Influence of global fluorination on chloramphenicol acetyltransferase activity and stability

Tatyana Panchenko, Wan Wen Zhu, Jin Kim Montclare

Research output: Contribution to journalArticlepeer-review

Abstract

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 acetyltransferase (CAT) with the analog, 5′,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 understandard 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 improves the 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.

Original languageEnglish (US)
Pages (from-to)921-930
Number of pages10
JournalBiotechnology and Bioengineering
Volume94
Issue number5
DOIs
StatePublished - Aug 5 2006

Keywords

  • Activity
  • Non-natural amino acid
  • Protein engineering
  • Residue-specific incorporation
  • Thermostability
  • Trifluoroleucine

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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