Covalent intermediate in the catalytic mechanism of the radical S-adenosyl-L-methionine methyl synthase RlmN trapped by mutagenesis.
Covalent intermediate in the catalytic mechanism of the radical S-adenosyl-L-methionine methyl synthase RlmN trapped by mutagenesis.
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在被诱变捕获的自由基S-腺苷-l-甲基合酶RLMN的催化机理中的共价中间体。
DOI：
10.1021/ja307855d
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发表时间：
2012-10-31
影响因子：
15
通讯作者：
Fujimori, Danica Galonic
中科院分区：
文献类型：
--
作者：
McCusker, Kevin P.;Medzihradszky, Katalin F.;Shiver, Anthony L.;Nichols, Robert J.;Yan, Feng;Maltby, David A.;Gross, Carol A.;Fujimori, Danica Galonic
The posttranscriptional modification of ribosomal RNA (rRNA) modulates ribosomal function and confers resistance to antibiotics targeted to the ribosome. The radical SAM (S-adenosyl-L-methionine) methyl synthases, RlmN and Cfr, both methylate A2503 within the peptidyl transferase center (PTC) of prokaryotic ribosomes, yielding 2-methyl- and 8-methyl-adenosine, respectively. The C2 and C8 positions of adenosine are unusual methylation substrates due to their electrophilicity. To accomplish this reaction, RlmN and Cfr proceed by a shared radical-mediated mechanism. However, in addition to the radical SAM CX3CX2C motif, both RlmN and Cfr contain two conserved cysteine residues required for in vivo function. These conserved cysteine residues are putatively involved in a covalent intermediate employed by RlmN and Cfr in order to achieve this challenging transformation. Currently, there is no direct evidence for this proposed covalent intermediate. We have further investigated the roles of these conserved cysteines in the mechanism of RlmN. Cysteine 118 mutants of RlmN are unable to resolve the covalent intermediate, either in vivo or in vitro, enabling us to isolate and characterize this intermediate. Additionally, tandem mass spectrometric analyses of mutant RlmN reveal a methylene-linked adenosine modification at cysteine 355. Employing deuterium-labeled SAM and RNA substrates in vitro has allowed us to further elucidate the mechanism of formation of this intermediate. Together, these experiments provide compelling evidence for the formation of a covalent intermediate species formed between RlmN and its rRNA substrate and the roles of the conserved cysteine residues in catalysis.
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自由基 SAM 酶进行甲基转移的结构基础。
DOI：
10.1126/science.1205358
发表时间：
2011-05-27
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Boal AK;Grove TL;McLaughlin MI;Yennawar NH;Booker SJ;Rosenzweig AC
通讯作者：
Rosenzweig AC
影响因子：
7.8
作者：
Booker, Squire J.
通讯作者：
Booker, Squire J.
影响因子：
14.8
作者：
Chatterjee, Abhishek;Li, Yue;Zhang, Yang;Grove, Tyler L.;Lee, Michael;Krebs, Carsten;Booker, Squire J.;Begley, Tadhg P.;Ealick, Steven E.
通讯作者：
Ealick, Steven E.
影响因子：
14.8
作者：
Fluehe, Eif;Knappe, Thomas A.;Marahiel, Mohamed A.
通讯作者：
Marahiel, Mohamed A.
DOI：
10.1007/b105586
发表时间：
2005-01-01
期刊：
FINE-TUNING OF RNA FUNCTIONS BY MODIFICATION AND EDITING
影响因子：
--
作者：
Douthwaite, S;Fourmy, D;Yoshizawa, S
通讯作者：
Yoshizawa, S