Mapping N- and C-terminals of Leishmania donovani tyrosine aminotransferase by gene truncation strategy: a functional study using in vitro and in silico approaches

Abstract Tyrosine aminotransferase (TAT) catalyzes the transamination of amino acids in Leishmania sp.. TAT from Leishmania donovani has been found to be extremely stable at extreme temperatures and pH conditions. This study was conceived to map the functions of the non-conserved N-terminal and cons...

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Autores principales:	Santanu Sasidharan, Prakash Saudagar
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/936c00f3dbe64414b436bbd4b80b5654
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spelling	oai:doaj.org-article:936c00f3dbe64414b436bbd4b80b56542021-12-02T16:06:39ZMapping N- and C-terminals of Leishmania donovani tyrosine aminotransferase by gene truncation strategy: a functional study using in vitro and in silico approaches10.1038/s41598-020-69512-y2045-2322https://doaj.org/article/936c00f3dbe64414b436bbd4b80b56542020-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-69512-yhttps://doaj.org/toc/2045-2322Abstract Tyrosine aminotransferase (TAT) catalyzes the transamination of amino acids in Leishmania sp.. TAT from Leishmania donovani has been found to be extremely stable at extreme temperatures and pH conditions. This study was conceived to map the functions of the non-conserved N-terminal and conserved C-terminal domain of TAT. N-terminal (NTAT) and C-terminal (CTAT) domain of TAT was truncated and cloned into the pET28a(+) vector. The truncated proteins were expressed, purified, and biochemically characterized. The K m of NTAT and CTAT for the tyrosine-pyruvate pair was determined to be 3.468 ± 0.796 mM and 4.581 ± 0.627 mM, repectively. Temperature and pH stability studies found NTAT to be stable like TAT but CTAT was extremely susceptible to temperature and pH changes. Upon docking and simulation for 100 ns, NTAT had lower SASA values. From UV spectroscopic study, PLP bound better to CTAT than NTAT because of the reduced SASA of NTAT. The sensitivity of CTAT was reasoned when the urea denaturation studies showed two-state denaturation which differed from NTAT’s and TAT’s biphasic folding mechanism. From this study, the authors hypothesize that the N-terminal is responsible for PLP stabilization and C-terminal protects the active site from extreme conditions.Santanu SasidharanPrakash SaudagarNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-15 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Santanu Sasidharan Prakash Saudagar Mapping N- and C-terminals of Leishmania donovani tyrosine aminotransferase by gene truncation strategy: a functional study using in vitro and in silico approaches
description	Abstract Tyrosine aminotransferase (TAT) catalyzes the transamination of amino acids in Leishmania sp.. TAT from Leishmania donovani has been found to be extremely stable at extreme temperatures and pH conditions. This study was conceived to map the functions of the non-conserved N-terminal and conserved C-terminal domain of TAT. N-terminal (NTAT) and C-terminal (CTAT) domain of TAT was truncated and cloned into the pET28a(+) vector. The truncated proteins were expressed, purified, and biochemically characterized. The K m of NTAT and CTAT for the tyrosine-pyruvate pair was determined to be 3.468 ± 0.796 mM and 4.581 ± 0.627 mM, repectively. Temperature and pH stability studies found NTAT to be stable like TAT but CTAT was extremely susceptible to temperature and pH changes. Upon docking and simulation for 100 ns, NTAT had lower SASA values. From UV spectroscopic study, PLP bound better to CTAT than NTAT because of the reduced SASA of NTAT. The sensitivity of CTAT was reasoned when the urea denaturation studies showed two-state denaturation which differed from NTAT’s and TAT’s biphasic folding mechanism. From this study, the authors hypothesize that the N-terminal is responsible for PLP stabilization and C-terminal protects the active site from extreme conditions.
format	article
author	Santanu Sasidharan Prakash Saudagar
author_facet	Santanu Sasidharan Prakash Saudagar
author_sort	Santanu Sasidharan
title	Mapping N- and C-terminals of Leishmania donovani tyrosine aminotransferase by gene truncation strategy: a functional study using in vitro and in silico approaches
title_short	Mapping N- and C-terminals of Leishmania donovani tyrosine aminotransferase by gene truncation strategy: a functional study using in vitro and in silico approaches
title_full	Mapping N- and C-terminals of Leishmania donovani tyrosine aminotransferase by gene truncation strategy: a functional study using in vitro and in silico approaches
title_fullStr	Mapping N- and C-terminals of Leishmania donovani tyrosine aminotransferase by gene truncation strategy: a functional study using in vitro and in silico approaches
title_full_unstemmed	Mapping N- and C-terminals of Leishmania donovani tyrosine aminotransferase by gene truncation strategy: a functional study using in vitro and in silico approaches
title_sort	mapping n- and c-terminals of leishmania donovani tyrosine aminotransferase by gene truncation strategy: a functional study using in vitro and in silico approaches
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/936c00f3dbe64414b436bbd4b80b5654
work_keys_str_mv	AT santanusasidharan mappingnandcterminalsofleishmaniadonovanityrosineaminotransferasebygenetruncationstrategyafunctionalstudyusinginvitroandinsilicoapproaches AT prakashsaudagar mappingnandcterminalsofleishmaniadonovanityrosineaminotransferasebygenetruncationstrategyafunctionalstudyusinginvitroandinsilicoapproaches
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Mapping N- and C-terminals of Leishmania donovani tyrosine aminotransferase by gene truncation strategy: a functional study using in vitro and in silico approaches

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