Abstract
Phytases hydrolyse phytate (myo-inositol hexakisphosphate), the principal form of phosphate stored in plant seeds to produce phosphate and lower phosphorylated myo-inositols. They are used extensively in the feed industry, and have been characterised biochemically and structurally with a number of structures in the PDB. They are divided into four distinct families: histidine acid phosphatases (HAP), β-propeller phytases, cysteine phosphatases and purple acid phosphatases and also split into three enzyme classes, the 3-, 5- and 6-phytases, depending on the position of the first phosphate in the inositol ring to be removed. We report identification, cloning, purification and 3D structures of 6-phytases from two bacteria, Hafnia alvei and Yersinia kristensenii, together with their pH optima, thermal stability, and degradation profiles for phytate. An important result is the structure of the H. alvei enzyme in complex with the substrate analogue myo-inositol hexakissulphate. In contrast to the only previous structure of a ligand-bound 6-phytase, where the 3-phosphate was unexpectedly in the catalytic site, in the H. alvei complex the expected scissile 6-phosphate (sulphate in the inhibitor) is placed in the catalytic site.
Highlights
IntroductionPlants use phytate (myo-inositol hexakisphosphate here called InsP6; Ins = inositol = cyclohexane-1,2,3,4,5,6-hexol) as their main storage form of phosphorous, inositol and a variety of minerals and it accounts for 75–80% of the total phosphorous in seeds such as those of cereals and legumes [1,2]
Plants use phytate as their main storage form of phosphorous, inositol and a variety of minerals and it accounts for 75–80% of the total phosphorous in seeds such as those of cereals and legumes [1,2]
We present structures of both enzymes: apo-Hafnia alvei phytase (HaPhy), its complexes with the phytate analogue myo-inositolhexakissulphate (MIHS-HaPhy) and the competitive inhibitor L(+)-tartrate and YkPhy with one of its products Pi (PiYkPhy)
Summary
Plants use phytate (myo-inositol hexakisphosphate here called InsP6; Ins = inositol = cyclohexane-1,2,3,4,5,6-hexol) as their main storage form of phosphorous, inositol and a variety of minerals and it accounts for 75–80% of the total phosphorous in seeds such as those of cereals and legumes [1,2]. The addition of phytase reduces the need to supplement the feed with Pi, a costly non-renewable resource that is estimated to be depleted within 50 years [9] [10]. It lessens the anti-nutritional effects and decreases the environmental burden from phosphate pollution in areas of intense animal farming by decreasing the amount of phosphate excreted by animals
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