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Tryptophan hydroxylase Introduction Tryptophan hydroxylase (TPH) is an enzyme belonging to the aromatic amino acid hydroxylase family. The enzymes belonging to this family are pterin dependent hydroxylases; pterin is a chemical compound which is composed of a pyrazine ring along with a pyrimidine ring. The aromatic amino acid hydroxylases catalyze the hydroxylation of aromatic side chains. Hydroxylation refers to the addition of -OH groups and these hydroxylases specifically add -OHs to the aromatic amino acids. There are a total of three aromatic amino acid hydroxylases each with a specific substrate: phenylalanine hydroxylase, tyrosine hydroxylase and tryptophan hydroxylase. Phenylalanine hydroxylase catalyzes the hydroxylation of phenylalanine, tyrosine hydroxylase catalyzes the addition of -OH onto tyrosine amino acid and tryptophan hydroxylase catalyzes the hydroxylation of tryptophan. TPH is known to catalyze the first step, which is also the rate limiting step in the formation of serotonin. Serotonin is an important hormone, and a neurotransmitter. Since TPH is responsible for the formation of serotonin, mutation(s) in the tryptophan hydroxylase enzyme coding gene has led to an increase in various psychoneurological disorders such as: schizophrenia, somatic anxiety, anger-related traits, bipolar disorder, suicidal behavior, and addictions among other traits (Jun et al., 2012).

Structure/Synthesis/Function Serotonin is first synthesized from the essential amino acid l-tryptophan. 1-tryptophan is now converted to 5-hydroxytryptophan by the enzyme tryptophan hydroxylase. 5-Hydroxytryptophan is then converted to serotonin by the enzyme aromatic amino acid (l-dihydroxyphenylalanine; l-DOPA) decarboxylase. Tryptophan is also absorbed into the bloodstream and circulated throughout the body in the pineal gland, it is hydroxylated by Tryptophan-5-Hydroxylase to 5-hydroxy-Trp, then decarboxylated by Aromatic Amino Acid Decarboxylase in the pineal cytosol. Then serotonin can be used to synthesis melatonin. (Mockus et al., 1998)

Enzymatic Mechanism In the body, tryptophan hydroxylase acts as the limiting rate step in the two step reaction of 5-hydroxytryptamine (Serotonin). First, BH4 (tetrahydrobiopterin an enzymatic cofactor), oxygen, and ferrous iron are used together to add a hydroxyl group to tryptophan. Next, a carboxyl group is removed by decarboxylase (Carkaci-Salli et al., 2006). These reactions produce serotonin. This enzyme has two isoforms: TPH1 and TPH2.

Molecular Characteristics of Tryptophan hydroxylase 1 and 2 These isoforms are encoded by two different TPH genes.Two studies conducted by Walter and colleagues indicated that numerous species share these isomers; human genes sharing 71% homology and rest on chromosomes 11 and 12 (Walther and Bader 2003; Walther et al., 2003 and McKinney et al., 2004). TPH1 enzyme is composed of 444 amino acids and these amino acids are highly preserved for both, TPH1 and TPH2 isoforms. TPH2, however, has a total of 485 amino acids, 41 additional amino acids at the N-terminal sequence when compared to TPH1 (Hasegawa et al., 2010). TPH1 has a molecular weight of 51 kDa and TPH2 has a molecular weight of 56 kDa weighing slightly more than the TPH1 isoform (Sakowski et al., 2006). Sakowski et al. tested mice to differentiate between the two isoforms. Their studies concluded that there is no overlapping between the two isoforms and TPH1 is the predominant form expressed in the pineal gland and in the mastocytoma cells. TPH2, on the other hand, is the predominant form expressed in brain cells in the following regions: mesencephalic tegmetum, stiatum and hippocampus. Therefore, TPH1 enzyme functions primarily in the pineal gland and in the gut, whereas, TPH2 enzyme is expressed mainly in the brain. Both of the enzymes are composed of three major domains consisting of: a C-terminal tetramerization sequence, central catalytic body for performing the hydroxylation catalysis and an N-terminal regulatory peptide. In addition to this, both enzymes are soluble and behave like a tetramer. Even though both enzymes are soluble TPH2 is slightly more soluble than TPH1 and is known to have different kinetic properties (McKinney et al., 2005). Both the isoforms are regulated by cAMP-dependent protein kinase A enzymes through phosphorylation (Kuhn et al., 2007). However, TPH2 has an additional phosphorylation site at serine at the 19th position (Ser19) which is not present in TPH1. Both the enzymes achieve the same function but multiple differences exist between TPH1 and TPH2 and these differences have very important implications for serotonin production and regulations in the brain and periphery. Although TPH1 and TPH2 function in different areas of the body and have different levels of activation, both forms have the same enzymatic mechanism. Having two types of isoforms gives additional control over serotonin regulation. In a comparative study presented by McKinney and associates, the group tested the physiological benefits of having 2 regulatory genes for the production of serotonin expressed in Escherichia coli. They found that both human enzymes have a tendency to clump together, mainly in the absence of a fusion partner: a tool typically used to increase protein expression and solubility in E.coli (Carter et al., 2009). Human TPH1 is less stable and will aggregate more readily than TPH2. The team also found that human TPH2 has a much larger N-terminal domain and has a lower affinity of Tryptophan, this was thought to be the cause of a more selective active cite cleft, resulting in decreased catalytic activity. The availability of Trp in living organisms (mainly mammals) has a direct effect on production rates of serotonin in the body. This limited availability could also be a reason for low affinity for Trp (McKinney et al., 2004). The decreased production of neural and peripheral serotonin has been linked to multiple biological abnormalities.

Abnormalities Resulting from TPH Malfunction Decarboxylase, the amino acid involved in the synthesis of serotonin(5-HT), is also the enzyme that converts L-DOPA to dopamine. Seasonal affective disorder (SAD) is a form of depression that is related to changes in seasons. Depression is a hereditary mental disorder caused by decreased concentrations of serotonin with a high mortality rate. SAD begins and ends at about the same times every year. Less often, SAD causes depression during fall/winter. Numerous pieces of evidence indicate the association of SAD with decreased brain neurotransmitter serotonin 5-HT(Van Praag, 2004); system functioning. Tryptophan is an essential amino acid, and the depletion could affect general protein synthesis which could possibly lead to depression. The lack of 5-HT in at least some depressed individuals, perhaps most convincingly when suicidality or aggression is present. 5-HT deficiency could possibly arise from multiple different defects in one or more of the various components of the 5-HT system. The multiple distinct mutations in TPH2 may be a representation of such a concept.

TPH2 has been strongly associated with numerous mental disorders. A recent study conducted by Chen et al., observed the effects of stress on TPH 1&2 gene expression and serotonin levels in a rat depression model; to determine if depression is caused by dysfunctions of TPH. Results from their study supported a correlation between TPH gene dysfunction and depression. In untreated depressed rats affected by stress, serotonin levels and enzyme expression displayed regression in collected brain tissue. Inhibition of TPH2 expressed in the brain and TPH1 in the liver and kidneys were associated with increased methylation of the promoter sequences (2017).

Conclusion: Tryptophan hydroxylase is one of many essential enzymes present in mammals. It has two isomers that utilizes the same enzymatic mechanism in the production of serotonin. TPH1 is predominantly a peripheral enzyme while TPH2 functions in the central nervous system. The dysfunction of this enzyme has been linked to numerous neuropsychological and physiological disorders. Continued research is needed to gain a better understanding of its effects and possible biochemical capabilities.

Resources Carkaci-Salli, N., Flanagan, J. M., Martz, M. K., Salli, U., Walther, D. J., Bader, M., & Vrana, K. E. (2006). Functional Domains of Human Tryptophan Hydroxylase 2 (hTPH2). Journal of Biological Chemistry, 281(38), 28105-28112. doi:10.1074/jbc.m602817200 Chen, Y., Xu, H., Zhu, M., Liu, K., Lin, B., Luo, R.,. . . Li, M. (2017). Stress inhibits tryptophan hydroxylase expression in a rat model of depression. Oncotarget, 8(38). doi:10.18632/oncotarget.18780 Jacobsen, J. P., Medvedev, I. O., & Caron, M. G. (2012). The 5-HT deficiency theory of depression: perspectives from a naturalistic 5-HT deficiency model, the tryptophan hydroxylase 2Arg439His knockin mouse. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 367(1601), 2444-59. Jane Carter, Jue Zhang, Thien-Lan Dang, Haruki Hasegawa, Janet D Cheng, Irene Gianan, Jason W O'Neill, Martin Wolfson, Sophia Siu, Sheldon Qu, David Meininger, Helen Kim, John Delaney, Christopher Mehlin.Fusion partners can increase the expression of recombinant interleukins via transient transfection in 2936E cells. Protein Sci. 2010 Feb; 19(2): 357–362. Published online 2009 Dec 15. doi: 10.1002/pro.307 McKinney, Jeffrey, et al. “Different Properties of the Central and Peripheral Forms of Human Tryptophan Hydroxylase.” Journal of Neurochemistry, Wiley/Blackwell (10.1111), 29 Nov. 2004, onlinelibrary.wiley.com/doi/epdf/10.1111/j.1471-4159.2004.02850.x. https://www.ncbi.nlm.nih.gov/gene?Db=gene&Cmd=DetailsSearch&Term=7166#phenotypes Sakowski, S. A., Kuhn, D. M., Geddes, Thomas, D. M., Levi, E., & Hatfield, J. S. (2006). Differential tissue distribution of tryptophan hydroxylase isoforms 1 and 2 as revealed with monospecific antibodies. Brain Research, 1085(1):11-8. doi:10.1016/j.brainres.2006.02.047 https://www.sciencedirect.com/topics/neuroscience/tryptophan-hydroxylase Kulikov, A. V. & Popova N. K. (2015). Tryptophan hydroxylase 2 in seasonal affective disorder: underestimated perspectives? Reviews in the Neurosciences. 26(6):679-90. doi: 10.1515/revneuro-2015-0013. http://www.jneurosci.org/content/jneuro/26/2/530.full.pdf https://www.ncbi.nlm.nih.gov/pubmed/10800597 (Fitzpatrick, 2000) Fitzpatrick, P. F. (2003). Mechanism of Aromatic Amino Acid Hydroxylation. Biochemistry, 42 (48), pp 14083–14091. doi: 10.1021/bi035656u http://www.hmdb.ca/metabolites/HMDB0000802 Kuhn, D. M., Sakowski, S. A., Geddes, T. J., Wilkerson, C., & Haycock, J. W. (2007). Phosphorylation and activation of tryptophan hydroxylase 2: identification of serine-19 as the substrate site for calcium, calmodulin-dependent protein kinase II. Journal of Neurochemistry, 103(4):1567-73. Walther, D. J., & Bader, M. (2003). A unique central tryptophan hydroxylase isoform. Biochemical Pharmacology, 66(9), 1673-1680. doi:10.1016/s0006-2952(03)00556-2 Walther, D. J. (2003). Synthesis of Serotonin by a Second Tryptophan Hydroxylase Isoform. Science,299(5603), 76-76. doi:10.1126/science.1078197 Jun, S. E., Kohen, R., Cain, K. C., Jarrett, M. E., & Heitkemper, M. M. (2012). TPH gene polymorphisms are associated with disease perception and quality of life in women with irritable bowel syndrome. Biological research for nursing, 16(1), 95-104. Mockus, S., & Vrana, K. (2007). Advances in the molecular characterization of              tryptophan hydroxylase. Journal of Molecular Neuroscience, 10(3), 163-179. Kulikov, A., & Popova, N. (2015). Tryptophan hydroxylase 2 in seasonal affective disorder: underestimated perspectives?. Reviews in the Neurosciences, 26(6),