Nonketotic Hyperglycinemia (NKH)
NKH is an autosomal recessive metabolic disease caused by a disruption of the glycine cleavage system. Mutations are typically found in the GLDC or AMT genes which code for proteins within the cleavage complex. Figure 3 from Swanson, et al. (2015) shows the wide variety of mutations identified in patients and families affected by NKH. Affected children carrying two different mutated alleles are not uncommon. Symptoms of the disease are related to the increased concentration of glycine in the central nervous system and include low muscle tone, seizures, apnea, lethargy, and coma. A significant number of affected children do not survive the neonatal period. Survivors have significant developmental delay and seizures which can be difficult to control.
Glycine metabolism
Glycine is the simplest of the amino acids and functions both as a building block for other amino acids and as a neurotransmitter in the brain. Glycine makes up 11.5% of the total amino acids in the human body, and it accounts for 20% of the total amino nitrogen in body proteins. 80% of the body's total glycine is typically required for protein synthesis in the developing human body (Razak, 2017). Glycine can be synthesized from carbon dioxide and ammonia and can be acquired in the diet, as shown in Figure 1 from Beyoglu and Idle’s 2012 paper. The pathways leading to glycine synthesis and to glycine degradation are both reversible. In contrast, the glycine deportation system is not reversible; once the glycine is bound to the benzoic acid carrier, it will be excreted through the kidneys.
Current Treatment for NKH
Treatment for NKH is primarily aimed at reducing the concentration of glycine in the blood and the cerebrospinal fluid. Administration of sodium benzoate takes advantage of the glycine deportation pathway shown in Figure 1 from Beyoglu and Idle’s 2012 paper at the lower right. The liver and the kidneys are the primary sites where glycine sequestration occurs. Benzoate is converted to benzoyl-CoA, and finally, combines with glycine to form hippuric acid which is then excreted into the urine.
Cinnamon to Sodium Benzoate: Treating Nonketotic Hyperglycinemia
Eating plain sodium benzoate irritates the digestive system. Interestingly, some compounds found in cinnamon can be metabolized to sodium benzoate by the liver. Thus, an adjunct treatment is to calculate the amount of cinnamon bark needed in mg cinnamon bark / kg of the patient’s weight. However, the amount of cinnamic acids in the cinnamon bark is not unified from area to area and between different barks, so there is no accurate dose for the medicine. The precise dosage is a critical problem that remains unsolved.
As stated in the paper "Metabolic engineering of Escherichia coli for the production of cinnamaldehyde," E. coli has the ability to carry out the pathway if modified to have the required genes that are responsible for the production of certain enzymes. We have plasmids containing the genes which code for the enzymes in the pathway and are in the process of expressing them in E. coli. Ideally, with the genetically modified E. coli, we will be able to produce cinnamic acids, cinnamoyl-CoA, and cinnamaldehyde. We are also looking into the chances of isolating the enzymes and testing them in cell-free conditions. This will allow us to produce each of the intermediates in the phenylalanine-to-cinnamaldehyde pathway. Ultimately, these products can be assessed for their ability to remove glycine from body fluids and reduce the symptoms of Nonketotic Hyperglycinemia.
Figure 2: Three enzymatic reactions (PAL, 4CL, and CCL) for the biosynthesis of cinnamaldehyde from l-phenylalanine (Bang et al., 2016).
References
Beyoğlu, D., & Idle, J. R. (2012). The glycine deportation system and its pharmacological consequences. Pharmacology & Therapeutics, 135(2), 151–167. https://doi.org/10.1016/j.pharmthera.2012.05.003
Bang, H. B., Lee, Y. H., Kim, S. C., Sung, C. K., & Jeong, K. J. (2016). Metabolic engineering of Escherichia coli for the production of cinnamaldehyde. Microbial Cell Factories, 15(1), 16. https://doi.org/10.1186/s12934-016-0415-9
Bjoraker, K. J., Swanson, M. A., Coughlin, C. R., Christodoulou, J., Tan, E. S., Fergeson, M., Dyack, S., Ahmad, A., Friederich, M. W., Spector, E. B., Creadon-Swindell, G., Hodge, M. A., Gaughan, S., Burns, C., & Van Hove, J. L. K. (2016). Neurodevelopmental outcome and treatment efficacy of benzoate and dextromethorphan in siblings with attenuated nonketotic hyperglycinemia. The Journal of Pediatrics, 170, 234–239. https://doi.org/10.1016/j.jpeds.2015.12.027
Hamosh, A., Maher, J. F., Bellus, G. A., Rasmussen, S. A., & Johnston, M. V. (1998). Long-term use of high-dose benzoate and dextromethorphan for the treatment of nonketotic hyperglycinemia. The Journal of Pediatrics, 132(4), 709–713. https://doi.org/10.1016/s0022-3476(98)70365-8
Ichikawa, K., Inami, Y., & Kaneko, K. (2020). Seventeen‐year long‐term survival of a case of neonatal nonketotic hyperglycinemia. Pediatrics International, 62(9), 1111–1113. https://doi.org/10.1111/ped.14254
Kukil, K., & Lindberg, P. (2022). Expression of phenylalanine ammonia lyases in synechocystis sp.. PCC 6803 and subsequent improvements of sustainable production of phenylpropanoids. Microbial Cell Factories, 21(1). https://doi.org/10.1186/s12934-021-01735-8
Mingoia, M., Conte, C., Di Rienzo, A., Dimmito, M. P., Marinucci, L., Magi, G., Turkez, H., Cufaro, M. C., Del Boccio, P., Di Stefano, A., & Cacciatore, I. (2022). Synthesis and biological evaluation of novel cinnamic acid-based antimicrobials. Pharmaceuticals, 15(2), 228. https://doi.org/10.3390/ph15020228
Okamura-Ikeda, K., Ohmura, Y., Fujiwara, K., & Motokawa, Y. (1993). Cloning and nucleotide sequence of the GCV operon encoding the escherichia coli glycine-cleavage system. European Journal of Biochemistry, 216(2), 539–548. https://doi.org/10.1111/j.1432-1033.1993.tb18172.x
Rangasamy, S. B., Raha, S., Dasarathy, S., & Pahan, K. (2021). Sodium benzoate, a metabolite of cinnamon and a food additive, improves cognitive functions in mice after controlled cortical impact injury. International Journal of Molecular Sciences, 23(1), 192. https://doi.org/10.3390/ijms23010192
Razak, M. A., Begum, P. S., Viswanath, B., & Rajagopal, S. (2017). Multifarious Beneficial Effect of Nonessential Amino Acid, Glycine: A Review. Oxidative medicine and cellular longevity, 2017, 1716701. https://doi.org/10.1155/2017/1716701
Riché, R., Liao, M., Pena, I. A., Leung, K.-Y., Lepage, N., Greene, N. D. E., Sarafoglou, K., Schimmenti, L. A., Drapeau, P., & Samarut, É. (2018). Glycine decarboxylase deficiency–induced motor dysfunction in zebrafish is rescued by counterbalancing glycine synaptic level. JCI Insight, 3(21). https://doi.org/10.1172/jci.insight.124642
Ruwizhi, N., & Aderibigbe, B. A. (2020). Cinnamic acid derivatives and their biological efficacy. International Journal of Molecular Sciences, 21(16), 5712. https://doi.org/10.3390/ijms21165712
Swanson, M. A., Coughlin, C. R., Scharer, G. H., Szerlong, H. J., Bjoraker, K. J., Spector, E. B., Creadon‐Swindell, G., Mahieu, V., Matthijs, G., Hennermann, J. B., Applegarth, D. A., Toone, J. R., Tong, S., Williams, K., & Van Hove, J. L. (2015). Biochemical and molecular predictors for prognosis in nonketotic hyperglycinemia. Annals of Neurology, 78(4), 606–618. https://doi.org/10.1002/ana.24485
Tian, S., Feng, J., Cao, Y., Shen, S., Cai, Y., Yang, D., Yan, R., Wang, L., Zhang, H., Zhong, X., & Gao, P. (2019). Glycine cleavage system determines the fate of pluripotent stem cells via the regulation of senescence and epigenetic modifications. Life Science Alliance, 2(5). https://doi.org/10.26508/lsa.201900413
Walczak-Nowicka, Ł. J., & Herbet, M. (2022). Sodium benzoate—harmfulness and potential use in therapies for disorders related to the nervous system: A Review. Nutrients, 14(7), 1497. https://doi.org/10.3390/nu14071497
Yilmaz, B., & Karabay, A. (2018). Food additive sodium benzoate (NAB) activates NFΚB and induces apoptosis in HCT116 cells. Molecules, 23(4), 723. https://doi.org/10.3390/molecules23040723