A new method to investigate the catalytic mechanism of YhdE pyrophosphatase by using a pyrophosphate fluorescence probe

Abstract YhdE is a Maf (multicopy associated filamentation) proteins from Escherichia coli which exhibits pyrophosphatase activity towards selected nucleotides, although its catalytic mechanism remains unclear. Herein we used a novel fluorescence probe (4-isoACBA–Zn(II) complex) to characterize the...

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Autores principales:	Qingya Shen, Hongwei Tan, Guo-wen Xing, Jimin Zheng, Zongchao Jia
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/91bceb3d4a0e41c5ac21ecf6090b2147
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spelling	oai:doaj.org-article:91bceb3d4a0e41c5ac21ecf6090b21472021-12-02T16:06:21ZA new method to investigate the catalytic mechanism of YhdE pyrophosphatase by using a pyrophosphate fluorescence probe10.1038/s41598-017-08368-12045-2322https://doaj.org/article/91bceb3d4a0e41c5ac21ecf6090b21472017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-08368-1https://doaj.org/toc/2045-2322Abstract YhdE is a Maf (multicopy associated filamentation) proteins from Escherichia coli which exhibits pyrophosphatase activity towards selected nucleotides, although its catalytic mechanism remains unclear. Herein we used a novel fluorescence probe (4-isoACBA–Zn(II) complex) to characterize the enzymatic properties of YhdE and its mutant, establishing a new method for assaying pyrophosphatase catalytic function. Our results reveal for the first time that the new fluorescence sensor confers high sensitivity and specificity and pyrophosphate (PPi) is the direct catalytic product of YhdE. Crystal structures of a mutant in the active-site loop (YhdE_E33A) show conformational flexibility implicated in the catalytic mechanism of YhdE. ITC experiments and computational docking further reveal that Asp70 and substrate dTTP coordinate Mn2+. Quantum mechanics calculations indicate that YhdE hydrolysis appears to follow a stepwise pathway in which a water molecule first attacks the α-phosphorus atom in the substrate, followed by the release of PPi from the pentavalent intermediate.Qingya ShenHongwei TanGuo-wen XingJimin ZhengZongchao JiaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-8 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Qingya Shen Hongwei Tan Guo-wen Xing Jimin Zheng Zongchao Jia A new method to investigate the catalytic mechanism of YhdE pyrophosphatase by using a pyrophosphate fluorescence probe
description	Abstract YhdE is a Maf (multicopy associated filamentation) proteins from Escherichia coli which exhibits pyrophosphatase activity towards selected nucleotides, although its catalytic mechanism remains unclear. Herein we used a novel fluorescence probe (4-isoACBA–Zn(II) complex) to characterize the enzymatic properties of YhdE and its mutant, establishing a new method for assaying pyrophosphatase catalytic function. Our results reveal for the first time that the new fluorescence sensor confers high sensitivity and specificity and pyrophosphate (PPi) is the direct catalytic product of YhdE. Crystal structures of a mutant in the active-site loop (YhdE_E33A) show conformational flexibility implicated in the catalytic mechanism of YhdE. ITC experiments and computational docking further reveal that Asp70 and substrate dTTP coordinate Mn2+. Quantum mechanics calculations indicate that YhdE hydrolysis appears to follow a stepwise pathway in which a water molecule first attacks the α-phosphorus atom in the substrate, followed by the release of PPi from the pentavalent intermediate.
format	article
author	Qingya Shen Hongwei Tan Guo-wen Xing Jimin Zheng Zongchao Jia
author_facet	Qingya Shen Hongwei Tan Guo-wen Xing Jimin Zheng Zongchao Jia
author_sort	Qingya Shen
title	A new method to investigate the catalytic mechanism of YhdE pyrophosphatase by using a pyrophosphate fluorescence probe
title_short	A new method to investigate the catalytic mechanism of YhdE pyrophosphatase by using a pyrophosphate fluorescence probe
title_full	A new method to investigate the catalytic mechanism of YhdE pyrophosphatase by using a pyrophosphate fluorescence probe
title_fullStr	A new method to investigate the catalytic mechanism of YhdE pyrophosphatase by using a pyrophosphate fluorescence probe
title_full_unstemmed	A new method to investigate the catalytic mechanism of YhdE pyrophosphatase by using a pyrophosphate fluorescence probe
title_sort	new method to investigate the catalytic mechanism of yhde pyrophosphatase by using a pyrophosphate fluorescence probe
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/91bceb3d4a0e41c5ac21ecf6090b2147
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A new method to investigate the catalytic mechanism of YhdE pyrophosphatase by using a pyrophosphate fluorescence probe

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