Part:BBa_K4140009

Phenylalanine hydroxylase (PAH)

Part Description

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 to 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 homones 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 convert phenylalanine to tyrosine and in case of its absence or deficiency lead 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

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(1)

Figure 1. (shows the mutational landscape of the PAH protein.)

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.

Fig. 2 shows the EPR spectra for the PHe in the native form and after the partial removal and total removal of iron.

References

1. Bloom, L. M., Gaffney, B. J., & Benkovic, S. J. (1986). characterization of phenylalanine hydroxylase. Biochemistry, 25(15), 4204-4210.‏

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]