Discovery of novel bacterial queuine salvage enzymes and pathways in human pathogens a,1 b,1 c,1 b d a Yifeng Yuan , Rémi Zallot , Tyler L. Grove , Daniel J. Payan , Isabelle Martin-Verstraete , Sara Sepic´ , Seetharamsingh Balamkundue, Ramesh Neelakandane, Vinod K. Gadie, Chuan-Fa Liue, Manal A. Swairjof,g, Peter C. Dedone,h,i, Steven C. Almoc, John A. Gerltb,j,k, and Valérie de Crécy-Lagarda,l,2 aDepartment of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611; bInstitute for Genomic Biology, University of Illinois at Urbana–Champaign, Urbana, IL 61801; cDepartment of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461; dLaboratoire de Pathogénèse des Bactéries Anaérobies, Institut Pasteur et Université de Paris, F-75015 Paris, France; eSingapore-MIT Alliance for Research and Technology, Infectious Disease Interdisciplinary Research Group, 138602 Singapore, Singapore; fDepartment of Chemistry and Biochemistry, San Diego State University, San Diego, CA 92182; gThe Viral Information Institute, San Diego State University, San Diego, CA 92182; hDepartment of Biological Engineering and Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; iCenter for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139; jDepartment of Biochemistry, University of Illinois at Urbana–Champaign, Urbana, IL 61801; kDepartment of Chemistry, University of Illinois at Urbana–Champaign, Urbana, IL 61801; and lUniversity of Florida Genetics Institute, Gainesville, FL 32610 Edited by Tina M. Henkin, The Ohio State University, Columbus, OH, and approved August 1, 2019 (received for review June 16, 2019) Queuosine (Q) is a complex tRNA modification widespread in 1A. The TGT enzyme, which is responsible for the base ex- eukaryotes and bacteria that contributes to the efficiency and accuracy change, is the signature enzyme in the Q biosynthesis pathway. of protein synthesis. Eukaryotes are not capable of Q synthesis and Major differences are found between bacterial and eukaryotic TGT rely on salvage of the queuine base (q) as a Q precursor. While enzymes (22). In eubacteria, TGT functions as a homodimer to many bacteria are capable of Q de novo synthesis, salvage of the incorporate preQ1 into the anticodon wobble of tRNAs (23, 24), prokaryotic Q precursors preQ0 and preQ1 also occurs. With the and maturation to Q is then needed. In contrast, the eukaryotic Escherichia coli exception of YhhQ, shown to transport preQ0 and TGT (eTGT) inserts the q base in tRNAs, yielding Q directly. preQ1, the enzymes and transporters involved in Q salvage and eTGTs are heterodimeric enzymes composed of a catalytic subunit recycling have not been well described. We discovered and char- (queuine tRNA-ribosyltransferase catalytic subunit 1 or QTRT1) acterized 2 Q salvage pathways present in many pathogenic and and an accessory subunit (queuine tRNA-ribosyltransferase acces- MICROBIOLOGY commensal bacteria. The first, found in the intracellular pathogen sory subunit 2 or QTRT2) (25). Key differences defining substrate Chlamydia trachomatis, uses YhhQ and tRNA guanine transglyco- specificities of the prokaryotic and eukaryotic TGTs are in the sylase (TGT) homologs that have changed substrate specificities to Zymomonas mobilis directly salvage q, mimicking the eukaryotic pathway. The second, substrate binding pocket where Val233 ( found in bacteria from the gut flora such as Clostridioides difficile, numbering) is replaced by a glycine and Cys158 is replaced by a valine. These changes allow for the accommodation of q in eTGT salvages preQ1 from q through an unprecedented reaction cata- lyzed by a newly defined subgroup of the radical-SAM enzyme asopposedtopreQ1 (24, 26, 27). family. The source of q can be external through transport by mem- Q synthesis is costly: it starts from GTP (Fig. 1A) and requires bers of the energy-coupling factor (ECF) family or internal through many cofactors (reviewed by Hutinet et al. [21]). Additionally, hydrolysis of Q by a dedicated nucleosidase. This work reinforces 6 of 8 enzymes in the pathway are metalloenzymes. Hence, even the concept that hosts and members of their associated microbiota in bacteria that can synthesize Q de novo, salvaging precursors is compete for the salvage of Q precursors micronutrients. Significance queuosine | nucleoside transport | sequence similarity network | comparative genomics | rSAM Queuosine (Q) is a tRNA modification found in eukaryotes and bacteria that plays an important role in translational efficiency he gut microbiome provides a variety of micronutrients im- and accuracy. Queuine (q), the Q nucleobase, is increasingly Tportant for human health (1). Among them is queuine (q), appreciated as an important micronutrient that contributes to recently designated “a longevity vitamin” (2). In eukaryotes, q is human health. We describe here that q salvage pathways exist the precursor base to the queuosine (Q) tRNA modification (3). in bacteria, including many pathogens and host-associated or- Mammals solely rely on q salvage from dietary sources and from ganisms, suggesting a direct competition for the q precursor in their gut microbiota to synthesize Q-modified tRNAs (4–6). To the human gut microbiome. We also show how a rational use of comparative genomics can lead to the discovery of novel types obtain Q34-tRNA, q is exchanged with the guanine (G) at the of enzymatic reactions, illustrated by the discovery of the queuine wobble position of tRNAs containing G34U35N36 anticodons (Asp, Asn, Tyr, and His) (7), in a reaction catalyzed by a eukaryotic type lyase enzyme. tRNA(34) guanine transglycosylase (TGT) (8, 9). The physiological Author contributions: Y.Y., R.Z., I.M.-V., S.C.A., J.A.G., and V.d.C.-L. designed research; importance of the Q modification is not fully understood. Al- Y.Y., R.Z., T.L.G., D.J.P., and I.M.-V. performed research; S.B., R.N., V.K.G., C.-F.L., and though it is not essential in tested cells under normal conditions P.C.D. contributed new reagents/analytic tools; Y.Y., R.Z., T.L.G., S.S., M.A.S., and (4, 10–14), Q plays a role in regulation of translation, cell pro- V.d.C.-L. analyzed data; and Y.Y., R.Z., T.L.G., I.M.-V., P.C.D., and V.d.C.-L. wrote the paper. liferation, stress response, and cell signaling (6, 15–17). Deficiency The authors declare no conflict of interest. of Q-tRNA level correlates with diseases including tumor growth This article is a PNAS Direct Submission. (reviewed in ref. 6), encephalomyelitis (18), and leukemia (19). Published under the PNAS license. Recently, Q-tRNA levels in human and mice cells have been Data deposition: The data reported in this paper have been deposited in the Protein Data shown to control translational speed of Q‐decoded codons as well Bank, https://www.rcsb.org (ID code 6P78). as near‐cognate codons (20). Taken together, q is a micronutrient 1Y.Y., R.Z., and T.L.G. contributed equally to this work. that links nutrition to translation efficiency. 2To whom correspondence may be addressed. Email: [email protected]. Unlike eukaryotes, most bacteria perform de novo synthesis of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Q via a pathway similar to the one elucidated in Escherichia coli, 1073/pnas.1909604116/-/DCSupplemental. recently summarized by Hutinet et al. (21) and presented in Fig. www.pnas.org/cgi/doi/10.1073/pnas.1909604116 PNAS Latest Articles | 1of10 Downloaded by guest on September 27, 2021 Fig. 1. Queuosine tRNA modification biosynthesis and predicted salvage pathways. (A) Biosynthesis of the Q modification at position 34 (Q34-tRNA) and preQ0/preQ1 salvage pathway in E. coli.(B) Predicted Q34-tRNA biosynthesis and queuine salvage pathway in C. trachomatis D/UW-3/CX. Red dashed arrows represent uncharacterized reactions. Molecule abbreviations and protein names are described in the main text. advantageous, as it reduces metabolic demand. Many organisms but lack all of the other genes encoding Q biosynthetic enzymes. have TGT encoding genes but lack those necessary for preQ0 This is the case for the intracellular human pathogen Chlamydia and preQ1 synthesis (28). In those organisms, precursors must be trachomatis D/UW-3/CX, even if the presence of Q has never been salvaged to obtain Q-modified tRNAs. Significant amounts of experimentally validated. The only predicted Q synthesis genes in free q have been detected in various plant and animal food C. trachomatis are the TGT homolog TGTCt (CT193; UniProt ID products (29), and preQ0 is certainly available in natural envi- O84196) and the YhhQ homolog YhhQCt (CT140; UniProt ID ronments (30). Specific transporters are expected to be involved O84142; Fig. 1B). Indeed, its environment (the mammalian cell) for salvage to occur. We previously demonstrated that a member does not contain preQ0/preQ1, but q or Q are supposedly available. of the COG1738 protein family (YhhQ) can transport preQ0 and We predict that the TGTCt substrate specificity must have preQ1 in E. coli (28). Transporters of the energy-coupling factor switched from preQ1 (classically observed for bacterial TGTs) to (ECF) family (QueT and QrtT) have been predicted to be in- q (observed for eTGTs). A structure-based multiple sequence volved in 7-deazapurine salvage, mainly in Gram-positive or- alignment was performed by using TGTs from C. trachomatis, ganisms, but have never been experimentally validated (31). Chlamydophila caviae, and Chlamydia psittaci (harboring the While tRNA turnover is ubiquitous and constant (32) and re- proposed eukaryotic type salvage pathway), typical bacterial leases the Q nucleoside and/or its monophosphate derivatives TGTs (preQ1 specific), and eTGTs (q specific). While sequences (6), the identities of the nucleotidases, nucleosidases, and other aligned well overall, the residues that accommodate the 7- enzymes involved in tRNA degradation for Q precursors salvage substituent group of the substrate differ for the organisms pre- remain elusive. dicted to salvage q (Fig.