Differential effects of isomeric incorporation of fluorophenylalanines into PvuII endonuclease

Abstract

Incorporation of fluorine into proteins has long served as a means of probing structure and function, yet there are few studies that examine the impact of fluorine substitution, particularly at locations distant from the active sites of enzymes. The flexibility of isomeric fluorine incorporation at Phe is used to explore subtle substitution effects on enzyme activity and conformation. The unnatural amino acids o-, m-, and p-fluorophenylalanines were incorporated biosynthetically into the representative PvuII restriction endonuclease. Interestingly, m-fluoro-Phe-PvuII endonuclease exhibits very similar conformational stability to that of the native enzyme, but it exhibits a reproducible, 2-fold higher average specific activity. Given the level of incorporation and the distribution of species, the species of modified enzyme responsible for this increase in specific activity is most likely even faster. Further, moving the fluorine atom from the meta- to the para-position of Phe results in a 4-fold decrease in specific activity and a decrease in conformational stability of 1.5 kcal/mol. Since none of the Phe residues in PvuII endonuclease lies near the DNA recognition or catalytic sites, this differential behavior alludes to the impact of subtle changes in enzyme conformation on endonuclease activity and suggests novel ways to influence catalytic behavior.

Unnatural amino acid mutagenesis-based enzyme engineering

“…Interestingly, 3-fluorophenylalanine, 9, gave a twofold increase in average specific activity, with a similar conformational stability to the wild type enzyme. Structural analysis of PvuII endonuclease revealed that the substituted phenylalanine residue was not located near the catalytic region or in a DNA-binding site [38]. Hence, this work showed that the incorporation of 9 at locations distant from the active site of PvuII endonuclease can alter its catalytic behavior through subtle changes in enzyme conformation.…”

Manipulation of enzyme properties by noncanonical amino acid incorporation

“…If such an isosteric NCAA is required for site-specific incorporation, the endogenous E. coli protein biosynthesis machinery should be modified to prevent global incorporation of the NCAA into unwanted sites. For example, the size and shape of (10) is very close to that of Phe and so (9) is easily introduced into a protein in a residuespecific manner [52,62]. However, we can easily imagine that there are situations in which only a single fluorine atom should be introduced into one site.…”

“…Replacing the phenylalanine residues in PvuII endonuclease with (8) resulted in a specific activity of the modified enzyme twice as high as that of the native enzyme. However, the introduction of (9) or (7) reduced the activity fourfold or did not affect the activity, respectively [52]. Second, phenylalanine residues replaced with fluorinated analogues were not located close to the DNA recognition and catalytic site.…”

“…To explore the impact of fluorine substitution on PvuII, phenylalanine residues were replaced with one of three fluorinated phenylalanine analogues (7), (8), (9) [52]. This study was different from the fluorine substitution in GST described above [50] in two aspects.…”

19F-Modified Proteins and 19F-Containing Ligands as Tools in Solution NMR Studies of Protein Interactions

“…A common approach is to utilize auxotrophic bacterial strains. For the introduction of 19 F-modified aromatic amino acids, an alternative approach is also available: adding glyphosate, an inhibitor of a key step in aromatic amino acid biosynthesis, during cellular growth, suppresses the production of tryptophan, tyrosine, and phenylalanine (Dominguez, Thornton, Melendez, & Dupureur, 2001;Evanics et al, 2006;Hoeltzli & Frieden, 1994;Kim et al, 1990).…”