Fig 1.
Degradation of phytate by AC1-2 MINPP.
Degradation was followed by HPLC at 30 min, 2h, 4h and 8h. Structures of inositol phosphate products (as pairs of enantiomers where appropriate, named by D-notation) are shown.
Fig 2.
Schematic detailing potential pathways of degradation of phytate by AC1-2 MINPP.
Predominant products are indicated by size of font; * indicates the predominant InsP5 impurity in the substrate; weight of arrow indicates likelihood of route of degradation.
Fig 3.
Predicted structural features of AC1-2 MINPP: U-loop and specificity pockets.
A, Alignment of the amino acid sequences of AC1-2 MINPP and BlMINPP in the region of the U-loop. A blue box delimits U-loop residues. Cysteine residues forming a disulphide bridge in the crystal structure of BlMINPP (PDB 6XRE) are highlighted yellow. Conserved residues (in red) are part of a MINPP-specific tetrapeptide motif. Positions of residues contributing to specificity pockets are indicated by inverted blue triangles. B, Molecular surface representations of the structures of apo- (left) and InsS6-bound (right) AC1-2 MINPP predicted by homology modelling. The U-loop residues are coloured green with the remainder of the molecule in cyan. Atoms of the substrate analogue inhibitor, InsS6, are shown as spheres and coloured red (oxygen), cyan (carbon) and orange (sulphur). C, Residues predicted to contribute to the specificity pockets of AC1-2 MINPP and selected histidine phytases. AC1-2, Bl and Bt are the MINPPs from Acinetobacter sp. AC1-2 (this study), Bifidobacterium longum (PDB 6XRE) and Bacteroides thetaiotaomicron (PDB 4FDU), respectively. Af is the histidine phytase from Aspergillus fumigatus (PDB 1SK8). Specificity pockets are labelled A-F as described by Acquistapace et al. (2000). Each alignment shows spatially equivalent residues in the specificity pockets of each enzyme, which lie within 5Å of the phosphorus of the corresponding phosphate group on the substrate (the positions of sulphate groups of the inhibitors in each structure are taken to be the same as the phosphate groups of phytate. Numbering is according to the AC1-2 sequence. Residues that are completely conserved are highlighted in bold with red text. Red arrows indicate residues that have the closest interactions with the substrate analogue inhibitor. Note that residues 298 and 301 contributing to the D-pocket are found on the U-loop. Residues marked with an asterisk in pockets D and E are predicted to not interact with the substrate when bound with the 4-phosphate in the A-pocket.
Fig 4.
Biochemical characterisation of AC1-2 MINPP.
A, The specificity of the enzyme towards different phosphate (P)—containing compounds assayed at 1mM. Significant differences between compound and InsP6 are indicated at P≤0.05, *; P≤0.01, **; P≤0.001, *** and P≤0.0001, ****. B, inhibition of activity by metal ions. Significant differences between metal ions and control are indicated at P≤0.05, *; P≤0.01, **; P≤0.001, *** and P≤0.0001, ****. C, pH-activity profile; D, thermostability. A-D, means and standard deviation of three measurements.
Fig 5.
Long-term stability of AC1-2 MINPP.
Stability was assessed in seven different stabilising solutions and a control (gel filtration buffer) during storage in ambient conditions. A, Isolation of AC1-2 MINPP through a two-step, Ni-affinity and size exclusion, purification. Aliquots of protein taken at different stages of the purification were subjected to SDS-PAGE on a 12% gel. Lanes labelled 1–9 are flanked, left, by molecular mass markers identified by mass (kDa). Lanes: 1 and 2, uninduced Rosetta ™ 2 (pLysS) cells; 3 and 4, 0.1 mM IPTG-induced cells; 5, crude lysate of concentrated, induced cells; 6, cell pellet; 7, clarified cell lysate (supernatant); 8, Amicon® Ultra-15 (10 kDa cut-off) -concentrated fraction, post Ni-affinity chromatography; 9, Amicon-concentrated fraction, post size exclusion chromatography. B, Enzyme activity of AC1-2 MINPP during prolonged storage at 4°C or ambient conditions (occasionally reaching 30–35°C). Protein was stored at a concentration of 4 μM in 50 mM Tris-HCl pH 7.5 300 mM NaCl (control) or in 25 mM Tris-HCl pH 7.5 150 mM NaCl with stabilising agent as indicated. Error bars show standard deviation of triplicate measurements. C. SDS-PAGE of AC1-2 MINPP stored for greater than three years at a concentration of 4 μM in 25 mM Tris-HCl pH 7.5 150 mM NaCl with stabilizing agents as indicated. Thereafter, aliquots of protein were subjected to SDS-PAGE on a 12% gel, Lanes 1–4, flanked on the left by molecular mass markers (kDa). Lanes, stabilising agents: 1, 30% w/v glycerol; 2, 30% w/v glycerol and 0.5 mg/ml BSA; 3, 30% w/v sucrose; 4, 30% w/v sucrose and 0.5 mg/ml BSA.
Fig 6.
Enantiospecificity of AC1-2 MINPP attack on phytate.
HPLC of: A, products of incubation of a purified and desalted D-and/or L-Ins(1,2,3,4,5)P5 [InsP5 4/6-OH] fraction generated by AC1-2 MINPP with AtITPK1; B, a no-enzyme control for A; C, products of incubation of InsP6 with AtITPK1; D, a no-enzyme control for C. E, products of incubation of D-Ins(1,2,3,5,6)P5 (InsP5 [4-OH]) with AtITPK1. F, a no-enzyme control for E. G, Products of incubation of D-Ins(1,2,3,4,5)P5 (InsP5 [6-OH]) with AtITPK1. H, a no-enzyme control for G. Approximately, one third of sample equivalent (to A-E, and H) was injected for G. The units and scales of panels (A-H) are identical.
Fig 7.
Kinetics parameters of AC1-2 MINPP activity against phytate.
Fig 8.
Validation of the primer sets and quantification of expression of the ac1-2 MINPP by qPCR.
A, Log-linearity of amplification with primer sets; B, ΔCt value of the LB and LB + InsP6 environments; C, ΔCt of the MM, MM + Pi and MM + InsP6 environments; D, Fold change, calculated using the 2-ΔΔCt method.