Influence of global fluorination on chloramphenicol acetyltransferase activity and stability

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 under standard 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.

Highly fluorinated amphiphilic molecules and self-assemblies with biomedical potential

“…Replacement of all the leucine residues by 5′,5′,5′-trifluoroleucines in the enzyme chloramphenicol acetyltransferase (CAT) preserved enzymatic activity at an ambient temperature as well as an enhancement in secondary structure [112]. However, chemical and thermal destabilizations were noted at elevated temperature that increased with increased trifluoroleucine incorporation.…”

“…Yet, remarkably, many studies indicate that in spite of notable changes related to the differences in steric demand, polarizability and electronegativity between CF 3 and CH 3 groups, and modifications in dipole moments and surface activity, substantially fluorinated proteins often retain the structure and activity of their native model [112][113][114]. Incorporation of trifluoromethyl alanine and other fluorinated amino acids in small chemotactic peptides preserved some of their aptitude at stimulating chemiluminescence in neutrophil cells [115].…”

Effects of Fluorination on the Folding Kinetics of a Heterodimeric Coiled Coil

“…As far as DfeGly and TfeGly are concerned, increasing degrees of fluorination apparently decrease the rate of coiled‐coil association. Although still awaiting direct experimental proof, we6a and others11 have proposed that this effect may be attributed to fluorine–fluorine contacts in the unfolded state. The assumption is that these contacts lead to an arrangement of the peptides that is inconsistent with correct folding and thus compete with structure formation.…”

Posttranslational Modification of Proteins Incorporating Nonnatural Amino Acids

“…Many functional groups including alkyne, alkene, ketone, and azides have been incorporated into the proteins via NAA, providing an orthogonal handle for protein modifications [122,223,224]. Halogenated NAAs, especially fluorinated amino acids, have made a significant impact on protein engineering [133,155,[225][226][227][228][229][230][231][232]. Repetitive protein polymers with fluorinated amino acid have shown distinct physicochemical properties, including an increase in thermal and chemical stability [155,228], as a result of the electronegativity and hydrophobicity of the fluorine atom [233,234].…”