Part:BBa_K4140009
Phenylalanine hydroxylase (PAH)
Part Description
The phenylalanine hydroxylase (PAH) gene is the part responsible for providing instructions to make phenylalanine hydroxylase enzyme. this enzyme is responsible for hydroxylating phenylalanine and turning it into tyrosine in the presence of tetrahydrobiopterin (BH4). and as phenylalanine is present in almost all proteins and some artificial sweeteners this process of hydroxylation is really important to prevent its accumulation.
also, the product of tyrosine is used to make various hormones and neurotransmitters.
Usage
PAH is a Member of the hydroxylase enzymes in the human body and it processes phenylalanine to be hydroxylated in order to produce tyrosine. PAH is a hydroxylase that needs biopterin that converts phenylalanine to tyrosine and in case of its absence or deficiency leads to a metabolic condition called Phenylketonuria that can occur in humans due to abnormalities in the gene that codes for it so we use this part to replace the deficient one and process phenylalanine into tyrosine. We used it in our therapeutic E.coli-based system to replace the deficient enzyme in these patients with an auto-regulatory switch. PAH has been improved by our team members to be tagged with KP-sp peptides to be exported extracellularly shown in figure 1.
Characterization of Mutational Landscape
After creating a multiple sequence alignment of the protein sequence and predicting mutational landscapes, the effect of these mutations on the evolutionary fitness of the protein is tested. The prediction of the mutational landscape by saturation mutagenesis of the PAH protein. The (G183E) mutation, as depicted in the chart, had the greatest score when compared to other mutations. On the other hand, it's clear that the (M180E) had the least evolutionary fitness for PAH protein. As displayed in Figure(2)
Literature Characterization
Following the removal and replacement of iron, a low-field region was designed for X-band EPR spectra of PAH. In (a), you can see the Native PAH EPR spectrum (160 pM, sp act. = 5.5, 1.1 iron/subunit). Instrument settings included modulation amplitude 20 G, receiver gain 1 X lo4, microwave power 0.1 mW, time constant 0.25 s, scan time 8 minutes, and scan range 0.4-2.4 kg. The values of the most prominent spectral features are given. (b) PAH EPR spectrum after partial iron removal (170 rM, sp act. = 0.6, 0.6 iron/subunit). The instrument settings are detailed in (a). (c) Reconstituted EPR spectrum of the sample described throughout (b). sp act. = 5.0 and 1.1 iron/subunit in the 88 pM reconstituted sample.. The instrument settings described for (a) were used except that the gain is 2 X lo4 as shown in figure 3.
Characterization by mathematical modeling
We are using mathematical modeling to detect the increased level of phenylalanine (phe) in phenylketonuria patients in our therapeutic platform. It depends on a E.coli-based system through a cascade of reactions to finally end by formation of PAH that is deficient in PKU patients as shown in graph (1).
Graph(1) illustrates a direct relation between phenylalanine and PAH ,so as the biomarker increases, the released amount of the enzyme increases till it reaches constant value after about 30 time units. Therefore, the maximum amount of the biomarker releases the maximum amount of PAH.
Experimental Characterization
This figure shows an experimental characterization of this part as it's validated through gel electrophoresis as it is in lane 10 (the last one). The run part (ordered from IDT) included KP-SP - PAH.
References
1. Bloom, L. M., Gaffney, B. J., & Benkovic, S. J. (1986). characterization of phenylalanine hydroxylase. Biochemistry, 25(15), 4204-4210.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 287
Illegal BamHI site found at 814
Illegal XhoI site found at 524 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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