Avian multiple inositol polyphosphate phosphatase is an active phytase that can be engineered to help ameliorate the planet's "phosphate crisis".
Contemporary phytase research is primarily concerned with ameliorating the problem of inadequate digestion of inositol hexakisphosphate (phytate; InsP6) in monogastric farm animal feed, so as to reduce the pollution that results from the high phosphate content of the manure. In the current study we pursue a new, safe and cost-effective solution. We demonstrate that the rate of hydrolysis of InsP6 by recombinant avian MINPP (0.7 micromol/mg protein/min) defines it as by far the most active phytase found to date in any animal cell (the corresponding activity of recombinant mammalian MINPP is only 0.006 micromol/mg protein/min). Although avian MINPP has less than 20% sequence identity with microbial phytases, we create a homology model of MINPP in which it is predicted that the structure of the phytase active site is well-conserved. This model is validated by site-directed mutagenesis and by use of a substrate analogue, scyllo-InsP6, which we demonstrate is only a weak MINPP substrate. In a model chicken cell line, we overexpressed a mutant form of MINPP that is secretion-competent. This version of the enzyme was actively secreted without affecting either cell viability or the cellular levels of any inositol phosphates. Our studies offer a genetic strategy for greatly improving dietary InsP6 digestion in poultry.
Cho
Jaiesoon
J
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, NIH, DHSS, Research Triangle Park, PO Box 12233, NC 27709, USA.
Choi
Kuicheon
K
Darden
Thomas
T
Reynolds
Paul R
PR
Petitte
James N
JN
Shears
Stephen B
SB
D010832:6-Phytase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D002645:Chickens / Q000378:metabolism*; D003247:Conservation of Natural Resources; D008297:Male; D010710:Phosphates / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D010833:Phytic Acid / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*
D010710:Phosphates; D010833:Phytic Acid; D010744:Phosphoric Monoester Hydrolases; D010832:6-Phytase; C064119:multiple inositol-polyphosphate phosphatase