Conserved Amino Acid Moieties of Candidatus Desulforudis audaxviator MazF Determine Ribonuclease Activity and Specificity

The toxin-antitoxin (TA) system, inherent to various prokaryotes, plays a critical role in survival and adaptation to diverse environmental stresses. The toxin MazF, belonging to the type II TA system, functions as a sequence-specific ribonuclease that recognizes 3 to 7 bases. In recent studies, cry...

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Autores principales:	Hiroko Tamiya-Ishitsuka, Masako Tsuruga, Naohiro Noda, Akiko Yokota
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	Candidatus Desulforudis audaxviator cell-free expression sequence-specific ribonuclease toxin-antitoxin system RNA binding Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/d93da86ccfd44f4c862a8db7828bc9d2
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spelling	oai:doaj.org-article:d93da86ccfd44f4c862a8db7828bc9d22021-11-15T19:08:42ZConserved Amino Acid Moieties of Candidatus Desulforudis audaxviator MazF Determine Ribonuclease Activity and Specificity1664-302X10.3389/fmicb.2021.748619https://doaj.org/article/d93da86ccfd44f4c862a8db7828bc9d22021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fmicb.2021.748619/fullhttps://doaj.org/toc/1664-302XThe toxin-antitoxin (TA) system, inherent to various prokaryotes, plays a critical role in survival and adaptation to diverse environmental stresses. The toxin MazF, belonging to the type II TA system, functions as a sequence-specific ribonuclease that recognizes 3 to 7 bases. In recent studies, crystallographic analysis of MazFs from several species have suggested the presence of amino acid sites important for MazF substrate RNA binding and for its catalytic activity. Herein, we characterized MazF obtained from Candidatus Desulforudis audaxviator (MazF-Da) and identified the amino acid residues necessary for its catalytic function. MazF-Da, expressed using a cell-free protein synthesis system, is a six-base-recognition-specific ribonuclease that preferentially cleaves UACAAA sequences and weakly cleaves UACGAA and UACUAA sequences. We found that MazF-Da exhibited the highest activity at around 60°C. Analysis using mutants with a single mutation at an amino acid residue site that is well conserved across various MazF toxins showed that G18, E20, R25, and P26 were important for the ribonuclease activity of MazF-Da. The recognition sequence of the N36A mutant differed from that of the wild type. This mutant cleaved UACAAG sequences in addition to UACAAA sequences, but did not cleave UACGAA or UACUAA sequences, suggesting that Asn36 affects the loosening and narrowing of MazF-Da cleavage sequence recognition. Our study posits UACAAA as the recognition sequence of MazF-Da and provides insight into the amino acid sites that are key to its unique enzymatic properties.Hiroko Tamiya-IshitsukaMasako TsurugaNaohiro NodaAkiko YokotaFrontiers Media S.A.articleCandidatus Desulforudis audaxviatorcell-free expressionsequence-specific ribonucleasetoxin-antitoxin systemRNA bindingMicrobiologyQR1-502ENFrontiers in Microbiology, Vol 12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Candidatus Desulforudis audaxviator cell-free expression sequence-specific ribonuclease toxin-antitoxin system RNA binding Microbiology QR1-502
spellingShingle	Candidatus Desulforudis audaxviator cell-free expression sequence-specific ribonuclease toxin-antitoxin system RNA binding Microbiology QR1-502 Hiroko Tamiya-Ishitsuka Masako Tsuruga Naohiro Noda Akiko Yokota Conserved Amino Acid Moieties of Candidatus Desulforudis audaxviator MazF Determine Ribonuclease Activity and Specificity
description	The toxin-antitoxin (TA) system, inherent to various prokaryotes, plays a critical role in survival and adaptation to diverse environmental stresses. The toxin MazF, belonging to the type II TA system, functions as a sequence-specific ribonuclease that recognizes 3 to 7 bases. In recent studies, crystallographic analysis of MazFs from several species have suggested the presence of amino acid sites important for MazF substrate RNA binding and for its catalytic activity. Herein, we characterized MazF obtained from Candidatus Desulforudis audaxviator (MazF-Da) and identified the amino acid residues necessary for its catalytic function. MazF-Da, expressed using a cell-free protein synthesis system, is a six-base-recognition-specific ribonuclease that preferentially cleaves UACAAA sequences and weakly cleaves UACGAA and UACUAA sequences. We found that MazF-Da exhibited the highest activity at around 60°C. Analysis using mutants with a single mutation at an amino acid residue site that is well conserved across various MazF toxins showed that G18, E20, R25, and P26 were important for the ribonuclease activity of MazF-Da. The recognition sequence of the N36A mutant differed from that of the wild type. This mutant cleaved UACAAG sequences in addition to UACAAA sequences, but did not cleave UACGAA or UACUAA sequences, suggesting that Asn36 affects the loosening and narrowing of MazF-Da cleavage sequence recognition. Our study posits UACAAA as the recognition sequence of MazF-Da and provides insight into the amino acid sites that are key to its unique enzymatic properties.
format	article
author	Hiroko Tamiya-Ishitsuka Masako Tsuruga Naohiro Noda Akiko Yokota
author_facet	Hiroko Tamiya-Ishitsuka Masako Tsuruga Naohiro Noda Akiko Yokota
author_sort	Hiroko Tamiya-Ishitsuka
title	Conserved Amino Acid Moieties of Candidatus Desulforudis audaxviator MazF Determine Ribonuclease Activity and Specificity
title_short	Conserved Amino Acid Moieties of Candidatus Desulforudis audaxviator MazF Determine Ribonuclease Activity and Specificity
title_full	Conserved Amino Acid Moieties of Candidatus Desulforudis audaxviator MazF Determine Ribonuclease Activity and Specificity
title_fullStr	Conserved Amino Acid Moieties of Candidatus Desulforudis audaxviator MazF Determine Ribonuclease Activity and Specificity
title_full_unstemmed	Conserved Amino Acid Moieties of Candidatus Desulforudis audaxviator MazF Determine Ribonuclease Activity and Specificity
title_sort	conserved amino acid moieties of candidatus desulforudis audaxviator mazf determine ribonuclease activity and specificity
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/d93da86ccfd44f4c862a8db7828bc9d2
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Conserved Amino Acid Moieties of Candidatus Desulforudis audaxviator MazF Determine Ribonuclease Activity and Specificity

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