title: Molecular basis of cobalamin-dependent RNA modification.
abstract: Queuosine (Q) was discovered in the wobble position of a transfer RNA (tRNA) 47 years ago, yet the final biosynthetic enzyme responsible for Q-maturation, epoxyqueuosine (oQ) reductase (QueG), was only recently identified. QueG is a cobalamin (Cbl)-dependent, [4Fe-4S] cluster-containing protein that produces the hypermodified nucleoside Q in situ on four tRNAs. To understand how QueG is able to perform epoxide reduction, an unprecedented reaction for a Cbl-dependent enzyme, we have determined a series of high resolution structures of QueG from Bacillus subtilis Our structure of QueG bound to a tRNATyranticodon stem loop shows how this enzyme uses a HEAT-like domain to recognize the appropriate anticodons and position the hypermodified nucleoside into the enzyme active site. We find Q bound directly above the Cbl, consistent with a reaction mechanism that involves the formation of a covalent Cbl-tRNA intermediate. Using protein film electrochemistry, we show that two [4Fe-4S] clusters adjacent to the Cbl have redox potentials in the range expected for Cbl reduction, suggesting how Cbl can be activated for nucleophilic attack on oQ. Together, these structural and electrochemical data inform our understanding of Cbl dependent nucleic acid modification.
authors.affiliation: Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.|Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Dowling
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Zachary D
authors.initials: ZD
authors.lastname: Miles
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Caroline
authors.initials: C
authors.lastname: Köhrer
authors.affiliation: Department of Chemistry, Boston University, Boston, MA 02215, USA.
authors.forename: Stephanie J
authors.initials: SJ
authors.lastname: Maiocco
authors.affiliation: Department of Chemistry, Boston University, Boston, MA 02215, USA.
authors.forename: Sean J
authors.identifier: http://orcid.org/0000-0003-0096-9551
authors.initials: SJ
authors.lastname: Elliott
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Vahe
authors.initials: V
authors.lastname: Bandarian
authors.affiliation: Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA cdrennan@mit.edu.|Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.|Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Drennan
mesh_terms: D000926:Anticodon; D001412:Bacillus subtilis / Q000235:genetics; D006860:Hydrogen Bonding; D007501:Iron / Q000737:chemistry; D008958:Models, Molecular; D008968:Molecular Conformation; D009690:Nucleic Acid Conformation; D009704:Nucleoside Q / Q000031:analogs & derivatives / Q000737:chemistry; D011485:Protein Binding; D012323:RNA Processing, Post-Transcriptional; D020871:RNA Stability; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism; D013455:Sulfur / Q000737:chemistry; D014805:Vitamin B 12 / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000926:Anticodon; D012329:RNA, Bacterial; C051634:nucleoside oQ; D009704:Nucleoside Q; D013455:Sulfur; D012343:RNA, Transfer; D007501:Iron; D012260:Ribonucleases; D014805:Vitamin B 12