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Protein engineering of class-A non-specific acid phosphatase (PhoN) of Salmonella typhimurium: Modulation of the pH-activity profile

Paper ID Volume ID Publish Year Pages File Format Full-Text
14173 1179 2006 5 PDF Available
Title
Protein engineering of class-A non-specific acid phosphatase (PhoN) of Salmonella typhimurium: Modulation of the pH-activity profile
Abstract

Engineering of the PhoN enzyme of Salmonella typhimurium due to its superior characteristics for bioremediation of heavy metals has been advocated by Macaskie and colleagues [Basnakova, G., Stephens, E.R., Thaller, M.C., Rossolini, G.M., Macaskie, L.E., 1998. The use of Escherichia coli bearing a phoN gene for the removal of uranium and nickel from aqueous flows. Appl. Microbiol. Biotechnol. 50, 266–272]. The native enzyme hydrolyzes disparate organophosphates and exhibits optimal phosphatase activity at pH 5.5, for instance, with substrate p-nitrophenyl phosphate. Structurally guided Ile-78 was mutated using site-directed mutagenesis to Ala, Asp and His residues, with an aim to shift the optimum pH of the PhoN enzyme. Encouragingly, the I78A mutant displays significantly higher (as high as 160 %) enzymatic efficiency over a broad pH range of 3.0–9.0, compared to the wild-type PhoN. The higher catalytic efficiency is due to the increase in kcat, and can be mainly attributed to a deshielding of catalytic His-158 from the bulk-solvent. The I78D mutant possesses nearly twice the specific activity at the optimum pH of 7.0. The alkaline shift of the pH-activity profile agrees well with reasoning based on electrostatics. An increase in Km, however, lowers the catalytic efficiency of the I78D mutant at the optimum pH. The I78H mutant, counter-intuitively, also exhibits an alkaline shift in the pH-optimum. Nonetheless, the active site scaffold in I78H mutant may not be disturbed, as similar steady-state kinetic parameters are observed for both I78H mutant and wild-type PhoN at their respective pH optima.

Keywords
Site-directed mutagenesis; PhoN protein of Salmonella typhimurium; Class-A bacterial non-specific acid phosphatase; Protein engineering; pH-optimum; Bioremediation
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Protein engineering of class-A non-specific acid phosphatase (PhoN) of Salmonella typhimurium: Modulation of the pH-activity profile
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biomolecular Engineering - Volume 23, Issue 5, October 2006, Pages 247–251
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
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Any Questions? feel free to contact us