title: Engineering Biological C-H Functionalization Leads to Allele-Specific Regulation of Histone Demethylases.
abstract: Oxidative C-H hydroxylation of methyl groups, followed by their removal from DNA, RNA, or histones, is an epigenetic process critical to transcriptional reprogramming and cell fate determination. This reaction is catalyzed by Fe(II)-dependent dioxygenases using the essential metabolite 2-ketoglutarate (2KG) as a cofactor. Given that the human genome encodes for more than 60 2KG-dependent dioxygenases, assigning their individual functions remains a significant challenge. Here we describe a protein-ligand interface engineering approach to break the biochemical degeneracy of these enzymes. Using histone lysine demethylase 4 as a proof-of-concept, we show that the enzyme active site can be expanded to employ bulky 2KG analogues that do not sensitize wild-type demethylases. We establish the orthogonality, substrate specificity, and catalytic competency of the engineered demethylation apparatus in biochemical assays. We further demonstrate demethylation of cognate substrates in physiologically relevant settings. Our results provide a paradigm for rapid and conditional manipulation of histone demethylases to uncloak their isoform-specific functions.
authors.affiliation: Department of Chemistry and ∥Department of Human Genetics, University of Pittsburgh , Pittsburgh, Pennsylvania 15260, United States.
authors.forename: Megan
authors.initials: M
authors.lastname: Breski
authors.affiliation: Department of Chemistry and ∥Department of Human Genetics, University of Pittsburgh , Pittsburgh, Pennsylvania 15260, United States.
authors.forename: Debasis
authors.initials: D
authors.lastname: Dey
authors.affiliation: Department of Chemistry and ∥Department of Human Genetics, University of Pittsburgh , Pittsburgh, Pennsylvania 15260, United States.
authors.forename: Sara
authors.initials: S
authors.lastname: Obringer
authors.affiliation: Department of Chemistry and ∥Department of Human Genetics, University of Pittsburgh , Pittsburgh, Pennsylvania 15260, United States.
authors.forename: Babu
authors.initials: B
authors.lastname: Sudhamalla
authors.affiliation: Department of Chemistry and ∥Department of Human Genetics, University of Pittsburgh , Pittsburgh, Pennsylvania 15260, United States.
authors.forename: Kabirul
authors.initials: K
authors.lastname: Islam