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 tRNATyr anticodon 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.

Vitamin B 12 in the spotlight again

“…In fact, the open-faced nature of Cbl may be key to the chemistry performed (see below). Reductive dehalogenases were previously proposed to form this third class [24,25], but they are now joined by epoxyqueuosine (oQ) reductases (QueGs) [20,22,26,27], and CblCs, the enzymes responsible for decyanation/dealkylation of Cbl [21]. In addition to lacking an upper ligand, Cbl bound to these enzymes also lacks a lower ligand.…”

“…QueG catalyzes reduction of epoxyqueuosine to form the RNA nucleoside queuosine (Q) that is present in the wobble position of certain tRNAs [26]. A recent structure of QueG from Bacillus subtilis co-crystallized with an oQ-modified tRNA Tyr anticodon stem loop reveals that Q binds directly above Cbl (Figure 3a) [20]. These structural data, along with electrochemical data that show the [4Fe-4S] clusters have redox potentials sufficient to generate the Co(I)-Cbl state, are consistent with a mechanism in which QueG catalyzes the conversion of oQ to Q through nucleophilic attack of Co(I)-Cbl on the substrate epoxide (Figure 3b–c) [20].…”

“…(a) Structure of QueG (yellow) with an oQ-modified tRNA Tyr anticodon stem loop bound (displayed as sticks with green carbons, left panel) [20]. The modified RNA base is inserted into a cavity in the protein, binding directly above Co of Cbl (displayed as sticks with dark pink carbons, right panel) [20]. Arg 141 (yellow carbons) of QueG blocks access to the lower axial face of Cbl.…”

“…(b) The proposed mechanism of QueG. Nucleophilic attack of Co(I)-Cbl species on the oQ substrate forms an organocobalt adduct that can be converted to product and cob(II)alamin through reduction and protonation [20]. (c) An overlay of Cbls and two [4Fe-4S] clusters (orange and yellow spheres) from QueG (carbons in dark pink) and the reductive dehalogenase enzymes PceA (cyan) and Np RdhA (light pink).…”

QueE: A Radical SAM Enzyme Involved in the Biosynthesis of 7-Deazapurine Containing Natural Products

“…Despite the structural diversity, it now appears the deazapurine moiety is biosynthesized in three steps from guanosine triphosphate (GTP) (McCarty et al, 2012; McCarty, Krebs, & Bandarian, 2012; McCarty, Somogyi, & Bandarian, 2009a; McCarty, Somogyi, Lin, Jacobsen, & Bandarian, 2009b; Miles, Roberts, McCarty, & Bandarian, 2014; Reader, Metzgar, Schimmel, & de Crécy-Lagard, 2004), and modified as appropriate by additional species-specific tailoring enzymes (McCarty et al, 2012; Bai, Fox, Lacy, Van Lanen, & Iwata-Reuyl, 2000; Dowling et al, 2016; Frey, McCloskey, Kersten, & Kersten, 1988; Lee, Van Lanen, & Iwata-Reuyl, 2007; Miles, McCarty, Molnar, & Bandarian, 2011; Miles, Myers, Kincannon, Britt, & Bandarian, 2015; Nelp & Bandarian, 2015; Nelp, Astashkin, Breci, McCarty, & Bandarian, 2014; Nelp, Song, Wysocki, & Bandarian, 2016; Okada, Harada, & Nishimura, 1976; Okada et al, 1979; Phillips et al, 2008; Reuter, Slany, Ullrich, & Kersten, 1991; Shindo-Okada, Okada, Ohgi, Goto, & Nishimura, 1980; Song, Nelp, Bandarian, & Wysocki, 2015; Van Lanen et al, 2005; Watanabe et al, 1997) (Fig. 1).…”