Labeling Substrates of Protein Arginine Methyltransferase with Engineered Enzymes and Matched <i>S</i>-Adenosyl-<scp>l</scp>-methionine Analogues

Wang, Rui; Zheng, Weihong; Yu, Honglin; Deng, Haiteng; Luo, Minkui

doi:10.1021/ja2006719

Cited by 121 publications

(202 citation statements)

References 37 publications

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“…Multiple SAM analogs have been reported as active cofactors for native and engineered PMTs (10,13,(23)(24)(25)(26). Most of these compounds contain a characteristic sulfonium-β-sp 2 carbon to promote the enzymatic transalkylation reaction (10).…”

Section: Resultsmentioning

confidence: 99%

Defining efficient enzyme–cofactor pairs for bioorthogonal profiling of protein methylation

Islam¹,

Chen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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116

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Protein methyltransferase (PMT)-mediated posttranslational modification of histone and nonhistone substrates modulates stability, localization, and interacting partners of target proteins in diverse cellular contexts. These events play critical roles in normal biological processes and are frequently deregulated in human diseases. In the course of identifying substrates of individual PMTs, bioorthogonal profiling of protein methylation (BPPM) has demonstrated its merits. In this approach, specific PMTs are engineered to process S-adenosyl-L-methionine (SAM) analogs as cofactor surrogates and label their substrates with distinct chemical modifications for target elucidation. Despite the proof-of-concept advancement of BPPM, few efforts have been made to explore its generality. With two cancer-relevant PMTs, EuHMT1 (GLP1/KMT1D) and EuHMT2 (G9a/ KMT1C), as models, we defined the key structural features of engineered PMTs and matched SAM analogs that can render the orthogonal enzyme-cofactor pairs for efficient catalysis. Here we have demonstrated that the presence of sulfonium-β-sp 2 carbon and flexible, medium-sized sulfonium-δ-substituents are crucial for SAM analogs as BPPM reagents. The bulky cofactors can be accommodated by tailoring the conserved Y1211/Y1154 residues and nearby hydrophobic cavities of EuHMT1/2. Profiling proteomewide substrates with BPPM allowed identification of >500 targets of EuHMT1/2 with representative targets validated using native EuHMT1/2 and SAM. This finding indicates that EuHMT1/2 may regulate many cellular events previously unrecognized to be modulated by methylation. The present work, therefore, paves the way to a broader application of the BPPM technology to profile methylomes of diverse PMTs and elucidate their downstream functions.epigenetic | bump-hole | posttranslation | proteomics

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Section: Resultsmentioning

confidence: 99%

Defining efficient enzyme–cofactor pairs for bioorthogonal profiling of protein methylation

Islam¹,

Chen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

116

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“…For example, incorporation of different transferrable amine moieties ( 21 ,4, 18 22 ,4, 19 23 ,4, 19b Table S2) can be followed by a coupling reaction with the N ‐hydroxysuccinimide (NHS) ester of an amine‐reactive probe 18. In addition, several examples exist on the application of AdoMet analogues with a transferrable terminal alkyne ( 5 ,20 Scheme 6; 20 ,20g,20h 24 ,20b,20c,20g and 25 ,4, 19b Table S2) or azide ( 26 g ,20, 21 27 ) 4. The transferred alkynes and azides can be further functionalized by using the biocompatible and highly‐efficient azide–alkyne cycloaddition reaction (one of the “click” series of reactions).…”

Section: Labeling Strategies Using Adomet‐dependent Mtasesmentioning

confidence: 99%

“…The transferred alkynes and azides can be further functionalized by using the biocompatible and highly‐efficient azide–alkyne cycloaddition reaction (one of the “click” series of reactions). However, some of these analogues, such as compound 20 (Table S2), which features a terminal alkyne, are highly unstable 4, 20g,20h. Other AdoMet analogues, such as the AdoEnyYn compound ( 5 , Scheme 6), are more stable, thus making them more suitable for use in bioorthogonal ligations 20b–20h…”

Section: Labeling Strategies Using Adomet‐dependent Mtasesmentioning

confidence: 99%

“…The focus of their work has been the human protein MTases G9a (also known as EuHMT2), GLP1 (also known as EuHMT1), and PRMT1 (for a complete overview, see Table 2). Enzyme‐mediated transalkylation reactions using an azido‐modified AdoMet analogue with engineered G9a and GLP121, 60 and an alkyne‐modified AdoMet analogue with engineered G9a20c and PRMT1 have been demonstrated 20g. Additionally, a selenium‐based SAM analogue with an alkyne linker was effectively employed in transalkyation reactions directed and catalyzed by the native protein MTases GLP1, G9a and SUV39H2 28, 60, 61…”

Section: Labeling Strategies Using Adomet‐dependent Mtasesmentioning

confidence: 99%

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Methyltransferase‐Directed Labeling of Biomolecules and its Applications

et al. 2017

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Methyltransferases (MTases) form a large family of enzymes that methylate a diverse set of targets, ranging from the three major biopolymers to small molecules. Most of these MTases use the cofactor S‐adenosyl‐l‐Methionine (AdoMet) as a methyl source. In recent years, there have been significant efforts toward the development of AdoMet analogues with the aim of transferring moieties other than simple methyl groups. Two major classes of AdoMet analogues currently exist: doubly‐activated molecules and aziridine based molecules, each of which employs a different approach to achieve transalkylation rather than transmethylation. In this review, we discuss the various strategies for labelling and functionalizing biomolecules using AdoMet‐dependent MTases and AdoMet analogues. We cover the synthetic routes to AdoMet analogues, their stability in biological environments and their application in transalkylation reactions. Finally, some perspectives are presented for the potential use of AdoMet analogues in biology research, (epi)genetics and nanotechnology.

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“…Although the presented cofactors 6BAz and SeAdoYn are designed to have small reactive groups, some MTases may not readily accept them. In these cases the active sites could be enlarged by protein engineering as has been demonstrated for DNA 35 , RNA 23 and protein MTases [25][26][27][28] with sterically more demanding double activated AdoMet analogues.…”

Section: Double Activated Cofactormentioning

confidence: 99%

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Hanz

Jung

Giesbertz

et al. 2014

JoVE

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S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5'-ATCGAT-3' sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection. Video LinkThe video component of this article can be found at

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Labeling Substrates of Protein Arginine Methyltransferase with Engineered Enzymes and Matched S-Adenosyl-l-methionine Analogues

Cited by 121 publications

References 37 publications

Defining efficient enzyme–cofactor pairs for bioorthogonal profiling of protein methylation

Defining efficient enzyme–cofactor pairs for bioorthogonal profiling of protein methylation

Methyltransferase‐Directed Labeling of Biomolecules and its Applications

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

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