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Title: An engineered variant of SETD3 methyltransferase alters target specificity from histidine to lysine methylation

Abstract

Most characterized SET domain (SETD) proteins are protein lysine methyltransferases, but SETD3 was recently demonstrated to be a protein (i.e. actin) histidine-N3 methyltransferase. Human SETD3 shares a high structural homology with two known protein lysine methyltransferases—human SETD6 and the plant LSMT—but differs in the residues constituting the active site. In the SETD3 active site, Asn255 engages in a unique hydrogen-bonding interaction with the target histidine of actin that likely contributes to its >1300-fold greater catalytic efficiency (kcat/Km) on histidine than on lysine. In this work, we engineered active-site variants to switch the SETD3 target specificity from histidine to lysine. Substitution of Asn255 with phenylalanine (N255F), together with substitution of Trp273 with alanine (W273A), generated an active site mimicking that of known lysine methyltransferases. The doubly substituted SETD3 variant exhibited a 13-fold preference for lysine over histidine. We show, by means of X-ray crystallography, that the two target nitrogen atoms—the N3 atom of histidine and the terminal ϵ-amino nitrogen of lysine—occupy the same position and point toward and are within a short distance of the incoming methyl group of SAM for a direct methyl transfer during catalysis. In contrast, SETD3 and its Asn255 substituted derivatives did not methylate glutamine (another potentiallymore » methylated amino acid). However, the glutamine-containing peptide competed with the substrate peptide, and glutamine bound in the active site, but too far away from SAM to be methylated. Our results provide insight into the structural parameters defining the target amino acid specificity of SET enzymes.« less

Authors:
 [1];  [1];  [2]; ORCiD logo [2];  [1]; ORCiD logo [1]
  1. Univ. of Texas M.D. Anderson Cancer Center, Houston, TX (United States)
  2. Stanford Univ., CA (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH); Cancer Prevention and Research Institute of Texas (CPRIT)
OSTI Identifier:
1615846
Grant/Contract Number:  
GM114306; GM133051; RR160029
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 295; Journal Issue: 9; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; S-adenosylmethionine (SAM); protein methylation; structural biology; enzyme catalysis; enzyme kinetics; histidine; glutamine methylation; histidine methylation; lysine methylation

Citation Formats

Dai, Shaobo, Horton, John R., Wilkinson, Alex W., Gozani, Or, Zhang, Xing, and Cheng, Xiaodong. An engineered variant of SETD3 methyltransferase alters target specificity from histidine to lysine methylation. United States: N. p., 2020. Web. https://doi.org/10.1074/jbc.RA119.012319.
Dai, Shaobo, Horton, John R., Wilkinson, Alex W., Gozani, Or, Zhang, Xing, & Cheng, Xiaodong. An engineered variant of SETD3 methyltransferase alters target specificity from histidine to lysine methylation. United States. https://doi.org/10.1074/jbc.RA119.012319
Dai, Shaobo, Horton, John R., Wilkinson, Alex W., Gozani, Or, Zhang, Xing, and Cheng, Xiaodong. Tue . "An engineered variant of SETD3 methyltransferase alters target specificity from histidine to lysine methylation". United States. https://doi.org/10.1074/jbc.RA119.012319. https://www.osti.gov/servlets/purl/1615846.
@article{osti_1615846,
title = {An engineered variant of SETD3 methyltransferase alters target specificity from histidine to lysine methylation},
author = {Dai, Shaobo and Horton, John R. and Wilkinson, Alex W. and Gozani, Or and Zhang, Xing and Cheng, Xiaodong},
abstractNote = {Most characterized SET domain (SETD) proteins are protein lysine methyltransferases, but SETD3 was recently demonstrated to be a protein (i.e. actin) histidine-N3 methyltransferase. Human SETD3 shares a high structural homology with two known protein lysine methyltransferases—human SETD6 and the plant LSMT—but differs in the residues constituting the active site. In the SETD3 active site, Asn255 engages in a unique hydrogen-bonding interaction with the target histidine of actin that likely contributes to its >1300-fold greater catalytic efficiency (kcat/Km) on histidine than on lysine. In this work, we engineered active-site variants to switch the SETD3 target specificity from histidine to lysine. Substitution of Asn255 with phenylalanine (N255F), together with substitution of Trp273 with alanine (W273A), generated an active site mimicking that of known lysine methyltransferases. The doubly substituted SETD3 variant exhibited a 13-fold preference for lysine over histidine. We show, by means of X-ray crystallography, that the two target nitrogen atoms—the N3 atom of histidine and the terminal ϵ-amino nitrogen of lysine—occupy the same position and point toward and are within a short distance of the incoming methyl group of SAM for a direct methyl transfer during catalysis. In contrast, SETD3 and its Asn255 substituted derivatives did not methylate glutamine (another potentially methylated amino acid). However, the glutamine-containing peptide competed with the substrate peptide, and glutamine bound in the active site, but too far away from SAM to be methylated. Our results provide insight into the structural parameters defining the target amino acid specificity of SET enzymes.},
doi = {10.1074/jbc.RA119.012319},
journal = {Journal of Biological Chemistry},
number = 9,
volume = 295,
place = {United States},
year = {2020},
month = {1}
}

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