Effects of N-Terminal and C-Terminal Polyhistidine Tag on the Stability and Function of the Thermophilic P450 CYP119

Clicks: 208
ID: 7877
2019
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Abstract
Biocatalysts are sought-after in synthesis of pharmaceuticals and agrochemicals due to their high regioselectivity and enantioselectivity. Among biocatalysts, heme-containing cytochrome P450 (P450) oxygenases are an attractive target since they catalyze oxidation of “unactivated” carbon-hydrogen bonds with high efficiency. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius, which has the potential to be widely used as a biocatalyst since it shows activity at high temperatures and low pH. Polyhistidine tags (His-tags) are widely used to simplify purification of proteins. However, His-tags can cause changes to protein structure and function. Here, we demonstrate the effects of His-tags on CYP119. To this end, the His-tags were cloned at the N-terminus or C-terminus of the CYP119, and His-tagged proteins were expressed and isolated. The thermostability and peroxidase activity of His-tagged CYP119s were tested and compared to wild type CYP119. Results indicated that while addition of His-tags increased the yield and simplified isolation of CYP119, they also influenced the electronic structure of active site and the activity of the protein. We show that N-terminal His-tagged CYP119 has desirable properties and potential to be used in industrial applications, but mechanistic studies using this protein need careful interpretation since the His-tag affects electronic properties of the active site heme iron.
Reference Key
yaprak2019effectsbioinorganic Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Aslantas, Yaprak;Surmeli, Nur Basak;Aslantas, Yaprak;Surmeli, Nur Basak;
Journal bioinorganic chemistry and applications
Year 2019
DOI
10.1155/2019/8080697
URL
https://www.hindawi.com/journals/bca/2019/8080697/abs/
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