Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/14/2010
Publication Date: 8/4/2010
Citation: Ullah, A.H., Sethumadhavan, K., Boone, S.A., Mullaney, E.J. Non-essential A69E mutations in the active site of Aspergillus niger phyA phytase leads to myriads of changes (Abstract).
Technical Abstract: 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 has 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 have 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 that are characteristics of the fungal phytase. The optimum temperature shifted from 58º C to 55º C. However, the greatest difference was observed in the mutant protein’s reaction to Gu.Cl 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 Gu.Cl. The kinetics performed at higher concentration of Gu.Cl also contrasted the difference between the wild type and mutant phytase. While Km was least affected, the Vmax 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.