Degradation of Phytate by the 6-Phytase from Hafnia alvei: A Combined Structural and Solution Study

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.

Soil Enzyme Activity as Affected by Tillage and Residue Management Practices under Diverse Cropping Systems

“…Phytase (myo-inositol hexakisphosphate phosphohydrolases) catalyses the hydrolysis of inositol phosphates and are potentially important in the soil for their role in phosphorus mineralization due to conversion of organic-P from phytate to plant available form (Ariza et al, 2013). The stability and activity of phytases in soil is affected by sorption on soil particle surfaces which may reduce the potential for interaction with substrates.…”

A Novel Phytase Derived from an Acidic Peat-Soil Microbiome Showing High Stability under Acidic Plus Pepsin Conditions

“…In SGF pH 2.0, 2.5 and 3.5, rPhyPt4 demonstrates similar or slightly higher stability than the Y. rohdei phytase. rPhyPt4 is thermostable at 60 ° C, as its activity decline is slower than several thermostable phytases reported previously, such as the HaPhy [Ariza et al, 2013], Ymphytase mutant Omni1 [Shivange et al, 2014], EcAppA mutant Mut1/2/4 [Wu et al, 2014], EcAppA mutant Q258N/Q349N [Yao et al, 2013] and Aspergillus terreus phytase mutant HyA [Jermutus et al, 2001]. However, its thermostability at 80 ° C has little advantage over other phytases.…”

Purification, biochemical characterization, and genetic cloning of the phytase produced by <i>Burkholderia</i> sp. strain a13

“…The alignment showed that the mature portion of this enzyme contains eight Cys residues, of which five are conserved in bacterial HAP phytases examined and the remaining three are conserved in six phytases, including H. alvei phytase. In the crystal structure of H. alvei phytase (Ariza et al, 2013), it has been reported that the eight conserved Cys residues formed four disulfide bridges with each other (Fig. 5).…”

“…Some of the bacterial HAP phytases were reported to be produced in the periplasmic space in their original hosts (Greiner, 2004;Greiner et al, 1993) and most of the genes possess sequences similar to the signal sequence at their N-termini (Ariza et al, 2013;Sajidan et al, 2004;TamayoRamos et al, 2012). Though we could not detect any phytase activity in the cultural broth of strain a13, it may be possible that the phytase is secreted into the periplasmic space in strain a13.…”

“…For the production of the recombinant bacterial HAP phytases, the yeast Pichia pastoris and the fungus Aspergillus oryzae were used as host strains in the case of phytases from Yersinia intermedia (Huang et al, 2006) and H. alvei (Ariza et al, 2013), respectively. In the case of Dickya paradisiaca phytase, the E. coli pET system was successfully used, but the level of production was rather low (Gu et al, 2009).…”