Part:BBa_K1751000

Background and priniciples

The gene RgPAL1 can express Phenylalanine ammonia lyase（PAL）, which is an enzyme that catalyzes a reaction converting L-phenylalanine to ammonia and trans-cinnamic acid.PAL is the first and committed step in the phenyl propanoid pathway and is therefore involved in the biosynthesis of the polyphenol compounds. In our project, PAL plays an important role in L-phenylalanine’s deaminization to produce trans-cinnamic acid.

Phenylalanine ammonia lyase (PAL) is an enzyme that catalyzes a reaction converting L-phenylalanine toammonia and trans-cinnamic acid. Phenylalanine ammonia lyase (PAL) is the first and committed step in the phenyl propanoid pathway and is therefore involved in the biosynthesis of the polyphenol compounds such as flavonoids,phenylpropanoids, and lignin in plants. Phenylalanine ammonia lyase is found widely in plants, as well as someyeast and fungi, with isoenzymes existing within many different species. It has a molecular mass in the range of 270-330 kDa. The activity of PAL is induced dramatically in response to various stimuli such as tissue wounding,pathogenic attack, light, low temperatures, and hormones. PAL has recently been studied for possible therapeutic benefits in humans afflicted with phenylketonuria. It has also been used in the generation of L-phenylalanine as precursor of the sweetener aspartame.

The enzyme is a member of the ammonia lyase family, which cleaves carbon-nitrogen bonds. Like other lyases, phenylalanine requires only one substrate for the forward reaction, but two for the reverse. It is thought to be mechanistically similar to the related enzyme histidine ammonia-lyase (HAL). The systematic name of this enzyme class is L-phenylalanine ammonia-lyase (trans-cinnamate-forming). Previously, it was designated PAL, but that class has been redesignated as phenylalanine ammonia-lyases, tyrosine ammonia-lyases, and phenylalanine/tyrosine ammonia-lyases. Other names in common use include tyrase,phenylalanine deaminase, tyrosine ammonia-lyase, L-tyrosine ammonia-lyase, phenylalanine ammonium-lyase, PAL, andL-phenylalanine ammonia-lyase.

Enzyme Mechanism

Phenylalanine ammonia lyase is specific for L-Phe, and to a lesser extent, L-Tyrosine. The reaction catalyzed by PAL is the spontaneous, non-oxidative deamination of L-phenylalanine to yield trans-cinnamic acid and ammonia.

L-phenylalanine --> trans-cinnamate + NH3

The cofactor 3,5-dihydro-5-methyldiene-4H-imidazol-4-one (MIO) is involved in the reaction and sits atop the positive pole of three polar helices in the active site, which helps to increase its electrophilicity.MIO is reported to attack the aromatic ring of L-Phe in a Friedel-Crafts-type reaction, which activates the C-H bond and leads to cleavage of the bond. The carbanionintermediate formed by this mechanism is stabilized by partial positive regions in the active site. The mechanism of the reaction of PAL is thought to be similar to the mechanism of the related enzyme histidine ammonia lyase.PAL is inhibited by trans-cinnamic acid, and, in some species, may be inhibited trans-cinnamic acid derivatives.D-Phe and D-Tyr are competitive inhibitors.

Useful Resources

（1）https://en.wikipedia.org/wiki/Phenylalanine_ammonia-lyase