Journal
BIOTECHNOLOGY AND BIOENGINEERING
Volume 94, Issue 5, Pages 921-930Publisher
WILEY
DOI: 10.1002/bit.20910
Keywords
protein-engineering; trifluoroleucine; activity; thermostability; non-natural amino acid; residue-specific incorporation
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Funding
- NIGMS NIH HHS [GM 62523, 5F32 GM 67375-02] Funding Source: Medline
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Varied levels of fluorinated amino acid have been introduced biosynthetically to test the functional limits of global substitution on enzymatic activity and stability. Replacement of all the leucine (LEU) residues in the enzyme chloramphenicol acetyltransf erase (CAT) with the analog, 51,5',5-trifluoroleucine (TFL), results in the maintenance of enzymatic activity under ambient temperatures as well as an enhancement in secondary structure but loss in stability against heat and denaturants or organic co-solvents. Although catalytic activity of the fully substituted CAT is preserved understanclard reaction conditions compared to the wild-type enzyme both in vitro and in vivo, as the incorporation levels increase, a concomitant reduction in thermostability and chemostability is observed. Circular dichroism (CD) studies reveal that although fluorination greatly improvesthe secondary structure of CAT, a large structural destabilization upon increased levels of TFL incorporation occurs at elevated temperatures. These data suggest that enhanced secondary structure afforded by TFL incorporation does not necessarily lead to an improvement in stability. (c) 2006 Wiley Periodicals, Inc.
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