Probing the role of the residues in the active site of the transaminase from Thermobaculum terrenum.

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 intuitiv...

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Autores principales:	Ekaterina Yu Bezsudnova, Alena Yu Nikolaeva, Alina K Bakunova, Tatiana V Rakitina, Dmitry A Suplatov, Vladimir O Popov, Konstantin M Boyko
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/8d9e7e54227c47c895f595f8bffbc68c
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spelling	oai:doaj.org-article:8d9e7e54227c47c895f595f8bffbc68c2021-12-02T20:08:57ZProbing the role of the residues in the active site of the transaminase from Thermobaculum terrenum.1932-620310.1371/journal.pone.0255098https://doaj.org/article/8d9e7e54227c47c895f595f8bffbc68c2021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0255098https://doaj.org/toc/1932-6203Creating 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.Ekaterina Yu BezsudnovaAlena Yu NikolaevaAlina K BakunovaTatiana V RakitinaDmitry A SuplatovVladimir O PopovKonstantin M BoykoPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 7, p e0255098 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ekaterina Yu Bezsudnova Alena Yu Nikolaeva Alina K Bakunova Tatiana V Rakitina Dmitry A Suplatov Vladimir O Popov Konstantin M Boyko Probing the role of the residues in the active site of the transaminase from Thermobaculum terrenum.
description	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.
format	article
author	Ekaterina Yu Bezsudnova Alena Yu Nikolaeva Alina K Bakunova Tatiana V Rakitina Dmitry A Suplatov Vladimir O Popov Konstantin M Boyko
author_facet	Ekaterina Yu Bezsudnova Alena Yu Nikolaeva Alina K Bakunova Tatiana V Rakitina Dmitry A Suplatov Vladimir O Popov Konstantin M Boyko
author_sort	Ekaterina Yu Bezsudnova
title	Probing the role of the residues in the active site of the transaminase from Thermobaculum terrenum.
title_short	Probing the role of the residues in the active site of the transaminase from Thermobaculum terrenum.
title_full	Probing the role of the residues in the active site of the transaminase from Thermobaculum terrenum.
title_fullStr	Probing the role of the residues in the active site of the transaminase from Thermobaculum terrenum.
title_full_unstemmed	Probing the role of the residues in the active site of the transaminase from Thermobaculum terrenum.
title_sort	probing the role of the residues in the active site of the transaminase from thermobaculum terrenum.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/8d9e7e54227c47c895f595f8bffbc68c
work_keys_str_mv	AT ekaterinayubezsudnova probingtheroleoftheresiduesintheactivesiteofthetransaminasefromthermobaculumterrenum AT alenayunikolaeva probingtheroleoftheresiduesintheactivesiteofthetransaminasefromthermobaculumterrenum AT alinakbakunova probingtheroleoftheresiduesintheactivesiteofthetransaminasefromthermobaculumterrenum AT tatianavrakitina probingtheroleoftheresiduesintheactivesiteofthetransaminasefromthermobaculumterrenum AT dmitryasuplatov probingtheroleoftheresiduesintheactivesiteofthetransaminasefromthermobaculumterrenum AT vladimiropopov probingtheroleoftheresiduesintheactivesiteofthetransaminasefromthermobaculumterrenum AT konstantinmboyko probingtheroleoftheresiduesintheactivesiteofthetransaminasefromthermobaculumterrenum
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Probing the role of the residues in the active site of the transaminase from Thermobaculum terrenum.

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