Bound for catalysis: Crystal structures of Escherichia coli phytase and its complex with phytic acid

Phytases catalyze the hydrolysis of inorganic phosphate from phytic acid (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and are able to improve the nutritional quality of phytate-rich diets. Escherichia coli phytase, a member of the histidine acid phosphatase family has the highest specific activity of all phytases characterized. Histidine acid phosphatases have acidic pH optima. and the conserved active site sequence motif: RHGXRXP. Catalysis involves an initial nucleophilic attack by the histidine of the active site motif, followed by hydrolysis of the phospho-histidine intermediate. Crystallographic studies were undertaken to investigate the structural basis for the high specific activity of E. coli phytase. A number of crystal forms of E. coli phytase have been grown and characterized. Crystals of the acidic form were grown by a batch method under conditions in which the enzyme exhibits the highest activity for phytate hydrolysis. Crystals of the high cadmium form provided the highest resolution data and demonstrated the rather large potential of this protein for metal cation binding. Crystals of the mercury form demonstrated the utility of Hg2+ to both promote crystallization by acting as an intermolecular bridge and provide useful phasing information in multiwavelength anomalous diffraction (MAD) experiments. In addition, a tungstate derivative obtained for the mercury crystal form provided a heavy atom derivative with exceptionally strong anomalous scattering and allowed the structure to be determined by a two-wavelength MAD method. Furthermore, the tungstate derivative also provided a useful phosphate analog complex structure. The structure of E. coli phytase was determined to a maximum resolution of 1.7 A. Despite a lack of sequence homology, the structure is homologous to the overall fold of other histidine acid phosphatases, namely the rat and human prostatic acid phosphatases and the Aspergillus niger PhyA phytase. E. coli phytase has an overall globular fold with a large prominent substrate binding pocket dividing the structure into an a domain and a larger alpha/beta domain. The alpha/beta domain is well conserved among the three structures and consists of a central twisted mixed beta sheet with a helices on each side. The active site is located at the C-terminal end of the parallel strands of the main beta sheet. Based on the phytate and tungstate complex structures of E. coli phytase, a unique "molecular switch" mechanism is proposed, in which catalysis is enhanced by substrate-induced conformational changes that increase the acidity of the general acid catalyst.