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(Oq) Reductase Reveals Parallels Between Halorespiration and Trna Modification

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(Oq) Reductase Reveals Parallels Between Halorespiration and Trna Modification Discovery of epoxyqueuosine (oQ) reductase reveals parallels between halorespiration and tRNA modification Zachary D. Miles, Reid M. McCarty, Gabriella Molnar, and Vahe Bandarian1 Department of Chemistry and Biochemistry, University of Arizona, 1041 East Lowell Street, Tucson, AZ 85721-0088 Edited by Christopher T. Walsh, Harvard Medical School, Boston, MA, and approved March 10, 2011 (received for review December 15, 2010) Transfer RNA is one of the most richly modified biological mole- This in vitro reconstitution of the 7-deazapurine core was aided cules. Biosynthetic pathways that introduce these modifications by the discovery of the toyocamycin biosynthetic gene cluster in are underexplored, largely because their absence does not lead Streptomyces rimosus, which contains three genes that had been to obvious phenotypes under normal growth conditions. Queuo- previously shown to be required for the biosynthesis of queuosine sine (Q) is a hypermodified base found in the wobble positions in Bacillus subtilis (10–13). The cluster also contained a GTP of tRNA Asp, Asn, His, and Tyr from bacteria to mankind. Using cyclohydrolase I (GCH I) homolog, which hinted at GTP as being liquid chromatography MS methods, we have screened 1,755 sin- the primary metabolic precursor. GCH I catalyzes the conversion gle gene knockouts of Escherichia coli and have identified the key of GTP to 7,8-dihydroneopterin triphosphate (H2NTP) in the final step in the biosynthesis of Q. The protein is homologous to first step in the biosynthesis of both folic acid and biopterin. B12-dependent iron-sulfur proteins involved in halorespiration. GCH I was subsequently shown to be required for the biosynth- The recombinant Bacillus subtilis epoxyqueuosine (oQ) reductase esis of Q in Escherichia coli (14). In vitro reconstitution was catalyzes the conversion of oQ to Q in a synthetic substrate, as well accomplished by the successive actions of GCH I, 6-carboxy- as undermodified RNA isolated from an oQ reductase knockout 5,6,7,8-tetrahydropterin (CPH4) synthase, 7-carboxy-7-deazagua- strain. The activity requires inclusion of a reductant and a redox nine (CDG) synthase, and preQ0 synthetase, demonstrating that mediator. Finally, exogenously supplied cobalamin stimulates the four enzymatic transformations are required to convert GTP activity. This work provides the framework for studies of the bio- to preQ0, a previously identified intermediate in the biosynthesis synthesis of other modified RNA components, where lack of acces- of queuosine (see Fig. 1). At this point, the biosynthetic pathway sible phenotype or obvious gene clustering has impeded discovery. toward toyocamycin diverges from that of queuosine with preQ0 Moreover, discovery of the elusive oQ reductase protein completes being elaborated to toyocamycin in a series of steps that are ana- the biosynthetic pathway of Q. logous to those in purine biosynthesis/salvage. By contrast, the biosynthetic pathway to queuosine entails the NADPH-depen- ∣ ∣ biochemistry queuosine reductive dehalogenation dent conversion of preQ0 to 7-amino-7-deazaguanine (preQ1)by preQ0 reductase (15, 16). PreQ1 base is then exchanged for gua- early 100 modifications have been identified in RNA, many nine in the anticodon loop of tRNA by tRNA:guanine transgly- Nof which are found in tRNA and are common to eukaryotes, cosylase (Tgt) (6, 17, 18) and elaborated to oQ by addition of a bacteria, and archaea (1). Most of these modifications are likely cyclopentanediol epoxide derived from S-adenosyl-L-methionine not essential under normal laboratory conditions, making dis- by oQ synthase (QueA) (7). The penultimate intermediate covery of biosynthetic pathways by phenotypic methods impossi- to queuosine, oQ, was isolated and characterized 23 y ago (19) ble. Queuosine (Q), a hypermodified RNA base containing a and B12 was subsequently implicated in its conversion to Q (20). 7-deazapurine core, is among the more complex RNA modifica- The identity of the enzyme that catalyzes the transformation and tions described to date. Q replaces the guanine in the wobble in vitro reconstitution of oQ reductase activity are reported in this positions of the subset of tRNA molecules with a 5′-GUN-3′ manuscript. sequence in their anticodon loops (His, Asp, Asn, and Tyr). Con- servation of Q in RNA of organisms in nearly all kingdoms of life Results and Discussion (2) suggests that the modification may be of cardinal importance. Screening of the Keio Collection. The availability of a series of iso- A physiological role for Q has eluded discovery partly because of genic variants of E. coli K-12, the Keio collection (21), in which gaps in understanding of the biosynthetic pathway. De novo bio- each of the approximate 4,000 nonessential genes are individually synthesis of Q occurs in bacteria whereas eukaryotes acquire the disrupted by a kanamycin resistance cassette, provided a valuable free base, queuine, from dietary sources (3–5). The bacterial opportunity to mount a search for the elusive oQ reductase. We pathway for biosynthesis of Q has been elucidated up to the identified 1,755 knockout strains carrying deletions in genes of penultimate intermediate, epoxyqueuosine (oQ) (6, 7). However, unknown function, annotated as “y” genes in the National Center the enzyme that catalyzes the final step in the pathway, conver- for Biotechnology Information genome database (see Dataset S1 sion of oQ to Q, had yet to be identified. Because no selectable for a complete list of deletion strains examined in this study), as phenotypes have been demonstrated for the modification, discov- potential candidates for genes that encode oQ reductase. Each ery of the final step in the pathway required a different approach that melded modern analytical methods and emerging tools in Author contributions: Z.D.M., R.M.M., and V.B. designed research; Z.D.M., R.M.M., G.M., bacterial genetics. This methodology has led to successful iden- and V.B. performed research; Z.D.M., R.M.M., and V.B. contributed new reagents/analytic tification of the final step and can be generalized to other RNA tools; Z.D.M., R.M.M., and V.B. analyzed data; and Z.D.M., R.M.M., and V.B. wrote modifications where lack of phenotype has hindered discovery of the paper. the biosynthetic pathway. The authors declare no conflict of interest. Structural parallels between the 7-deazapurine core of queuo- This article is a PNAS Direct Submission. sine and 7-deazapurine-containing antibiotic toyocamycin were 1To whom correspondence should be addressed. E-mail: [email protected]. noted long before the various elements of the biosynthetic path- This article contains supporting information online at www.pnas.org/lookup/suppl/ way leading to either compound was reconstructed in vitro (7–9). doi:10.1073/pnas.1018636108/-/DCSupplemental. 7368–7372 ∣ PNAS ∣ May 3, 2011 ∣ vol. 108 ∣ no. 18 www.pnas.org/cgi/doi/10.1073/pnas.1018636108 Downloaded by guest on September 26, 2021 Fig. 1. Biosynthetic pathway for production of queuosine with the oQ reductase step elucidated in this manuscript boxed. The block arrow denotes a series of steps analogous to purine salvage/repair, which convert the 7-cyano-7-deazaguanine base to toyocamycin. deletion strain was grown in Lennox broth, RNA was isolated from each culture, digested, and dephosphorylated (22–24). Nucleosides were resolved by reverse phase HPLC and detected by tandem UV-visible spectrophotometry and mass spectrometry. Under these conditions, oQ and Q, which are present in minis- cule amounts, are readily detected and identified. Representative UV-visible and extracted ion chromatograms obtained from wild-type E. coli as processed and described above are shown in Fig. 2A. Queuosine and epoxyqueuosine typically elute at approximately 30–32 min in chromatograms. Extracted ion chromatograms monitoring oQ (m∕z ¼ 426) and Q (m∕z ¼ 410) show that under normal growth conditions both forms are present in RNA (Fig. 2B). However, the intensity of the peak for oQ is typically 0–30% of that of Q. RNA isolated from any strain carrying deletions up to and including Tgt would be pre- dicted to be completely devoid of Q and oQ (6). Indeed, deletion of CPH4 synthase (b2765), CDG synthase (b2777), preQ0 synthethase (b0444), preQ0 reductase (b2794), and Tgt (b0406) lead to disappearance of both Q and oQ from RNA. GCH I is an essential gene for E. coli because it also catalyzes the first step in biosynthesis of folic acid (25, 26), and is therefore not included Fig. 2. Analysis of RNA from Keio collection deletion strains for presence in the Keio collection. Interestingly, although oQ or Q are absent and absence of Q and oQ. A shows a representative UV-visible trace of the RNA samples, highlighting the region where oQ and Q elute. Wild-type in extracted ion chromatograms of RNA from the QueA (b0405) E. coli B m∕z ¼ contain very little oQ when grown in LB. shows extracted ion chro- deletion strain, the precursor to oQ, preQ1 nucleoside ( matograms of wild-type and deletion strains corresponding to each step in 312), is clearly present (see Fig. 1 for structure). the pathway. Deletion of any gene up to and including Tgt leads to complete Liquid chromatography (LC) MS analysis of y genes in the absence of oQ (m∕z ¼ 426)orQ(m∕z ¼ 410). Deletion of queA leads to Keio collection revealed a single gene, yjeS (b4166), which is accumulation of preQ1 nucleoside (m∕z ¼ 312), as would be expected (see required for conversion of oQ to Q. RNA from the ΔyjeS strain Fig. 1). Deletion of queG leads to complete absence of Q and accumulation clearly lacks Q and accumulates oQ, as would be expected if YjeS of oQ. A seven-point data smoothing algorithm was applied to all of the extracted ion chromatograms. catalyzes conversion of oQ to Q in vivo. We observed the identical phenotype with the deletion of tonB (see Fig.
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