Open AccessProbing the role of the residues in the active site of the transaminase from Thermobaculum terrenum
Author(s) -
Ekaterina Yu. Bezsudnova,
A.Y. Nikolaeva,
Alina K. Bakunova,
Tatiana V. Rakitina,
Dmitry A. Suplatov,
В. О. Попов,
Konstantin M. Boyko
Publication year - 2021
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0255098
Subject(s) - transamination , active site , transaminase , amination , biochemistry , enzyme , amine gas treating , stereochemistry , amino acid , mutagenesis , chemistry , substrate (aquarium) , pyridoxal phosphate , biology , cofactor , catalysis , organic chemistry , mutation , ecology , gene
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.