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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Published in | PloS one Vol. 8; no. 11; p. e80967 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
United States
Public Library of Science
04.11.2013
Public Library of Science (PLoS) |
Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Conceived and designed the experiments: ASL SYX SHC. Performed the experiments: ASL SHC. Analyzed the data: SYX SHC. Contributed reagents/materials/analysis tools: ASL SYX SHC. Wrote the manuscript: SYX SHC. Competing Interests: Authors Shuang-yong Xu and Siu-Hong Chan have an affiliation to a commercial funder of this research (New England Biolabs, Inc.). Author Shuang-yong Xu is an Academic Editor for PLOS ONE. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. Current address: Institut National Des Sciences Appliquées de Toulouse, Toulouse, France |
ISSN: | 1932-6203 1932-6203 |
DOI: | 10.1371/journal.pone.0080967 |