The role of the methyltransferase domain of bifunctional restriction enzyme RM.BpuSI in cleavage activity.

Restriction enzyme (REase) RM.BpuSI can be described as a Type IIS/C/G REase for its cleavage site outside of the recognition sequence (Type IIS), bifunctional polypeptide possessing both methyltransferase (MTase) and endonuclease activities (Type IIC) and endonuclease activity stimulated by S-adeno...

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Autores principales:	Arthur Sarrade-Loucheur, Shuang-yong Xu, Siu-Hong Chan
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/41b818833a534067b4ec76d48a4dc26a
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spelling	oai:doaj.org-article:41b818833a534067b4ec76d48a4dc26a2021-11-18T08:48:24ZThe role of the methyltransferase domain of bifunctional restriction enzyme RM.BpuSI in cleavage activity.1932-620310.1371/journal.pone.0080967https://doaj.org/article/41b818833a534067b4ec76d48a4dc26a2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24224063/?tool=EBIhttps://doaj.org/toc/1932-6203Restriction enzyme (REase) RM.BpuSI can be described as a Type IIS/C/G REase for its cleavage site outside of the recognition sequence (Type IIS), bifunctional polypeptide possessing both methyltransferase (MTase) and endonuclease activities (Type IIC) and endonuclease activity stimulated by S-adenosyl-L-methionine (SAM) (Type IIG). The stimulatory effect of SAM on cleavage activity presents a major paradox: a co-factor of the MTase activity that renders the substrate unsusceptible to cleavage enhances the cleavage activity. Here we show that the RM.BpuSI MTase activity modifies both cleavage substrate and product only when they are unmethylated. The MTase activity is, however, much lower than that of M1.BpuSI and is thought not to be the major MTase for host DNA protection. SAM and sinefungin (SIN) increase the Vmax of the RM.BpuSI cleavage activity with a proportional change in Km, suggesting the presence of an energetically more favorable pathway is taken. We further showed that RM.BpuSI undergoes substantial conformational changes in the presence of Ca(2+), SIN, cleavage substrate and/or product. Distinct conformers are inferred as the pre-cleavage/cleavage state (in the presence of Ca(2+), substrate or both) and MTase state (in the presence of SIN and substrate, SIN and product or product alone). Interestingly, RM.BpuSI adopts a unique conformation when only SIN is present. This SIN-bound state is inferred as a branch point for cleavage and MTase activity and an intermediate to an energetically favorable pathway for cleavage, probably through increasing the binding affinity of the substrate to the enzyme under cleavage conditions. Mutation of a SAM-binding residue resulted in altered conformational changes in the presence of substrate or Ca(2+) and eliminated cleavage activity. The present study underscores the role of the MTase domain as facilitator of efficient cleavage activity for RM.BpuSI.Arthur Sarrade-LoucheurShuang-yong XuSiu-Hong ChanPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 11, p e80967 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Arthur Sarrade-Loucheur Shuang-yong Xu Siu-Hong Chan The role of the methyltransferase domain of bifunctional restriction enzyme RM.BpuSI in cleavage activity.
description	Restriction enzyme (REase) RM.BpuSI can be described as a Type IIS/C/G REase for its cleavage site outside of the recognition sequence (Type IIS), bifunctional polypeptide possessing both methyltransferase (MTase) and endonuclease activities (Type IIC) and endonuclease activity stimulated by S-adenosyl-L-methionine (SAM) (Type IIG). The stimulatory effect of SAM on cleavage activity presents a major paradox: a co-factor of the MTase activity that renders the substrate unsusceptible to cleavage enhances the cleavage activity. Here we show that the RM.BpuSI MTase activity modifies both cleavage substrate and product only when they are unmethylated. The MTase activity is, however, much lower than that of M1.BpuSI and is thought not to be the major MTase for host DNA protection. SAM and sinefungin (SIN) increase the Vmax of the RM.BpuSI cleavage activity with a proportional change in Km, suggesting the presence of an energetically more favorable pathway is taken. We further showed that RM.BpuSI undergoes substantial conformational changes in the presence of Ca(2+), SIN, cleavage substrate and/or product. Distinct conformers are inferred as the pre-cleavage/cleavage state (in the presence of Ca(2+), substrate or both) and MTase state (in the presence of SIN and substrate, SIN and product or product alone). Interestingly, RM.BpuSI adopts a unique conformation when only SIN is present. This SIN-bound state is inferred as a branch point for cleavage and MTase activity and an intermediate to an energetically favorable pathway for cleavage, probably through increasing the binding affinity of the substrate to the enzyme under cleavage conditions. Mutation of a SAM-binding residue resulted in altered conformational changes in the presence of substrate or Ca(2+) and eliminated cleavage activity. The present study underscores the role of the MTase domain as facilitator of efficient cleavage activity for RM.BpuSI.
format	article
author	Arthur Sarrade-Loucheur Shuang-yong Xu Siu-Hong Chan
author_facet	Arthur Sarrade-Loucheur Shuang-yong Xu Siu-Hong Chan
author_sort	Arthur Sarrade-Loucheur
title	The role of the methyltransferase domain of bifunctional restriction enzyme RM.BpuSI in cleavage activity.
title_short	The role of the methyltransferase domain of bifunctional restriction enzyme RM.BpuSI in cleavage activity.
title_full	The role of the methyltransferase domain of bifunctional restriction enzyme RM.BpuSI in cleavage activity.
title_fullStr	The role of the methyltransferase domain of bifunctional restriction enzyme RM.BpuSI in cleavage activity.
title_full_unstemmed	The role of the methyltransferase domain of bifunctional restriction enzyme RM.BpuSI in cleavage activity.
title_sort	role of the methyltransferase domain of bifunctional restriction enzyme rm.bpusi in cleavage activity.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/41b818833a534067b4ec76d48a4dc26a
work_keys_str_mv	AT arthursarradeloucheur theroleofthemethyltransferasedomainofbifunctionalrestrictionenzymermbpusiincleavageactivity AT shuangyongxu theroleofthemethyltransferasedomainofbifunctionalrestrictionenzymermbpusiincleavageactivity AT siuhongchan theroleofthemethyltransferasedomainofbifunctionalrestrictionenzymermbpusiincleavageactivity AT arthursarradeloucheur roleofthemethyltransferasedomainofbifunctionalrestrictionenzymermbpusiincleavageactivity AT shuangyongxu roleofthemethyltransferasedomainofbifunctionalrestrictionenzymermbpusiincleavageactivity AT siuhongchan roleofthemethyltransferasedomainofbifunctionalrestrictionenzymermbpusiincleavageactivity
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The role of the methyltransferase domain of bifunctional restriction enzyme RM.BpuSI in cleavage activity.

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