationship of enzymes in bacteria and animals and suggests that animals encode alternative enzymes of the cycle that differ in function from known MS and ICL in non-metazoan species.

Similarities with TCA Cycle

The glyoxylate cycle utilizes three of the five enzymes associated with the tricarboxylic acid cycle and shares many of its intermediate steps. The two cycles vary when, in the gylcoxylate cycle, isocitrate lyase (ICL) converts isocitrate into glyoxylate and succinate instead of α-ketogluterate as seen in the TCA cycle. This bypasses decarboxylation steps that take place in the TCA cycle, allowing simple carbon compounds to be used in the later synthesis of macromolecules, including glucose. The glyoxylate cycle then continues on, using glyoxylate and acetyl-CoA to produce malate.

Role in gluconeogenesis

Fatty acids from lipids are commonly used as an energy source by vertebrates via degradation by beta oxidation into acetate molecules. This acetate, bound to the active thiol group of coenzyme A, enters the citric acid cycle (TCA cycle) where it is fully oxidized to carbon dioxide. This pathway thus allows cells to obtain energy from fat. To utilize 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 as plants, fungi, and bacteria, require very large amounts of carbohydrates during growth for the biosynthesis of complex structural polysaccharides, such as cellulose, glucans, and chitin. In these organisms, in the absence of available carbohydrates (for example, in certain microbial environments or during seed 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 into succinate and glyoxylate (the latter gives the cycle its name). Glyoxylate condenses with acetyl-CoA (a step catalyzed by malate synthase), yielding malate. Both malate and oxaloacetate can be converted into phosphoenolpyruvate, which is the substrate of phosphoenolpyruvate carboxykinase, the first enzyme in gluconeogenesis. 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.

In plants the glyoxylate cycle occurs in special peroxisomes which are called glyoxysomes. Vertebrates were once thought to be unable to perform this cycle because there was no evidence for its two key enzymes, isocitrate lyase and malate synthase. However some research has suggested that this pathway exists for at least some if not all vertebrates. Some publications conflict in this area: for example one paper has stated that the glyoxalate cycle is active in hibernating bears, but this report was disputed in a later paper. On the other hand, no functional genes related to known forms of malate synthase or isocitrate lyase have been identified in placental mammal genomes, while malate synthase appears to be functional in some non-placental mammals and other vertebrates. Vitamin D may regulate this pathway in vertebrates.

Engineering Concepts

The prospect of engineering various metabolic pathways into mammals for which they are not native is of a topic of great interest for bioengineers today. The glyoxylate cycle is one of these pathways which engineers have attempted to engineer 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 synthesize glucose through the cycle, allowing for increased production of wool. Mammals are incapable of executing the pathway due to the lack of two enzymes, isocitrate lyase, and malate synthase, which are needed in order for the cycle to take place.

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 bacteria E.Coli, from which the AceA gene, responsible for encoding for isocitrate lyase, and the AceB gene, responsible for encoding for malate synthase were sequenced. Engineers have been able to successfully incorporate the gene into mammalian cells in culture, and the cells were successful in being able to translate and transcribe 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, as while the DNA has been expressed in some tissues, including the liver, and small intestine, the level of expression is not high, and found to not 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 as epithelial cells.

Efforts to engineer the pathway into more complex animals, such as sheep have not been effective, showing that much more research would be needed in order to incorporate the pathway into more complex mammals, and it is possible that a high expression of the cycle in animals would not be tolerated by the chemistry of the cell. Efforts to engineer the cycle into mammals will benefit from advances in nuclear transfer technology, which will enable engineers to examine and access the pathway for functional integration within the genome before its transfer to animals.

External links

• Comparative Analysis of Glyoxylate Cycle Key Enzyme Isocitrate Lyase from Organisms of Different Systematic Groups