Expanding Cofactor Repertoire of Protein Lysine Methyltransferase for Substrate Labeling

Islam, Kabirul; Zheng, Weihong; Yu, Honglin; Deng, Haiteng; Luo, Minkui

doi:10.1021/cb2000567

Cited by 101 publications

(190 citation statements)

References 36 publications

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“…37,38 Usually, the wild-type enzymes were tested for promiscuous activity on SAM analogs and directed evolution was limited to semi-rational approaches. 39 We also show how our setup can be readily implemented for screening of methyltransferase inhibitors. As a proof of concept, we tested three known methyltransferase inhibitors and found that only (-)-epigallocatechin gallate inhibited hTGS1.…”

Section: Resultsmentioning

confidence: 99%

An enzyme-coupled high-throughput assay for screening RNA methyltransferase activity inE. Colicell lysate

Schulz

Rentmeister²

2012

RNA Biology

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Section: Resultsmentioning

confidence: 99%

An enzyme-coupled high-throughput assay for screening RNA methyltransferase activity inE. Colicell lysate

Schulz

Rentmeister²

2012

RNA Biology

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“…After processing the reaction at ambient temperature (22°C) for specific time intervals, the peptide sample was enriched by C18 ZipTip (Millipore), washed with 0.1% TFA/ddH 2 O (vol/vol) 10 times, and eluted out with 50% acetonitrile/0.1% TFA, according to the manufacturer's instructions. The resultant peptide products were subjected to MALDI-MS as described previously (42). Briefly, to prepare MALDI-TOF-MS samples, 2 μL of the purified peptide was mixed with 1 μL of saturated α-cyano-hydroxy-cinnamic acid solution (50% acetonitrile/0.1% TFA) on a MALDI sample plate and allowed to dry at ambient temperature (22°C).…”

Section: Methodsmentioning

confidence: 99%

“…The biological relevance of SET8, as well as our general interest in PKMTs, inspired us to leverage BIE and KIE studies of SET8 to explore bond motions along the reaction path of the PKMT-catalyzed methylation. Given the convenience of MS in characterizing protein methylation (42,43), we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically labeled H4K20 peptide. With this approach, BIEs and KIEs of SAM and SeAM (Se-adenosyl-L-selenomethionine, a more reactive SAM surrogate) were determined for SET8-catalzyed H4K20 methylation.…”

Section: Significancementioning

confidence: 99%

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

Linscott

Kapilashrami

Wang

et al. 2016

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

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Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)-PKMT-substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (S N 2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MSbased method to measure KIEs and binding isotope effects (BIEs) of the cofactor S-adenosyl-L-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic 13 C KIE of 1.04, an inverse intrinsic α-secondary CD 3 KIE of 0.90, and a small but statistically significant inverse CD 3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical S N 2 transition state with the C-N and C-S distances of 2.35-2.40 Å and 2.00-2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steadystate kinetics with the SAM analog Se-adenosyl-L-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.S tepwise progression of an enzyme-catalyzed chemical reaction is accompanied by changes of bond orders and vibrational modes involved with specific atoms of the reactant(s) (1, 2). Such changes can be traced experimentally by measuring the ratios of turnover rates [kinetic isotope effects (KIEs)] or binding affinities [binding isotope effects (BIEs)] of the reactant(s) when the relevant atoms are replaced by heavy isotopes (3, 4). KIEs and BIEs are thus useful parameters for elucidating transition-state (TS) structures and catalytic mechanisms, which sometimes cannot be elucidated readily through sole measurement of steady-state kinetics (5-9). A sufficient set of KIEs and BIEs at the positions involved with bond motions can afford electrostatic and geometric constraints, when combined with computational modeling, to define an enzymatic TS (10-12). This information provides not only the atomic resolution of the transient structure at the highest energy summit along the reaction path, but also structural guidance for designing tight-binding TS analog inhibitors (13...

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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 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%

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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Expanding Cofactor Repertoire of Protein Lysine Methyltransferase for Substrate Labeling

Cited by 101 publications

References 36 publications

An enzyme-coupled high-throughput assay for screening RNA methyltransferase activity inE. Colicell lysate

An enzyme-coupled high-throughput assay for screening RNA methyltransferase activity inE. Colicell lysate

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

Methyltransferase‐Directed Labeling of Biomolecules and its Applications

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