Journal of Biological Chemistry
Volume 285, Issue 41, 8 October 2010, Pages 31849-31858
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Enzymology
SET7/9 Catalytic Mutants Reveal the Role of Active Site Water Molecules in Lysine Multiple Methylation*

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SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine ϵ-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di- and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine ϵ-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated ϵ-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes.

Enzyme Mechanisms
Histone Methylation
Protein Methylation
S-Adenosylmethionine
X-ray Crystallography
Chromatin Modification
Isothermal Titration Calorimetry
SET Domain Methyltransferase
TAF10
Transcription

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The atomic coordinates and structure factors (codes 3M53, 3M54, 3M55, 3M56, 3M57, 3M58, 3M59, and 3M5A) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

*

This work was supported, in whole or in part, by National Institutes of Health Grant R01 GM073839 (to R. C. T.) and National Institutes of Health Administrative Supplement GM073839-04S1 (to R. C. T.) funded through the American Recovery and Reinvestment Act. This work was also supported by Department of Energy Grant DE-FG02-92ER20075 (to R. L. H.).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1.

1

Supported by a Canadian Institutes of Health Research postdoctoral fellowship.