Kinetic Characterization of O-Phospho-L-Tyrosine Phosphohydrolase Activity of Two Fungal Phytases.

Authors: Ullah, Abul; Sethumadhavan, Kandan; Mullaney, Edward

Submitted to: Journal of Agriculture and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/18/2008
Publication Date: 7/16/2008
Citation: Ullah, A.H., Sethumadhavan, K., Mullaney, E.J. 2008. Kinetic characterization of o-phospho-L-tyrosine phosphohydrolase activity of two fungal phytases. Journal of Agriculture and Food Chemistry. 56(16):7467-7471.

Interpretive Summary: Phytases are a group of enzymes that breakdown phytic acid, a known plant metabolite, which is also considered an antinutrient. Phytic acid is abundantly present in oil crop seeds such as cotton, soybean, and a variety of legumes, which binds minerals and robs animals' body of essential metals such as iron, calcium, copper, etc. Even though simple-stomached animals including humans lack the ability to breakdown phytic acid, a variety of microorganisms have the ability to digest this antinutrient because they have the enzymes, phytase, to do so. This creates an environmental problem. When phytic acid rich diet is fed to farm animals, the undigested antinutrient is excreted with the fecal materials. Microbes then degrade phytic acid liberating phosphates, which contaminate a variety of waterways. In warm summer months, algae feast on excess phosphate in nitrogen rich waters engendering 'harmful algal bloom' (HAB) popularly known as red tide. In recent years, phytase produced by fungus and bacteria are mixed with animal feed to release phosphate in animal gut to contain the excess phosphate inside animal body; thereby, reducing the release of free phosphates in waterways. Our lab was instrumental in developing the technology to clone a hyperactive phytase for feed supplementation. We found a second phytase that was even more active albeit at lower pH, which limits its application to digest phytic acid in animals' gut. In this communication, we present evidence to show that this phytase could breakdown a phosphorylated amino acid, tyrosine. This modified amino acid is present in many proteins and scientists think that it serves as a signaling agent. The presence of excess amounts of phosphorylated tyrosine is implicated in cancer; therefore, the breakdown of this compound will be beneficial and therapeutic. The second phytase has the unique ability to breakdown phospho-tyrosine quite efficiently. And this is what is the conclusion of this scientific study. If the medical community is looking for a fast acting phospho-tyrosine to act as a therapeutic agent, then, this phytase will perform very well. Genetic engineering of this enzyme may even make it a potent degrading agent to breakdown phospho-tyrosine into tyrosine (a normal amino acid found in the animal and plant kingdom) and free phosphate (a nutrient).

Technical Abstract: Fungal phytases belonging to 'Histidine Acid Phosphatase' or HAP class of phosphomonoesterase that catalyzes the hydrolysis of phytic acid could also hydrolyze O-phospho-tyrosine. Two phytases from Aspergillus niger and Aspergillus awamori with pH optima 2.5 were tested for phospho-tyrosine hydrolase activity. Both enzymes cleaved the phosphomonoester bond of O-phospho-tyrosine efficiently at acidic pH. The Km for O-phospho-tyrosine ranged from 465 to 590 µM as opposed to 135 to 160 µM for phytate. The Vmax, however, is 2 to 4 times higher for O-phospho-tyrosine than it is for phytate. The catalytic efficiency of phytase for O-phospho-tyrosine is on the same order as it is for phytate (3.5 x 106 to 1.6 x 107 M-1sec-1); the pH versus activity profile for O-phospho-tyrosine is, however, different from what it is for phytate. The temperature optima shifted 5ºC higher to 70ºC when O-phospho-tyrosine was used as the substrate. Taken together, the kinetic data show that fungal HAPs that are known as PhyB are capable of cleaving the phospho-monoester bond in O-phospho-tyrosine. This is the first time that O-phospho-tyrosine hydrolase activity has been reported to be possible in the subgroup of HAP known as phytase.