Part:BBa_K200008

Phenylalanine hydroxylase

[http://en.wikipedia.org/wiki/Phenylalanine_hydroxylase Phenylalanine hydroxylase (PAH)] is the enzyme that breaks down [http://en.wikipedia.org/wiki/Phenylalanine phenylalanine] to [http://en.wikipedia.org/wiki/Tyrosine tyrosine]. Deficiency of this enzyme activity has been identified as one of the causes of the disorder called [http://en.wikipedia.org/wiki/Phenylketonuria phenylketonuria (PKU)]#PAH1. Because our project aims to deliver such enzyme to the small intestine, we have also submitted a protease resistant version (BBa_K200028).

Characterization of Mutational Landscape by AFCM-Egypt 2022 team

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)

Figure 2. (shows the mutational landscape of the PAH protein.)

Literature Characterization by AFCM-Egypt 2022 team

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.

Fig. 3 shows the EPR spectra for the PHe in the native form and after the partial removal and total removal of iron.

Characterization by mathematical modeling by AFCM-Egypt 2022 team

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 by AFCM-Egypt 2022 team

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.

Usage and Biology

PAH works well at a PH of around 7.0 (similar to the small intestine), and it is a non-heme iron-dependent enzyme. The enzyme binds three substrates: reduced pterin (PH4), phenylalanine, and oxygen, and releases two products: tyrosine and oxidized pterin #PAH2. Furthermore, the presence of oxygen and an additional cofactor (TPNH) is required for the hydrolysis to take place #PAH4. The enzyme is stable between 7 and 40 degrees celsius#PAH2 and in this range activity increases exponentially with temperature but Km (Michaelis-Menten constant) does not vary significantly between 20 and 40 degrees. The enzyme can be rendered protease resistant via full phosphorylation.#PAH3
Phenylketonuria is a condition whose prevalence varies considerably with populations. In Northern Europe for example the incidence is estimated to 1 in 10000#PAH6. At the other end of the spectrum, Turkey is estimated to have an incidence of 1 in 2600 births#PAH6. This condition is due to the inability of the individuals to catabolise essential amino acid Phenylalanine into Tyrosine. This leads to hyperphenylalaninemia in the blood which, in turn, causes damage to the brain resulting in loss of cognitive abilities and mental retardation ([http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=261600 OMIM]). There are two different types of the disease: classical PKU and BH₄-responsive PKU. The former form of the disease is linked to a deficient PAH enzyme sequence that renders the resulting protein non functional. The latter is due to a deficient co-factor BH₄. Today, only the BH₄ can be tackled, thus encompassing only half of the phenylketonuriac population#PAH5. As part of the Imperial iGEM 2009 [http://2009.igem.org/Team:Imperial_College_London The E.ncapsulator] project, we propose a new, broader therapy strategy where both the functional enzyme and cofactor are delivered to the patient.

Requirements

• Works for PH 7
• Works between 7 to 40 degrees

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 287
  Illegal BamHI site found at 814
  Illegal XhoI site found at 524
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]