Abstract
The maximum activity of Yersinia enterocolitica phytase (YeAPPA) occurs at pH 5.0 and 45 °C, and notably, its specific activity (3.28 ± 0.24 U mg−1) is 800-fold less than that of its Yersinia kristeensenii homolog (YkAPPA; 88% amino acid sequence identity). Sequence alignment and molecular modeling show that the arginine at position 79 (Arg79) in YeAPPA corresponding to Gly in YkAPPA as well as other histidine acid phosphatase (HAP) phytases is the only non-conserved residue near the catalytic site. To characterize the effects of the corresponding residue on the specific activities of HAP phytases, Escherichia coli EcAPPA, a well-characterized phytase with a known crystal structure, was selected for mutagenesis—its Gly73 was replaced with Arg, Asp, Glu, Ser, Thr, Leu, or Tyr. The results show that the specific activities of all of the corresponding EcAPPA mutants (17–2,400 U mg−1) were less than that of the wild-type phytase (3,524 U mg−1), and the activity levels were approximately proportional to the molecular volumes of the substituted residues’ side chains. Site-directed replacement of Arg79 in YeAPPA (corresponding to Gly73 of EcAPPA) with Ser, Leu, and Gly largely increased the specific activity, which further verified the key role of the residue at position 79 for determining phytase activity. Thus, a new determinant that influences the catalytic efficiency of HAP phytases has been identified.
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Acknowledgements
This research was supported by the Key Program of Transgenic Plant Breeding (2009ZX08019-002B) and the Agricultural Science and Technology Conversion Funds (2008GB23260388) and the Earmarked Fund for Modern Agro-industry Technology Research System (NYCYTX-42-G2-05).
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Dawei Fu and Zhongyuan Li contributed equally to this paper.
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Fu, D., Li, Z., Huang, H. et al. Catalytic efficiency of HAP phytases is determined by a key residue in close proximity to the active site. Appl Microbiol Biotechnol 90, 1295–1302 (2011). https://doi.org/10.1007/s00253-011-3171-0
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DOI: https://doi.org/10.1007/s00253-011-3171-0