Insertion of an unnatural amino acid into the protein structure: preparation and properties of 3-fluorotyrosine-containing organophosphate hydrolase

Abstract

Five Tyr residues present in the native organophosphate hydrolase (OPH) containing a hexahistidine tag at the N terminus of the protein molecule (His 6 OPH) were replaced by fluorine containing analogs using a biosynthetic approach. The modified enzyme had an extended pH optimum of action shifted to acidic pH and an enhanced thermal stability in the alkaline pH region.

Chemical Synthesis of Site‐Specifically 2′‐Azido‐Modified RNA and Potential Applications for Bioconjugation and RNA Interference

“…Incorporating NCAA can also facilitate NMR spectroscopic studies of proteins (reviewed in [16]). In particular, labeling a protein with a NCAA containing a 19 F atom is a very popular method to characterize protein structural changes by NMR spectroscopy [17–22]. Third, NCAAs containing a heavy atom, such as selenium, bromine, or iodine, are widely used to facilitate X‐ray crystal‐structure determination of proteins [23–26].…”

Biocatalysis with Unnatural Amino Acids: Enzymology Meets Xenobiology

“…Similarly,V otchitseva et al [120] demonstrated that the global replacement of Tyrb yi ts monofluorinated analogue mFY in organophosphate hydrolase (OPH) improved the stability of protein towards high temperature and alkaline pH conditions.M oreover,t he pH-optimum of the variant was shifted to more acidic values,thereby conferring asignificant catalytic advantage over the WT under acidic conditions. Montclare and co-workers [121] replaced all 13 leucine (Leu) residues in chloramphenicol acetyl transferase (CAT) by 5,5,5-trifluoroleucine (Tfl, Figure 4) through SPI.…”

Unnatural amino acid mutagenesis-based enzyme engineering

“…Further, organophosphate hydrolase engineered by substituting tyrosine with 8 had an extended pH optimum of activity at acidic pH and improved thermal stability at alkaline pH. In addition, 40% residual activity was maintained in the fluorinated enzyme after heating at 558C, whereas the wild type enzyme retained only 13% activity [36]. More recently, engineering v-transaminase (v-TA) by global substitution of tyrosine by 8 yielded enhanced activity up to 20% higher than that of the wild type enzyme.…”