Modulating the pK_a of a Tyrosine in KlenTaq DNA Polymerase that Is Crucial for Abasic Site Bypass by in Vivo Incorporation of a Non‐canonical Amino Acid

Abstract

It is estimated that about 10,000 abasic sites are formed per day per cell. Abasic sites impose a significant challenge for bypass synthesis by DNA polymerases. Recently, a tyrosine in KlenTaq DNA polymerase has been highlighted as being crucial for nucleotide selection opposite abasic sites. Structural data indicated a hydrogen bond between the tyrosine's hydroxy group and the N3 of an incoming ddATP opposite the abasic site. In order to further investigate abasic site bypass, we incorporated the unnatural amino acid 2,3,5-trifluorotyrosine at the position of the crucial tyrosine of KlenTaq DNA polymerase. Fluorine substitution at the tyrosine decreased the pka value of the tyrosine's hydroxy group and allowed its protonation state to be modulated. Single-nucleotide-incorporation experiments revealed reduced activity for the KlenTaq mutant compared to the wild-type when bypassing an abasic site analogue. The finding stresses the involvement of this tyrosine and its hydrogen bonding in abasic site bypass.

Towards applications of synthetic genetic polymers in diagnosis and therapy

“…Marx & colleagues have been very active in the field, even adopting the use of unnatural amino acids as part of the polymerase polypeptide, with potential applications to expand polymerase function [38]. Benner and coworkers, as part of their long-term goal of developing a high-fidelity expanded genetic alphabet as described above, recently evolved a Taq polymerase variant capable of efficiently amplifying sequences containing the artificial Z:P base pair, which relies on an unnatural hydrogenbonding pattern [39].…”

Unnatural amino acid mutagenesis-based enzyme engineering

“…Likewise, site-specific incorporation of 2,3, 5-trifluorotyrosine, 20, into ribonucleotide reductase was performed to investigate the significance of a stable tyrosine radical pathway for maintaining enzyme conformation and activity during catalysis [54]. Incorporating 20 site-specifically into the KlenTaq DNA polymerase active site allowed the investigators to probe the role and importance of specific hydrogen bond interactions in the abasic bypass site, based on the modulation of pKa values [55]. Further, site-specific incorporation of isotopically labeled UAA such as 2-nitrobenzyltyrosine, 21, 2-amino-3-(4-(trifluoromethoxy)phenyl)-propanoic acid, 22, and 4-methoxyphenylalanine, 23, at 11 different positions around the proposed binding site in the thioesterase domain of human fatty acid synthase demonstrated the utility of NMR-active UAAs as a tool to probe the structure, dynamics, and ligand binding of enzymes [56].…”

Biocatalysis with Unnatural Amino Acids: Enzymology Meets Xenobiology

“…Thes ubstitution dramatically reduced the activity,but it revealed the importance of the pK a value of the Ty rr esidue for hydrogen bonding with the target DNA. [105] Then itroreductase from E. coli (NTR) reduces the nitro group of various compounds.NTR is currently used as alocal prodrug activator for cancer therapy,a lthough it has the limitation of ah igh K m value towards the substrate CB1954 (Scheme 8).…”