Alternative catalytic residues in the active site of Esco acetyltransferases.
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ICR Authors
Authors
Ajam, T
De, I
Petkau, N
Whelan, G
Pena, V
Eichele, G
De, I
Petkau, N
Whelan, G
Pena, V
Eichele, G
Document Type
Journal Article
Date
2020-06-17
Date Accepted
2020-05-13
Abstract
Cohesin is a protein complex whose core subunits, Smc1, Smc3, Scc1, and SA1/SA2 form a ring-like structure encircling the DNA. Cohesins play a key role in the expression, repair, and segregation of eukaryotic genomes. Following a catalytic mechanism that is insufficiently understood, Esco1 and Esco2 acetyltransferases acetylate the cohesin subunit Smc3, thereby inducing stabilization of cohesin on DNA. As a prerequisite for structure-guided investigation of enzymatic activity, we determine here the crystal structure of the mouse Esco2/CoA complex at 1.8 Å resolution. We reconstitute cohesin as tri- or tetrameric assemblies and use those as physiologically-relevant substrates for enzymatic assays in vitro. Furthermore, we employ cell-based complementation studies in mouse embryonic fibroblast deficient for Esco1 and Esco2, as a means to identify catalytically-important residues in vivo. These analyses demonstrate that D567/S566 and E491/S527, located on opposite sides of the murine Esco2 active site cleft, are critical for catalysis. Our experiments support a catalytic mechanism of acetylation where residues D567 and E491 are general bases that deprotonate the ε-amino group of lysine substrate, also involving two nearby serine residues - S566 and S527- that possess a proton relay function.
Citation
Scientific reports, 2020, 10 (1), pp. 9828 - ?
Source Title
Publisher
NATURE PORTFOLIO
ISSN
2045-2322
eISSN
2045-2322
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Research Team
Mechanisms and regulation of pre-mRNA splicing