Author(s)

Abul H. J. Ullah, Kandan Sethumadhavan, Stephanie Boone, Edward J. Mullaney

Southern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, New Orleans, USA.

The active site motif of proteins belonging to "Histidine Acid Phosphatase" (HAP) contains a hepta-peptide region, RHGXRXP. A close comparison among fungal and yeast HAPs revealed the fourth residue of the hepta-peptide to be E instead of A, which is the case with A. niger PhyA phytase. However, another phytase, PhyB, from the same microorganism has a higher turnover number and it shows E in this position. We mutated A69 residue to E in the fungal PhyA phytase. The mutant phytase shows a myriad of new kinetic properties. The pH profile shifted 0.5 pH unit in both 5.0 and 2.5 bi-hump peaks. The optimum temperature shifted down from 58℃ to 55℃. However, the greatest difference was observed in the mutant protein's reaction to GuCl at a concentration of 0.1 to 0.2 M. The activity of the mutant phytase jumped 100% while the wild type protein showed no activity enhancement in the same concentration range of GuCl. The kinetics performed at higher concentration of GuCl also contrasted the difference between the wild type and mutant phytase. While K_m was least affected, the V_max increased for the mutant and decreased for the wild type. The sensitivity towards myo-inositol hexasulfate, a potent inhibitor, was decreased by the mutation. All in all, A69E mutation has affected a multitude of enzymatic properties of the protein even though the residue was thought to be non-critical for phytase's catalytic function notwithstanding its location in the conserved hepta-peptide region of the biocatalyst.

Phytase; Histidine Acid Phosphatase; Aspergillus niger; Site-Directed Mutagenesis

A. H. J. Ullah, K. Sethumadhavan, S. Boone and E. J. Mullaney, "A Single Mutation in the Hepta-Peptide Active Site of Aspergillus niger PhyA Phytase Leads to Myriad Biochemical Changes," Advances in Microbiology, Vol. 2 No. 3, 2012, pp. 388-394. doi: 10.4236/aim.2012.23049.