Manipulation of enzyme properties by noncanonical amino acid incorporation

Abstract

Since wild-type enzymes do not always have the properties needed for various applications, enzymes are often engineered to obtain desirable properties through protein engineering techniques. In the past decade, complementary to the widely used rational protein design and directed evolution techniques, noncanonical amino acid incorporation (NCAAI) has become a new and effective protein engineering technique. Recently, NCAAI has been used to improve intrinsic functions of proteins, such as enzymes and fluorescent proteins, beyond the capacities obtained with natural amino acids. Herein, recent progress on improving enzyme properties through NCAAI in vivo is reviewed and the challenges of current approaches and future directions are also discussed. To date, both NCAAI methods-residue- and site-specific incorporation-have been primarily used to improve the catalytic turnover number and substrate binding affinity of enzymes. Numerous strategies used to minimize structural perturbation and stability loss of a target enzyme upon NCAAI are also explored. Considering the generality of NCAAI incorporation, we expect its application could be expanded to improve other enzyme properties, such as substrate specificity and solvent resistance in the near future.

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

“…[1,2] On the other hand, ncAAs which are orthogonal to the host cell translation system are usually incorporated sitespecifically by suppression of stop codons (SCS) using orthogonal aminoacyl-tRNA synthetase (aaRS) / tRNA pairs (o-pairs). [3] Although the incorporation of one single ncAA into proteins has provided fascinating insights into sequencefunction relationships [2][3][4] as well as potential applications in enzymology, [5][6][7] it is often desirable to introduce simultaneously different chemical functionalities by using two or more ncAAs. [8,9] For example, Budisa and coworkers introduced three ncAAs in parallel into the model protein barstar using the SPI method: [10] the incorporation of the Trp analogue 4-azatryptophan (4AzaTrp) endowed the protein with a blue fluorescent probe for bio-imaging, [11] the Met analogue homopropargylglycine (Hpg) equipped it with a biorthogonal handle for post-translational conjugation by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), [12] while the Pro analogue (4S)-FPro ((4S)-FPro) provided a stabilizing effect on its structure.…”

Unnatural amino acid mutagenesis-based enzyme engineering

“…For example, the residue-specific approach allows UAA incorporation at multiple sites, and this can have synergistic effects in the enzyme. The sitespecific method, by contrast, allows new chemical functionalities to be precisely introduced into enzymes very easily [17]. More recently, an increase in the diversity of UAAs and advances in incorporation methods have made it possible to overcome some existing challenges to engineering biocatalysts.…”

Manipulating the substrate specificity of murine dihydrofolate reductase enzyme using an expanded set of amino acids

“…As an emerging technique, enzyme engineering utilizing nonnatural amino acids as well as natural amino acids have been explored (reviewed in [8]). Such an expanded set of amino acids expands the protein sequence space beyond the chemical/physical limits set by nature [9].…”

“…If the incorporation site of a nonnatural amino acid is appropriately chosen, the perturbation of the enzyme structure and function can be minimized. So far, numerous non-natural amino acids have been introduced into a recombinant protein expressed in industrially important expression hosts, such as Escherichia coli, yeast, and mammalian cells in a site-specific manner [8,11]. Recently, over 500 mg/L of human growth hormone was produced in over 1000-L scale using SCS [30].…”