Capturing Unknown Substrates via in Situ Formation of Tightly Bound Bisubstrate Adducts: S‑Adenosyl-vinthionine as a Functional Probe for AdoMet-Dependent Methyltransferases
Identifying an enzyme’s substrates is essential to understand its function, yet it remains challenging. A fundamental impediment is the transient interactions between an enzyme and its substrates. In contrast, tight binding is often observed for multisubstrate-adduct inhibitors due to synergistic in...
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| Veröffentlicht in: | Journal of the American Chemical Society 2016-03, Vol.138 (9), p.2877-2880 |
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| container_title | Journal of the American Chemical Society |
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| creator | Qu, Wanlu Catcott, Kalli C Zhang, Kun Liu, Shanshan Guo, Jason J Ma, Jisheng Pablo, Michael Glick, James Xiu, Yuan Kenton, Nathaniel Ma, Xiaoyu Duclos, Richard I Zhou, Zhaohui Sunny |
| description | Identifying an enzyme’s substrates is essential to understand its function, yet it remains challenging. A fundamental impediment is the transient interactions between an enzyme and its substrates. In contrast, tight binding is often observed for multisubstrate-adduct inhibitors due to synergistic interactions. Extending this venerable concept to enzyme-catalyzed in situ adduct formation, unknown substrates were affinity-captured by an S-adenosyl-methionine (AdoMet, SAM)-dependent methyltransferase (MTase). Specifically, the electrophilic methyl sulfonium (alkyl donor) in AdoMet is replaced with a vinyl sulfonium (Michael acceptor) in S-adenosyl-vinthionine (AdoVin). Via an addition reaction, AdoVin and the nucleophilic substrate form a covalent bisubstrate-adduct tightly complexed with thiopurine MTase (2.1.1.67). As such, an unknown substrate was readily identified from crude cell lysates. Moreover, this approach is applicable to other systems, even if the enzyme is unknown. |
| doi_str_mv | 10.1021/jacs.5b05950 |
| format | Article |
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A fundamental impediment is the transient interactions between an enzyme and its substrates. In contrast, tight binding is often observed for multisubstrate-adduct inhibitors due to synergistic interactions. Extending this venerable concept to enzyme-catalyzed in situ adduct formation, unknown substrates were affinity-captured by an S-adenosyl-methionine (AdoMet, SAM)-dependent methyltransferase (MTase). Specifically, the electrophilic methyl sulfonium (alkyl donor) in AdoMet is replaced with a vinyl sulfonium (Michael acceptor) in S-adenosyl-vinthionine (AdoVin). Via an addition reaction, AdoVin and the nucleophilic substrate form a covalent bisubstrate-adduct tightly complexed with thiopurine MTase (2.1.1.67). As such, an unknown substrate was readily identified from crude cell lysates. Moreover, this approach is applicable to other systems, even if the enzyme is unknown.</description><identifier>ISSN: 0002-7863</identifier><identifier>EISSN: 1520-5126</identifier><identifier>DOI: 10.1021/jacs.5b05950</identifier><identifier>PMID: 26901520</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Chromatography, High Pressure Liquid ; Click Chemistry ; Ethionine - analogs & derivatives ; Ethionine - chemistry ; Ethionine - metabolism ; Humans ; Methyltransferases - chemistry ; Methyltransferases - metabolism ; S-Adenosylmethionine - chemistry ; S-Adenosylmethionine - metabolism ; Spectrophotometry, Ultraviolet ; Substrate Specificity</subject><ispartof>Journal of the American Chemical Society, 2016-03, Vol.138 (9), p.2877-2880</ispartof><rights>Copyright © 2016 American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a324t-dd47f7e2e58b227b53a7ef77ec5d5b874e5a2c392f2773b5b1c61cac7946fac13</citedby><cites>FETCH-LOGICAL-a324t-dd47f7e2e58b227b53a7ef77ec5d5b874e5a2c392f2773b5b1c61cac7946fac13</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/jacs.5b05950$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/jacs.5b05950$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,776,780,2752,27053,27901,27902,56713,56763</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26901520$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Qu, Wanlu</creatorcontrib><creatorcontrib>Catcott, Kalli C</creatorcontrib><creatorcontrib>Zhang, Kun</creatorcontrib><creatorcontrib>Liu, Shanshan</creatorcontrib><creatorcontrib>Guo, Jason J</creatorcontrib><creatorcontrib>Ma, Jisheng</creatorcontrib><creatorcontrib>Pablo, Michael</creatorcontrib><creatorcontrib>Glick, James</creatorcontrib><creatorcontrib>Xiu, Yuan</creatorcontrib><creatorcontrib>Kenton, Nathaniel</creatorcontrib><creatorcontrib>Ma, Xiaoyu</creatorcontrib><creatorcontrib>Duclos, Richard I</creatorcontrib><creatorcontrib>Zhou, Zhaohui Sunny</creatorcontrib><title>Capturing Unknown Substrates via in Situ Formation of Tightly Bound Bisubstrate Adducts: S‑Adenosyl-vinthionine as a Functional Probe for AdoMet-Dependent Methyltransferases</title><title>Journal of the American Chemical Society</title><addtitle>J. Am. Chem. Soc</addtitle><description>Identifying an enzyme’s substrates is essential to understand its function, yet it remains challenging. A fundamental impediment is the transient interactions between an enzyme and its substrates. In contrast, tight binding is often observed for multisubstrate-adduct inhibitors due to synergistic interactions. Extending this venerable concept to enzyme-catalyzed in situ adduct formation, unknown substrates were affinity-captured by an S-adenosyl-methionine (AdoMet, SAM)-dependent methyltransferase (MTase). Specifically, the electrophilic methyl sulfonium (alkyl donor) in AdoMet is replaced with a vinyl sulfonium (Michael acceptor) in S-adenosyl-vinthionine (AdoVin). Via an addition reaction, AdoVin and the nucleophilic substrate form a covalent bisubstrate-adduct tightly complexed with thiopurine MTase (2.1.1.67). As such, an unknown substrate was readily identified from crude cell lysates. Moreover, this approach is applicable to other systems, even if the enzyme is unknown.</description><subject>Chromatography, High Pressure Liquid</subject><subject>Click Chemistry</subject><subject>Ethionine - analogs & derivatives</subject><subject>Ethionine - chemistry</subject><subject>Ethionine - metabolism</subject><subject>Humans</subject><subject>Methyltransferases - chemistry</subject><subject>Methyltransferases - metabolism</subject><subject>S-Adenosylmethionine - chemistry</subject><subject>S-Adenosylmethionine - metabolism</subject><subject>Spectrophotometry, Ultraviolet</subject><subject>Substrate Specificity</subject><issn>0002-7863</issn><issn>1520-5126</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkcFu1DAQhi0Eokvhxhn5yIEU24njLLftli1IRSC1PUeOM-l6ydqLx261N16BJ-GdeJI66hYunKyxvu8faX5CXnN2wpng7zfa4InsmJxL9oTMuBSskFzUT8mMMSYK1dTlEXmBuMljJRr-nByJes4mcEZ-L_UupmDdDb12352_c_QydRiDjoD01mpq84-Nia582OpovaN-oFf2Zh3HPT31yfX01OKjQxd9n0zED_Tyz89fix6cx_1Y3FoX19m1DqhGqukqOTOF6ZF-C74DOviQXf8FYnEGO3DZjDRP6_2Ygx0OEDQCviTPBj0ivDq8x-R69fFq-am4-Hr-ebm4KHQpqlj0faUGBQJk0wmhOllqBYNSYGQvu0ZVILUw5VwMQqmykx03NTfaqHlVD9rw8pi8fcjdBf8jAcZ2a9HAOGoHPmHLlRK8qmXZZPTdA2qCRwwwtLtgtzrsW87aqaJ2qqg9VJTxN4fk1G2h_ws_dvJv9WRtfAr5SPj_rHtnaJ9T</recordid><startdate>20160309</startdate><enddate>20160309</enddate><creator>Qu, Wanlu</creator><creator>Catcott, Kalli C</creator><creator>Zhang, Kun</creator><creator>Liu, Shanshan</creator><creator>Guo, Jason J</creator><creator>Ma, Jisheng</creator><creator>Pablo, Michael</creator><creator>Glick, James</creator><creator>Xiu, Yuan</creator><creator>Kenton, Nathaniel</creator><creator>Ma, Xiaoyu</creator><creator>Duclos, Richard I</creator><creator>Zhou, Zhaohui Sunny</creator><general>American Chemical Society</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20160309</creationdate><title>Capturing Unknown Substrates via in Situ Formation of Tightly Bound Bisubstrate Adducts: S‑Adenosyl-vinthionine as a Functional Probe for AdoMet-Dependent Methyltransferases</title><author>Qu, Wanlu ; Catcott, Kalli C ; Zhang, Kun ; Liu, Shanshan ; Guo, Jason J ; Ma, Jisheng ; Pablo, Michael ; Glick, James ; Xiu, Yuan ; Kenton, Nathaniel ; Ma, Xiaoyu ; Duclos, Richard I ; Zhou, Zhaohui Sunny</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a324t-dd47f7e2e58b227b53a7ef77ec5d5b874e5a2c392f2773b5b1c61cac7946fac13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Chromatography, High Pressure Liquid</topic><topic>Click Chemistry</topic><topic>Ethionine - analogs & derivatives</topic><topic>Ethionine - chemistry</topic><topic>Ethionine - metabolism</topic><topic>Humans</topic><topic>Methyltransferases - chemistry</topic><topic>Methyltransferases - metabolism</topic><topic>S-Adenosylmethionine - chemistry</topic><topic>S-Adenosylmethionine - metabolism</topic><topic>Spectrophotometry, Ultraviolet</topic><topic>Substrate Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Qu, Wanlu</creatorcontrib><creatorcontrib>Catcott, Kalli C</creatorcontrib><creatorcontrib>Zhang, Kun</creatorcontrib><creatorcontrib>Liu, Shanshan</creatorcontrib><creatorcontrib>Guo, Jason J</creatorcontrib><creatorcontrib>Ma, Jisheng</creatorcontrib><creatorcontrib>Pablo, Michael</creatorcontrib><creatorcontrib>Glick, James</creatorcontrib><creatorcontrib>Xiu, Yuan</creatorcontrib><creatorcontrib>Kenton, Nathaniel</creatorcontrib><creatorcontrib>Ma, Xiaoyu</creatorcontrib><creatorcontrib>Duclos, Richard I</creatorcontrib><creatorcontrib>Zhou, Zhaohui Sunny</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of the American Chemical Society</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Qu, Wanlu</au><au>Catcott, Kalli C</au><au>Zhang, Kun</au><au>Liu, Shanshan</au><au>Guo, Jason J</au><au>Ma, Jisheng</au><au>Pablo, Michael</au><au>Glick, James</au><au>Xiu, Yuan</au><au>Kenton, Nathaniel</au><au>Ma, Xiaoyu</au><au>Duclos, Richard I</au><au>Zhou, Zhaohui Sunny</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Capturing Unknown Substrates via in Situ Formation of Tightly Bound Bisubstrate Adducts: S‑Adenosyl-vinthionine as a Functional Probe for AdoMet-Dependent Methyltransferases</atitle><jtitle>Journal of the American Chemical Society</jtitle><addtitle>J. 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| source | ACS Publications; MEDLINE |
| subjects | Chromatography, High Pressure Liquid Click Chemistry Ethionine - analogs & derivatives Ethionine - chemistry Ethionine - metabolism Humans Methyltransferases - chemistry Methyltransferases - metabolism S-Adenosylmethionine - chemistry S-Adenosylmethionine - metabolism Spectrophotometry, Ultraviolet Substrate Specificity |
| title | Capturing Unknown Substrates via in Situ Formation of Tightly Bound Bisubstrate Adducts: S‑Adenosyl-vinthionine as a Functional Probe for AdoMet-Dependent Methyltransferases |
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