Figure 1.
Agranoff’s rule for inositol numbering.
(a) Agranoff’s turtle with the head up in the 2-phosphate position. (b) Phytate numbered according to Agranoff’s nomenclature, with the 2-phosphate axial and pointing upwards, with the carbon atoms numbered anticlockwise around the ring.
Figure 2.
The branches of Escherichia (purple), Citrobacter (light green) and Yersinia (red) are colour coded. The wheel represents a neighbour-joining of pairwise Smith-Waterman alignment scores. A full list of the UniProt entries can be found in the Supporting Information S1. The grey triangles indicate PDB entries, while the red triangle is YkPhy and the blue triangle is HaPhy.
Figure 3.
Structure-based sequence alignment for four representative phytases: HaPhy, YkPhy, EcPhy and AnPhy.
The alignment is based on the 3D structures of HaPhy, EcPhy and AnPhy using the T-Coffee Expresso web server [67], [68] – three is the maximum number of structures allowed by the server. The secondary structure elements of HaPhy are shown above, and of AnPhy below, the alignment. Fully conserved resides are shown in red boxes. The Figure was generated using ESPript [69], [70].
Figure 4.
The digestion profile of phytate (InsP) by H. alvei phytase.
The InsP profile at pH 4.0 is shown after incubation with HaPhy at 37°C for 0, 5, 10, 15, 20, 25, 30, and 45 min (1 FYT/ml, 5 mM Na-phytate).
Table 1.
Crystallographic statistics.
Figure 5.
(a) Ribbon representation of the HaPhy fold. The α domain (residues 25–45 and 137–264) is shown in coral, the α/β domain is in blue. The four disulphide bridges are in sphere format and lie in surface loops. The active site lies at the interface between the two domains – viz. the position of the active site bound ligand from the MIHS-HaPhy complex. (b) Mono view of the binding of MIHS to the active site of HaPhy, with the density contoured at the 1σ level for the MIHS. The six sulphates are labelled S1–S6. The side chains of three residues (His19, Asp307 and Thr308– the latter from the wild type structure) which form key interactions are shown as cylinders, with the carbons atoms coloured green for the complex structure, and coral for the wild type. The extensive additional interactions of the MIHS with the protein and water molecules are not shown for clarity. (c) Stereo view of the protein surface around the ligand labelled as in (b). (d) Schematic representation of the interactions in the active site. (a–c) were drawn using CCP4mg [71], and (d) using LigPlot+ [72].
Figure 6.
Stereo view of binding site comparisons for the HAPP phytases.
(a) Superposition of the binding site of the MIHS-HaPhy complex (green) on the corresponding region of tartrate bound HaPhy (grey) and phosphate-bound YkPhy (yellow). (b) Superposition of the binding site of the MIHS-HaPhy complex (green) on the corresponding region of the EcPhy phytate complex (blue). (c) Superposition of the binding site of the MIHS-HaPhy complex on the corresponding region of the MIHS-AnPhy complex (protein in purple, MIHS grey). The ligand-binding residues are shown in ball and stick.