Abstract
Creating biocatalysts for (R)-selective amination effectively is highly desirable in organic synthesis. Despite noticeable progress in the engineering of (R)-amine activity in pyridoxal-5’-phosphate-dependent transaminases of fold type IV, the specialization of the activity is still an intuitive task, as there is poor understanding of sequence-structure-function relationships. In this study, we analyzed this relationship in transaminase from Thermobaculum terrenum, distinguished by expanded substrate specificity and activity in reactions with L-amino acids and (R)-(+)-1-phenylethylamine using α-ketoglutarate and pyruvate as amino acceptors. We performed site-directed mutagenesis to create a panel of the enzyme variants, which differ in the active site residues from the parent enzyme to a putative transaminase specific to (R)-primary amines. The variants were examined in the overall transamination reactions and half-reaction with (R)-(+)-1-phenylethylamine. A structural analysis of the most prominent variants revealed a spatial reorganization in the active sites, which caused changes in activity. Although the specialization to (R)-amine transaminase was not implemented, we succeeded in understanding the role of the particular active site residues in expanding substrate specificity of the enzyme. We showed that the specificity for (R)-(+)-1-phenylethylamine in transaminase from T. terrenum arises without sacrificing the specificity for L-amino acids and α-ketoglutarate and in consensus with it.
Highlights
MethodsThe TaTT variants were created through site-directed mutagenesis using the modified QuikChange protocol as described in [33]
We suggested that TaTT as a transaminase with an expanded substrate specificity can be changed more effectively than canonical branched-chain L-amino acid transaminases (BCATs) or D-amino acid aminotransferases (DAATs) and chose a strategy to increase the R-TA-like activity of TaTT by changing residues in the active site
We chose the sites for mutagenesis based on the structural similarity between BCATs and R-TAs (S3 Table) and the significance of subfamily-specific positions (SSP) that may contribute to the selective recognition of substrates (Table 2)
Summary
The TaTT variants were created through site-directed mutagenesis using the modified QuikChange protocol as described in [33]. Oligonucleotides used as primers for mutagenesis and mutation verification are listed in S1 Table. Eighteen cycles of PCR amplification were performed on an expression plasmid carrying a wild-type TaTT (WT TaTT) gene using the Tersus Plus PCR kit (Evrogen, Russia) and mutagenesis primers (S1 Table). Clones carrying target mutations were identified by a colony PCR assay performed using Taq DNA polymerase and a pair of primers—a check primer specified in S1 Table and the corresponding T7 universal primer. The combination of several mutations was achieved by step-by-step point mutagenesis as described above. All selected clones were sequenced on ABI 3730xl DNA Analyzer (Applied Biosystems, USA)
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