A Single Mutation in the Hepta-Peptide Active Site of Aspergillus niger PhyA Phytase Leads to Myriad Biochemical Changes

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.

DOI: 10.4236/aim.2012.23049                

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 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.

Keywords

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

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	T. R. Shieh and J. H. Ware, “Survey of Microorganisms for the Production of Extracellular Phytase,” Applied Microbiology, Vol. 16, No. 9, 1968, pp. 1348-1351. http://aem.asm.org/content/16/9/1348.full.pdf
[2]	A. H. J. Ullah and D. M. Gibson, “Extracellular Phytase (E.C. 3.1.3.8) from Aspergillus ficuum NRRL 3135: Purification and characterization,” Preparative Biochemistry, Vol. 17, No. 1, 1987, pp. 63-91. doi:10.1080/00327488708062477
[3]	S. Haefner, A. Knietsch, E. Scholten, J. Braun, M. Lohscheidt and O. Zelder, “Biotechnological Production and Applications of Phytases,” Applied Microbiology and Biotechnology, Vol. 68, No. 5, 2005, pp. 588-997. doi:10.1007/s00253-005-0005-y
[4]	R. J. Wodzinski and A. H. J. Ullah, “Phytase,” Advances in Applied Microbiology, Vol. 42, 1996, pp. 263-302. doi:10.1016/S0065-2164(08)70375-7
[5]	A. H. J. Ullah and B. Q. Phillippy, “Immobilization of Aspergillus ficuum Phytase: Product Characterization of the Bioreactor,” Preparative Biochemistry, Vol. 18, No. 4, 1988, pp. 483-489. doi:10.1080/00327488808062546
[6]	A. H. J. Ullah, K. Sethumadhavan and E. J. Mullaney, “Salt Effect on the pH Profile and Kinetic Parameters of Microbial Phytases,” Journal of Agricultural and Food Chemistry, Vol. 56, No. 9, 2008, pp. 3398-3402. doi:10.1021/jf073137i
[7]	J. D. Weaver, A. H. J. Ullah, K. Sethumadhavan, E. J. Mullaney and X. G. Lei, “Impact of Assay Conditions on Activity Estimate and Kinetics Comparison of Aspergillus niger PhyA and Escherichia coli AppA2 Phytases,” Journal of Agricultural and Food Chemistry, Vol. 57, No. 12, 2009, pp. 5315-5320. doi:10.1021/jf900261n
[8]	A. H. J. Ullah, B. J. Cummins and H. C. Dischinger, Jr., “Cyclohexanedione Modification of Arginine at the Active Site of Aspergillus ficuum Phytase,” Biochemical and Bio- physical Research Communications, Vol. 178, No. 1, 1991, pp. 45-53. doi:10.1016/0006-291X(91)91777-A
[9]	R. L. Van Etten, R. Davidson, P. E. Stevis, H. MacArthur and D. L. Moore, “Covalent Structure, Disulfide Bonding, and Identification of Reactive Surface and Active Site Residues of Human Prostatic Acid Phosphatase,” Journal of Biological Chemistry, Vol. 266, No. 4, 1991, pp. 2313-2319. http://www.jbc.org/content/266/4/2313.full.pdf+html
[10]	A. H. J. Ullah and H. C. Dischinger, Jr., “Identification of Active Site Residues in Aspergillus ficuum Extracellular pH 2.5 Optimum Acid Phosphatase,” Biochemical and Biophysical Research Communications, Vol. 192, No. 2, 1993, pp. 754-759. doi:10.1006/bbrc.1993.1478
[11]	W. Zhang, E. J. Mullaney and X. G. Lei, “Adopting Selected Hydrogen Bonding and Ionic Interacttions from Aspergillum fumigatus Phytase Structure Improves the Thermostability of Aspergillus niger PhyA Phytase,” Applied and Environmental Microbiology, Vol. 73, No. 9, 2007, pp. 3069-3076. doi:10.1128/AEM.02970-06
[12]	T. Kim, E. J. Mullaney, J. M. Porres, R. Roneker, S. Crowe, S. Rice, T. Ko, A. H. J. Ullah, C. B. Daly, R. Welch and X. G. Lei, “Shifting the pH Profile of Aspergillus niger PhyA Phytase to Match the Stomach pH Enhances Its Effectiveness as an Animal Feed Additive,” Applied and Environmental Microbiology, Vol. 72, No. 6, 2006, pp. 4397-4403. doi:10.1128/AEM.02612-05
[13]	E. J. Mullaney, H. Locovare, K. Sethumadhavan, S. Boone, X. G. Lei and A. H. J. Ullah, “Site-Directed Mutagenesis of Disulfide Bridges in Aspergillus niger NRRL 3135 Phytase (PhyA), Their Expression in Pichia pastoris and Catalytic Characterization,” Applied Microbiology and Biotechnology, Vol. 87, No. 4, 2010, pp. 1367-1372. doi:10.1007/s00253-010-2542-2
[14]	A. H. J. Ullah, K. Sethumadhavan and E. J. Mullaney, “Monitoring of Unfolding and Refolding in Fungal Phytase (PhyA) by Dynamic Light Scattering,” Biochemical and Biophysical Research Communications, Vol. 327, No. 4, 2005, pp. 993-998. doi:10.1016/j.bbrc.2004.12.111
[15]	J. K. Heinonen and R. J. Lahti, “A New and Convenient Calorimetric Determination of Inorganic Orthophosphate and Its Application to the Assay of Inorganic Pyrophosphatase,” Analytical Biochemistry, Vol. 113, No. 2, 1981, pp. 313-317. doi:10.1016/0003-2697(81)90082-8
[16]	A. H. J. Ullah and K. Sethumadhavan, “Myo-Inositol Hexasulfate Is a Potent Inhibitor of Aspergillus ficuum Phytase,” Biochemical and Biophysical Research Communications, Vol. 251, No. 1, 1998, pp. 260-263. doi:10.1006/bbrc.1998.9456
[17]	D. Kostrewa, F. Grueninger-Leitch, A. D’Arcy, C. Broger, D. Mitchell and A. P. G. M. Van Loon, “Crystal Structure of Phytase from Aspergillus ficuum at 2.5 ? Resolution,” Nature Structural Biology, Vol. 4, No. 3, 1997, pp. 185-190. doi:10.1038/nsb0397-185
[18]	D. Kostrewa, M. Wyss, A. D’Arcy and A. P. G. M. Van Loon, “Crystal Structure of Aspergillus niger pH 2.5 Acid Phosphatase at 2.4 ? Resolution,” Journal of Molecular Biology, Vol. 288, No. 5, 1999, pp. 965-974. doi:10.1006/jmbi.1999.2736
[19]	D. Lim, S. Golova, C. W. Forsberg and Z. Jia,” Crystal Structures of Escherichia coli Phytase and Its Complex with Phytate,” Nature Structural Biology, Vol. 7, No. 2, 2000, pp. 108-113. doi:10.1038/72371
[20]	K. B?hm, T. Herter, J. Müller, R. Borriss and U. Heinemann, “Crystal Structure of Klebsiella sp. ASR1 Phytase Suggests Substrate Binding to a Preformed Active Site that Meets the Requirements of a Plant Rhizosphere Enzyme” Federation of European Biochemical Societies Journal, Vol. 277, No. 5, 2000, pp. 1284-1296. doi:10.1111/j.1742-4658.2010.07559.x
[21]	E. J. Mullaney, C. B. Daly, T. Kim, J. M. Porres, X. G. Lei, K. Sethumadhavan and A. H. J. Ullah, “Site-Directed Mutagenesis of Aspergillus niger NRRL 3135 Phytase at Residue 300 to Enhance Catalysis at pH 4.0,” Biochemical Biophysical Research Communications, Vol. 297, No. 4, 2002, pp. 1016-1020. doi:10.1016/S0006-291X(02)02325-2
[22]	Y. S. Tian, R. H. Peng, J. Xu, W. Zhao, F. Gao, X. Fu, A. S. Xiong and Q. H. Yao, “Semi-Rational Site-Directed Mutagenesis of phyi1s from Aspergillus niger 113 at Two Residue to Improve Its Phytase Activity,” Molecular Biology Reports, Vol. 38, No. 2, 2011, pp. 977-982. doi:10.1007/s11033-010-0192-1
[23]	A. J. Oakley, “The Structure of Aspergillus niger Phytase PhyA in Complex with a Phytate Mimetic,” Biochemical and Biophysical Research Communications, Vol. 397, No. 4, 2010, pp. 745-749. doi:10.1016/j.bbrc.2010.06.024
[24]	D. C.-C. Lim, “Bound for Catalysis. Crystal Structures of Escherichia coli Phytase and Its Complex with Phytic Acid,” M.S. Thesis, Queen’s University, Kingston, 1999.
[25]	A. H. J. Ullah, K. Sethumadhavan and E. J. Mullaney, “Unfolding and Refolding of Aspergillus niger PhyB Phytase: Role of Disulfide Bridges,” Journal of Agricultural and Food Chemistry, Vol. 56, No. 17, 2008, pp. 8179-8183. doi:10.1021/jf8013712
[26]	M. S. Kim and X. G. Lei, “Enhancing Thermostability of Escherichia coli Phytase AppA2 by Error-Prone PCR,” Applied Microbiology and Biotechnology, Vol. 79, No. 1, 2008, pp. 69-75. doi:10.1007/s00253-008-1412-7
[27]	W. Zhu, D. Qiao, M. Huang, G. Yang, H. Xu and Y. Cao, “Modifying Thermostability of AppA from Escherichia coli,” Current Microbiology, Vol. 61, No. 4, 2010, pp. 267-273. doi:10.1007/s00284-010-9606-5