title: Discovery of epoxyqueuosine (oQ) reductase reveals parallels between halorespiration and tRNA modification.
abstract: Transfer RNA is one of the most richly modified biological molecules. Biosynthetic pathways that introduce these modifications are underexplored, largely because their absence does not lead to obvious phenotypes under normal growth conditions. Queuosine (Q) is a hypermodified base found in the wobble positions of tRNA Asp, Asn, His, and Tyr from bacteria to mankind. Using liquid chromatography MS methods, we have screened 1,755 single gene knockouts of Escherichia coli and have identified the key final step in the biosynthesis of Q. The protein is homologous to B(12)-dependent iron-sulfur proteins involved in halorespiration. The recombinant Bacillus subtilis epoxyqueuosine (oQ) reductase catalyzes the conversion of oQ to Q in a synthetic substrate, as well as undermodified RNA isolated from an oQ reductase knockout strain. The activity requires inclusion of a reductant and a redox mediator. Finally, exogenously supplied cobalamin stimulates the activity. This work provides the framework for studies of the biosynthesis of other modified RNA components, where lack of accessible phenotype or obvious gene clustering has impeded discovery. Moreover, discovery of the elusive oQ reductase protein completes the biosynthetic pathway of Q.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, 1041 East Lowell Street, Tucson, AZ 85721-0088, USA.
authors.forename: Zachary D
authors.initials: ZD
authors.lastname: Miles
authors.forename: Reid M
authors.initials: RM
authors.lastname: McCarty
authors.forename: Gabriella
authors.initials: G
authors.lastname: Molnar
authors.forename: Vahe
authors.initials: V
authors.lastname: Bandarian
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D055786:Gene Knockout Techniques; D013058:Mass Spectrometry; D015394:Molecular Structure; D009704:Nucleoside Q / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000378:metabolism; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry*; D014805:Vitamin B 12
chemical_list: D017931:DNA Primers; C051634:nucleoside oQ; D009704:Nucleoside Q; D012343:RNA, Transfer; D010088:Oxidoreductases; D014805:Vitamin B 12