Kinetic Mechanism of Protein N-terminal Methyltransferase 1

Richardson, Stacie L.; Mao, Yunfei; Zhang, Gang; Hanjra, Pahul; Peterson, Darrell L.; Huang, Rong

doi:10.1074/jbc.m114.626846

Cited by 45 publications

(93 citation statements)

References 25 publications

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“…We performed kinetic analysis for NTMT1 by determining the Michaelis–Menten constants for RCC1-10 peptide and AdoMet (Table 3). Kinetic constants obtained from DRQ–MALDI–MS are comparable with results from a fluorescence assay, which demonstrates the application of our method in enzyme kinetic studies and further validates the accuracy of the results [41]. For more extensive assay analysis, such as kinetics, automation of data analysis is necessary.…”

Section: Resultssupporting

confidence: 55%

“…Synthetic peptides SCL1-12 (SGAAAASAAGYE), human RCC1-10 (SPKRIAKRRS), and SET1α-10 (APKRQSPLPP) were prepared using standard Fmoc chemistry with a CEM Liberty microwave peptide synthesizer. Methylated and acetylated peptides were prepared from RCC1-10 according to the literature [39–41]. All peptides were purified by reverse-phase HPLC (Waters) and quantified.…”

Section: Methodsmentioning

confidence: 99%

“…Curve-fitting analysis was performed with Prism GraphPad. The human NTMT1 (EC 2.1.1.244, Addgene) and NatA (EC 2.3.1.88) were expressed and purified according to the literature [32,38,41]. …”

Section: Methodsmentioning

confidence: 99%

“…Peptide methylation was performed under the following conditions: 0.2 µM NTMT1, 25 mM Tris, pH 7.5, 50 mM KOAc, 2 mM DTT and peptide substrate at either 30 or 37 °C for 5 min [41]. AdoMet was added to initiate the reaction.…”

Section: Methodsmentioning

confidence: 99%

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A direct, ratiometric, and quantitative MALDI–MS assay for protein methyltransferases and acetyltransferases

Richardson

Hanjra

Zhang

et al. 2015

Analytical Biochemistry

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Protein methylation and acetylation play important roles in biological processes, and misregulation of these modifications is involved in various diseases. Therefore, it is critical to understand the activities of the enzymes responsible for these modifications. Herein we describe a sensitive method for ratiometric quantification of methylated and acetylated peptides via MALDI-MS by direct spotting of enzymatic methylation and acetylation reaction mixtures without tedious purification procedures. The quantifiable detection limit for peptides with our method is approximately 10 fmol. This is achieved by increasing the signal-to-noise ratio through the addition of NH4H2PO4 to the matrix solution and reduction of the matrix α-cyanohydroxycinnamic acid concentration to 2 mg/ml. We have demonstrated the application of this method in enzyme kinetic analysis and inhibition studies. The unique feature of this method is the simultaneous quantification of multiple peptide species for investigation of processivity mechanisms. Its wide buffer compatibility makes it possible to be adapted to investigate the activity of any protein methyltransferase or acetyltransferase.

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Section: Resultssupporting

confidence: 55%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

A direct, ratiometric, and quantitative MALDI–MS assay for protein methyltransferases and acetyltransferases

Richardson

Hanjra

Zhang

et al. 2015

Analytical Biochemistry

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“…METTL11B was suggested to be primarily a monomethyltransferase that may prime substrates for the action of NTMT1 (55). Crystal structures of human NTMT1 in complex with substrate peptides have been solved (56,57) that support the substrate specificity of this enzyme determined from kinetic (58,59) and inhibitor studies (60). A variety of functions have been proposed for XPK N-terminal methylation of eukaryotic proteins (61), including regulating the affinity of protein binding to DNA (62,63), DNA repair (64-66) and protection from aminopeptidase attack (49).…”

Section: Protein N-terminal Methyltransferasesmentioning

confidence: 95%

The ribosome: A hot spot for the identification of new types of protein methyltransferases

Clarke¹

2018

Journal of Biological Chemistry

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Cellular physiology depends on the alteration of protein structures by covalent modification reactions. Using a combination of bioinformatic, genetic, biochemical, and mass spectrometric approaches, it has been possible to probe ribosomal proteins from the yeast Saccharomyces cerevisiae for posttranslationally methylated amino acid residues and for the enzymes that catalyze these modifications. These efforts have resulted in the identification and characterization of the first protein histidine methyltransferase, the first N-terminal protein methyltransferase, two unusual types of protein arginine methyltransferases, and a new type of cysteine methylation. Two of these enzymes may modify their substrates during ribosomal assembly because the final methylated histidine and arginine residues are buried deep within the ribosome with contacts only with RNA. Two of these modifications occur broadly in eukaryotes, including humans, while the others demonstrate a more limited phylogenetic range. Analysis of strains where the methyltransferase genes are deleted has given insight into the physiological roles of these modifications. These reactions described here add diversity to the modifications that generate the typical methylated lysine and arginine residues previously described in histones and other proteins. Regulation of biological function by protein methylation reactionsIt is more and more apparent just how much of biological function is dependent upon posttranslational modifications (1). The human genome encodes some 900 enzymes catalyzing just protein phosphorylation or ubiquitination (2,3). However, it is now clear that methyl groups can stand beside phosphate groups and ubiquitin as major players in controlling the physiological functions of proteins. We are beginning to understand how the much greater diversity of protein methylation reactions can give rise to a greater diversity of function (1,4-8). We are also learning the importance of crosstalk between protein and DNA methylation reactions (9) and among protein methylation, phosphorylation, acetylation, and ubiquitination reactions (10-12). Finally, we are discovering how alterations in protein methylation pathways can lead to human pathology, particularly in cancer (13-15).The collection of over sixty human protein arginine and lysine methyltransferases that leave histone "marks" recognized by reader proteins to guide gene expression are perhaps the poster children for the importance of protein methyltransferases (16-17). However, recent work has emphasized the importance of methylating non-histone substrates at these residues, particularly ribosomal proteins, translation factors, and transcription factors in a wide variety of organisms (7,8,15,18,19). Furthermore, the methylation of lysine and arginine residues represents just the tip of the iceberg in protein methylation -modifications have also been established at histidine, modified histidine (diphthamide), glutamate, glutamine, asparagine, Lisoaspartate, D-aspartate, cysteine, isoprenylcysteine, me...

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Fifty years of DNA “Breathing”: Reflections on old and new approaches

2013

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Summary The coding sequences for genes, and much other regulatory information involved in genome expression, are located ‘inside’ the DNA duplex. Thus the ‘macromolecular machines’ that read-out this information from the base sequence of the DNA must somehow access the DNA ‘interior’. Double-stranded (ds) DNA is a highly structured and cooperatively stabilized system at physiological temperatures, but is also only marginally stable and undergoes a cooperative ‘melting phase transition’ at temperatures not far above physiological. Furthermore, due to its length and heterogeneous sequence, with AT-rich segments being less stable than GC-rich segments, the DNA genome ‘melts’ in a multistate fashion. Therefore the DNA genome must also manifest thermally driven structural (‘breathing’) fluctuations at physiological temperatures that should reflect the heterogeneity of the dsDNA stability near the melting temperature. Thus many of the breathing fluctuations of dsDNA are likely also to be sequence dependent, and could well contain information that should be ‘readable’ and useable by regulatory proteins and protein complexes in site-specific binding reactions involving dsDNA ‘opening’. Our laboratory has been involved in studying the breathing fluctuations of duplex DNA for about 50 years. In this ‘Reflections’ article we present a relatively chronological overview of these studies, starting with the use of simple chemical probes (such as hydrogen exchange, formaldehyde and simple DNA ‘melting’ proteins) to examine the local stability of the dsDNA structure, and culminating in sophisticated spectroscopic approaches that can be used to monitor the breathing-dependent interactions of regulatory complexes with their duplex DNA targets in ‘real time’.

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Kinetic Mechanism of Protein N-terminal Methyltransferase 1

Cited by 45 publications

References 25 publications

A direct, ratiometric, and quantitative MALDI–MS assay for protein methyltransferases and acetyltransferases

A direct, ratiometric, and quantitative MALDI–MS assay for protein methyltransferases and acetyltransferases

The ribosome: A hot spot for the identification of new types of protein methyltransferases

Fifty years of DNA “Breathing”: Reflections on old and new approaches

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