The role of the methyltransferase domain of bifunctional restriction enzyme RM.BpuSI in cleavage activity.
The role of the methyltransferase domain of bifunctional restriction enzyme RM.BpuSI in cleavage activity.
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DOI：
10.1371/journal.pone.0080967
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Chan SH
中科院分区：
文献类型：
--
作者：
Sarrade-Loucheur A;Xu SY;Chan SH
限制性内切酶（REase）RM.BpuSI可被描述为IIS/C/G型REase，因为其切割位点在识别序列之外（IIS型），具有甲基转移酶（MTase）和内切核酸酶活性（IIC型）以及由S-腺苷-L-甲硫氨酸（SAM）刺激的内切核酸酶活性（IIG型）的双功能多肽。SAM对切割活性的刺激作用提出了一个主要的矛盾：MTase活性的辅因子使底物对切割不敏感，从而增强了切割活性。在这里，我们表明，RM.BpuSI MTase活性修改切割底物和产品只有当它们是未甲基化的。然而，MTase活性远低于M1.BpuSI的活性，并且被认为不是用于宿主DNA保护的主要MTase。SAM和sinefungin（SIN）增加了RM.BpuSI切割活性的Vmax，Km成比例地变化，表明采用了能量上更有利的途径。我们进一步表明，RM.BpuSI在Ca ~（2+）、SIN、裂解底物和/或产物的存在下发生了显著的构象变化。不同的构象异构体被推断为预裂解/裂解状态（存在Ca 2+、底物或两者）和MTase状态（存在SIN和底物、SIN和产物或仅产物）。有趣的是，当只有SIN存在时，RM.BpuSI采用独特的构象。这种SIN结合状态被推断为切割和MTase活性的分支点，以及能量上有利的切割途径的中间体，可能通过在切割条件下增加底物与酶的结合亲和力。SAM结合残基的突变导致在底物或Ca 2+存在下构象改变，并消除了切割活性。本研究强调了MTase结构域作为RM. BpuSI有效切割活性的促进剂的作用。
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 Ca2+, SIN, cleavage substrate and/or product. Distinct conformers are inferred as the pre-cleavage/cleavage state (in the presence of Ca2+, 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 Ca2+ 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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影响因子：
3.7
作者：
Engler, Carola;Kandzia, Romy;Marillonnet, Sylvestre
通讯作者：
Marillonnet, Sylvestre
DOI：
10.1038/84104
发表时间：
2001-02-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Goedecke, K;Pignot, M;Weinhold, E
通讯作者：
Weinhold, E
影响因子：
14.9
作者：
Morgan, Richard D.;Bhatia, Tanya K.;Lovasco, Lindsay;Davis, Theodore B.
通讯作者：
Davis, Theodore B.
DOI：
10.1073/pnas.75.7.3099
发表时间：
1978-01-01
影响因子：
11.1
作者：
BICKLE, TA;BRACK, C;YUAN, R
通讯作者：
YUAN, R
影响因子：
11.4
作者：
MEISEL, A;MACKELDANZ, P;SCHROEDER, C
通讯作者：
SCHROEDER, C