In our project, the tryptophan hydroxylases (TPH) from human, mice, zebrafish, and rabbits have been selected and analyzed for their catalytic efficiency in the rate-limiting step of producing serotonin.
Therefore, the CDs from four types of TPH have been codon optimized and synthesized by our sponsor- ATANTARES biotech, and constructed onto the Ecoli expressing vector pET-28a using Gibson assembly.
The positive colony after colony PCR selection has been sent out for Sanger sequencing to avoid any potential mutation.
Purification of TPH protein
The expressing vector was then transformed into Ecoli strain BL21(DE3). After that, serious of experiments has been performed to adjust the optimum expressing condition. (Detailed protocols can be downloaded at Experiments.) The optimum expression condition in a 500ml flask is shown below.
After that, the His-tag affinity purification technique was used to purify the TPH protein from Ecoli culture.
We ran an SDS-PAGE gel for the final product of our concentrated purification product. Four bands were clearly observed on the gel. Showing that the protein has been successfully expressed in Ecoli cells.
In silico and in vivo selection of most active TPH
To better understand the TPH activity in the intestine, we decided to use a hybrid of in silico and in vivo experiments to investigate the TPH activity. The tryptophan hydroxylases (TPH) from human, mice, zebrafish, and rabbits have been selected and analyzed for their catalytic efficiency in the rate-limiting step of producing serotonin. In accordance with the kinetics of enzymatic reactions modeled by Michaelis-Menten equations, the rate of a substrate-enzyme catalysis is governed by the relative affinity of this enzyme to its substrate. Hence, by predicting the binding affinity of the TPHs to the tryptophan molecule,it would allow us to predict the catalytic effectiveness of the enzyme selected.
In silico binding affinity prediction
The full-length 3D structures of the four TPHs from human, mouse, zebrafish, and rabbit are acquired as the PDB files from the uniport database under the following entry numbers: P17752; P09810; Q6PBX4; P17290 for each host organism respectively. For entry numbers P09810, Q6PBX4, and P17290, the known x-ray/NMR structures of the proteins are missing. Instead, the Alphafold predicted full-length structure of the proteins was selected for further analysis.
The catalytic centers of each TPH molecule were identified by comparing to the TPH-tryptophan binding complex characterized by Windahl, Petersen et. al. ([2]). In which the 5’ carboxyl group of the tryptophan molecule is under interaction with a Ser residual deprotonated by an adjacent His residual. Similar structures were also identified in the three TPH selected in this project.
Molecular docking is performed with the TPHs set as the receptor and the tryptophan as the ligand. The polar hydrogen was added to the molecule and the overall Gasteiger energy of the structure has been calculated using the AutoDockTools software ([3]). The 40×40×40Å docking grid box is then drawn around the predicted active site. The docking is performed by the Autodock4 Genetic Algorithm with the following parameters: 10 random GA runs; population size of 150; 150000 for maximum evals; 17000 for maximum number of generations.
The TPH-Trp complexes generated as the autodock .pdbqt format is then ranked by the overall energy score form the most negative (the strongest binding) to the least negative value. The structure is then converted into .pdb format via VEGA ZZ software and the binding is of Trp with TPH at the active site is visualized with the PyMol program.
The docking results of different TPH demonstrate a varied binding affinity to the Trp substrate. In which the zebrafish TPH-Trp binding complex has a most negative binding energy of -8.03 kcal/mol among the three TPH from different hosts. The rabbit TPH on the other hand, has a lowest binding affinity to the substrate (-6.54kcl/mol), and it was further found that the protonatable Ser residual in the rabbit TPH active site, is not fully docked with the Trp substrate, that may result in the lowering of the binding affinity.
Result of in silico prediction
In all, although all TPH enzymes have a varied range of binding affinity (energy) to the tryptophan substrate, our docking simulations data as demonstrated strong evidence for the potential of binding and catalysis of each TPH to the tryptophan. In which the zebrafish TPH would hypothetically mediate the highest efficiency of tryptophan hydrolysis among the four selected TPH. Our wet lab experiments and results would, furthermore, help us identify the most potent TPH enzyme to be produced in E. coli expression and used for the final product.
In vitro enzyme activity testing
To measure the HTP activity, we followed the protocol in part BBa_K1598002 (http://parts.igem.org/Part:BBa_K1598002) by team UCL in 2015.
In their protocol, a continuous fluorometric assay for HTP activity based on the different spectral characteristics of tryptophan and 5hydroxytryptophan is presented. Hydroxylation of tryptophan at the 5position results in a large increase in the fluorescence of the molecule. The assay selectively monitors the fluorescence yield of 5hydroxytryptophan by exciting the reaction mix at 300 nm. The rate of increase of the emission signal was found to be directly proportional to the enzyme concentration. Inner filter effects due to quinonoid dihydropterin accumulation were eliminated by the inclusion of a thiol reductant. Activity measured using this assay method was found to be the same as that determined by established discontinuous HPLC assay methods.
The standard reaction conditions are: 60 uM tryptophan, 300 uM 6methyltetrahydropterin, 200 mM ammonium sulfate, 7 mM DTT, 25 ug/ml catalase, 25 uM ferrous ammonium sulfate, 50 mM Mes, pH 7.0, with atmospheric oxygen, 0400 nM (=0102 ug) of TPH1.
Based on the protocol, we have successfully obtained data for the HTP activity.
Final conclusion
Therefore, our wet lab experiments and results fit our in silico prediction of HTP activity and show that the zebrafish TPH has the highest efficiency of tryptophan hydrolysis among the four selected TPHs. Therefore, we will choose this strain of HTP in our final product.
Integrated the arabinose induced self lysis system
Moreover, to ensure the safety and containment of this synthetic biology approach, an arabinose-inducible suicide system has been integrated. This system serves as a fail-safe mechanism to prevent unintended biological contamination or release of genetically modified organisms into the environment. This is because rabinose was considered as a safe food ingredient. Therefore, we choose it to be our inducer of self-lysis system. The patient can take a pill of arabinose to start the system and eliminate the engineered bacteria in their intestine. The part (BBa_K112000) was registered in 2020 and we successfully repeat it’s function.
Reference
[1] Kuhn, D.M. and Hasegawa, H., 2020. Tryptophan hydroxylase and serotonin synthesis regulation. In Handbook of Behavioral Neuroscience (Vol. 31, pp. 239-256). Elsevier.
[2] Windahl, M.S., Petersen, C.R., Christensen, H.E. and Harris, P., 2008. Crystal structure of tryptophan hydroxylase with bound amino acid substrate. Biochemistry, 47(46), pp.12087-12094.
[3] Morris, G.M., Huey, R., Lindstrom, W., Sanner, M.F., Belew, R.K., Goodsell, D.S. and Olson, A.J., 2009. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. Journal of computational chemistry, 30(16), pp.2785-2791.
