Structure-guided mutational analysis reveals the functional requirements for product specificity of DOT1 enzymes.
DOT1 enzymes are conserved methyltransferases that catalyse the methylation of lysine 79 on histone H3 (H3K79). Most eukaryotes contain one DOT1 enzyme, whereas African trypanosomes have two homologues, DOT1A and DOT1B, with different enzymatic activities. DOT1A mediates mono- and dimethylation of H3K76, the homologue of H3K79 in other organisms, whereas DOT1B additionally catalyses H3K76 trimethylation. However, it is unclear how these different enzymatic activities are achieved. Here we employ a trypanosomal nucleosome reconstitution system and structure-guided homology modelling to identify critical residues within and outside the catalytic centre that modulate product specificity. Exchange of these residues transfers the product specificity from one enzyme to the other, and reveals the existence of distinct regulatory domains adjacent to the catalytic centre. Our study provides the first evidence that a few crucial residues in DOT1 enzymes are sufficient to catalyse methyl-state-specific reactions. These results might also have far-reaching consequences for the functional understanding of homologous enzymes in higher eukaryotes.
Department of Cell and Developmental Biology, Theodor-Boveri-Institute at the Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
Gülcin
G
Dindar
Gene Center, Department of Biochemistry, Ludwig-Maximilians-University Munich, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
Andreas M
AM
Anger
Department of Molecular Biology and Center for Integrated Protein Science Munich (CIPSM) at the Adolf-Butenandt-Institute, Ludwig-Maximilians-University Munich, Schillerstrasse 44, 80336 Munich, Germany.
Christine
C
Mehlhorn
Department of Molecular Biology and Center for Integrated Protein Science Munich (CIPSM) at the Adolf-Butenandt-Institute, Ludwig-Maximilians-University Munich, Schillerstrasse 44, 80336 Munich, Germany.
Sandra B
SB
Hake
Department of Cell and Developmental Biology, Theodor-Boveri-Institute at the Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
Christian J
CJ
Janzen
D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis / Q000379:methods; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008745:Methylation; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000378:metabolism; D017385:Sequence Homology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics / Q000502:physiology; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics / Q000378:metabolism
D009707:Nucleosomes; D011495:Histone-Lysine N-Methyltransferase