Discovery of Novel Bacterial Queuine Salvage Enzymes and Pathways in Human Pathogens

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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 catalyzed 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. 2A). Docking q in the active site of a Riboswitches are regulatory elements of messenger RNA mol- typical bacterial TGT (from Z. mobilis) is not possible: the ecules. Three classes of preQ1 riboswitches have been described V233 side chain restricts the space to allow only the small preQ1 upstream of known Q synthesis genes (33, 34). Exploring genes substrate in the binding pocket (Fig. 2B). The C. trachomatis downstream of predicted preQ1 riboswitches played a key role for enzyme is atypical in this aspect, as it allows docking of q in its the identification of YhhQ as preQ1/preQ0 transporter (28). In the active site: G235 allows the cyclopentenediol ring present in q to RegPrecise database (35), genes downstream of predicted preQ1 be accommodated, similarly to what is seen for eTGTs (Fig. 2B) riboswitches include queC, queD, queE, queF, yhhQ, and genes (26, 27). previously associated with Q metabolism but without experimental If the TGTCt substrate is q, YhhQCt must, unlike its E. coli validation: members of COG1957 (inosine-uridine nucleoside N- homolog, be transporting q and not preQ0/preQ1. No structure is ribohydrolase; IunH), COG4708 (predicted membrane protein, available for any member of the YhhQ family, and the residues QueT), and QrtT (substrate-specific component STY3230 of involved in substrate recognition and transport are yet to be queuosine-regulated ECF transporter) families (33, 36). identified. However, sequence similarity networks (SSNs) analyses In summary, while the de novo Q biosynthesis pathway is well using the EFI webtools (39, 40) allowed us to define sub- characterized, many open questions remain regarding the pos- groups within the YhhQ family that correlate well with the sibility of Q salvage in bacteria. The work presented here elu- various pathway configurations (Fig. 2C). This suggests a spe- cidates 2 salvage pathways in pathogenic bacteria. cialization of the transport activity for salvage of q, preQ0,or preQ1. The C. trachomatis YhhQCt transporter (Fig. 2C, blue Results arrow) is in a subgroup from organisms that harbor TGT but not The Chlamydia trachomatis TGT and YhhQ Homologs Are Involved in Q QueA or QueG/QueH homologs, and is hence predicted to be Salvage. The distribution of Q metabolism genes in more than involved in q salvage. 10,000 genomes is shown in the PubSEED subsystem (37) To test our hypothesis in vivo, we expressed predicted C. tra- “q_salvage_in_Bacteria” (http://pubseed.theseed.org//SubsysEditor. chomatis q salvage genes in an E. coli derivative auxotrophic for cgi?page=ShowSubsystem&subsystem=q_salvage_in_Bacteria). Q (Fig. 2D). While E. coli is among the organisms that harbor a Previous phylogenomic analyses suggested that an eukaryotic-type complete Q de novo pathway, it can also salvage preQ0 and salvage pathway, i.e., the ability to directly use q, must exist in preQ1, imported by YhhQEc (Fig. 1A) (28). E. coli is not pre- bacteria such as Chlamydia, Wolbachia, Corynebacterium, Actino- dicted to salvage q based on the substrate specificity of its TGTEc myces,andBifidobacterium species (28, 38), as they possess a TGT enzyme (41). We set out to test whether Q could be detected in encoding gene and sometimes predicted Q precursor transporters tRNAs extracted from an E. coli ΔqueD strain after feeding with

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Fig. 2. C. trachomatis salvages queuine in 2 steps. (A) Amino acid sequence alignment of select TGT proteins using PROMALS3D (74). The catalytic residues are shown in bold. The residues that accommodate the 7-substituent group of the substrate are shown in red. Dots indicate regions intentionally deleted for this figure. Dashes indicate gaps in the sequence alignment. UniProt IDs for proteins included in multiple alignment are as follows: Zymomonas mobilis (P28720), E. coli (P0A847), Shigella flexneri (Q54177), Homo sapiens QTRT1 (Q9BXR0), Caenorhabditis elegans (Q23623), C. trachomatis (A0A0E9DEF3), C.

caviae (Q822U8), and C. psittaci (A0A2D2DY33). (B) Comparison of substrate-binding pockets of TGT proteins. The binding pocket of TGTCt is modeled and docked with q (purple), compared with that of Z. mobilis TGT (green; crystal structure with docked q) and H. sapiens QTRT1 (orange; crystal structure bound to q).

The residues that accommodate the substrate’s 7-substituent moiety are shown in a stick model; S233LG235 in TGTCt,L231AVG234 in Z. mobilis TGT and L230SGG233 in H. sapiens QTRT1. Queuine is colored bright green. A steric clash in Z. mobilis TGT precludes binding of q (dashed circle). (C) Protein sequence similarity network of 6,187 YhhQ sequences that were retrieved from the PubSEED subsystem “q_salvage_in_Bacteria” and colored based on the predicted salvaged molecule in the

organism from which they originate: red for preQ0, yellow for preQ1, and dark blue and sky blue for queuine. Red and blue arrows indicate YhhQ homologs from E. coli and C. trachomatis,respectively.(D) Scheme of Q metabolism in the E. coli derivatives used to test the function of CT140 and CT193. Dashed arrows represent reactions that are being tested. Precursors in gray are not synthesized de novo in these strains. (E and F) Detection of Q-tRNA by the APB assay in Asp tRNA GUC in the presence of exogenous queuine (q) while expressing CT140/yhhQCt and/or CT193/tgtCt in different E. coli derivatives.

Yuan et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 27, 2021 exogenous q (10 nM or 100 nM; SI Appendix, Fig. S1). In this Northern-based assay, tRNAs containing Q migrate more slowly on a polyacrylamide gel containing 3-(acrylamido)phenylboronic acid (APB) than tRNAs lacking the modification (42). After transfer on a nylon membrane, a biotinylated probe specific for Asp tRNA GUC is used for detection (17, 43). On the obtained blot, tRNAs that are modified with Q (SI Appendix, Fig. S1,WT control) appear in a band located higher than unmodified tRNAs (SI Appendix, Fig. S1, Δtgt control). The presence of this specific band is used as a proxy for the presence of the Q modification in the tRNAs extracted. As shown in SI Appendix, Fig. S1, no Q-modified tRNAs were detected in bulk tRNA extracted from the ΔqueD strain in the presence of q. The same result was obtained in the E. coli ΔqueD ΔqueF strain (SI Ap- pendix, Fig. S1). As a positive control, the formation of Q was observed when preQ1 (10 nM) was added (SI Appendix, Fig. S1). These results confirmed that E. coli MG1655 salvages preQ0 and preQ1, but not q, a condition necessary to test our hypothesis. To test whether the YhhQ homolog (CT140/YhhQCt) and TGT homolog (CT193/TGTCt)ofC. trachomatis use q as a substrate, the corresponding genes were expressed in the E. coli ΔqueD strain (Fig. 2D) and the presence of Q was evaluated in extracted bulk tRNAs. Q was detected in the presence of exogenous q (10 nM) only when both genes were expressed (Fig. 2E). This result supports the prediction that YhhQCt transports q and that TGTCt catalyzes the base exchange between q and the target guanine in tRNA. To further explore the substrate specificities of YhhQCt and TGTCt, we deleted yhhQ or tgt in the E. coli ΔqueD ΔqueF strain expressing one or both Chlamydia genes and pro- bed for the presence of Q when different precursors (preQ0, preQ1, and q) are supplemented during growth (Fig. 2F). tRNA Δ Δ Δ extracted from the queD queF tgt pBAD24:tgtCt strain grown Fig. 3. CD1682, CD1683, and CD1684 are required for queuosine modifica- in the presence of any of the 3 precursors lacks Q (Fig. 2 F, Left), tion in tRNA of C. difficile.(A) Predicted Q-tRNA biosynthesis pathway in C. demonstrating that TGTCt does not use preQ1 or preQ0. How- difficile strain 630. The magnified subfigure shows a model of the ECF trans- ever, YhhQCt can transport preQ1, as tRNA extracted from porters that include 4 subunits: S, the substrate-specific transmembrane component (S component); T, the energy-coupling module consisting of a ΔqueD ΔqueF ΔyhhQ pBAD33:yhhQCt cells fed with preQ1 shows a low but detectable signal for Q (Fig. 2 F, Middle). transmembrane protein (T component); and A and A′, pairs of ABC ATPases (A This set of genetic experiments not only validated the func- proteins). Dashed arrows represent uncharacterized reactions. Molecule abbreviations and protein names are described in the text. (B) Detection of Q- tional hypothesis about the C. trachomatis q salvage genes but Asp tRNA by the APB assay in tRNA GUC of C. difficile 630 WT, 630 Δtgt,and630 also provided tools to test the function of predicted q salvage ΔCD1682-CD1684 strains. tRNA extracted from E. coli WT and Δtgt strains was genes from other species. used as control. (C) Representation of the genomic context of the radical SAM cluster. C. difficile 630 (accession: NC_009089.1), C. perfringens ATCC 13124 Identification and Experimental Validation of Clostridioides difficile Q (accession: NC_008261.1), Clostridium botulinum E1 str. “BoNT E Beluga” (acces- Salvage Genes. Clostridioides difficile is an enteropathogen that can sion: NZ_ACSC00000000.1), R. gnavus ATCC 29149 (accession: NZ_AAYG02000018.1), develop in the colon after an antibiotic exposure, leading to dys- and L. bacterium 2_1_58FAA (accession: ACTO00000000.1). Each gene is colored biosis of the gut microbiota. C. difficile is an example of a subgroup according to Pfam domain. Predicted promoters and rho-independent ter- minators are indicated by dashed arrows and dots, respectively. PreQ of organisms that lack the preQ1 synthesis genes (queD, queE, 1 queC,andqueF) but harbor genes encoding the remaining path- riboswitches are indicated by stem loops. way enzymes TGT, QueA, and QueG or QueH (28). A parsi- monious prediction is that preQ1 must be salvaged in these reported in a previous bioinformatic study (36). This riboswitch organisms (Fig. 3A) and that they harbor bacterial-type TGT en- is located ∼140 bp upstream the start site of CD1682 (UniProt zymes. The prediction about the substrate specificity of the TGT Cd ID Q186N9), the first gene of the operon, predicted to encode a enzyme of C. difficile (CD2802) was first tested. When expressing the nucleoside hydrolase (member of the IunH family, COG1957 corresponding gene in an E. coli ΔqueD ΔqueF Δtgt pBAD::yhhQCt strain that can transport all Q precursors, Q was detected in tRNAs and IPR036452). The last gene of the operon, CD1684 (UniProt ID Q186P0), is predicted to encode for an enzyme belonging only in the presence of preQ1, confirming that C. difficile TGTCd, to the radical SAM enzyme family (IPR006638; Fig. 3C). like its E. coli ortholog, incorporates preQ1 only in tRNA (SI Ap- Identical operons were also identified in other bacteria from pendix,Fig.S2).TheroleofTGTCd in Q synthesis was confirmed by deleting the corresponding gene in the C. difficile 630 genome (SI the gut microbiome such as Clostridium perfringens, Ruminococcus Appendix,Fig.S3). tRNA extracted from the tgt+ strain contained gnavus,andLachnospiraceae bacterium (Fig. 3C). The presence Q, while the Δtgt strain lacked the modification (Fig. 3B). of a preQ1 riboswitch suggested a role of the CD1682-CD1684 Because C. difficile does not encode a YhhQ homolog, another operon in Q salvage. It was confirmed genetically in C. difficile, transporter must be involved in salvaging Q precursors. One as tRNA extracted from the ΔCD1682-CD1684 strains lacked candidate is CD1683 (UniProt ID Q186P1), annotated as a Q(Fig.3B and SI Appendix,Fig.S3). substrate-specific component of the queuosine ECF transporter qrtT in the RegPrecise database (35). CD1683 is the second gene The Nucleoside Hydrolase CD1682 Hydrolyzes Queuosine to Queuine. of a 3-gene operon predicted to be under the control of a preQ1 CD1682 is a protein belonging to a nucleoside hydrolase family, riboswitch [Rfam accession RF00522 in RegPrecise (35)], as enzymes typically cleaving the N-glycosidic bond connecting

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1909604116 Yuan et al. Downloaded by guest on September 27, 2021 nucleobases to ribose (44) with a preference for inosine and sumed more rapidly than guanosine or inosine, while adenosine uridine (IunH family, also COG1957 and IPR036452). Nucleo- is nearly not consumed. Future investigations will explore the side hydrolase family members harbor a conserved motif detailed kinetic parameters and substrate preferences for this DxDxxxDD, located toward their N termini (44). The second and nucleoside hydrolase subgroup often identified embedded in Q + last Asp in the motif are involved in the chelation of a Ca2 ion at salvage operonic structures. the active site. Upon binding, the ribose moiety of substrate In vivo, the source of Q could be intracellular from degrada- nucleobases is coordinated by the metal ion, allowing for hydro- tion of tRNA or exogenous if a Q transporter is present in C. lysis. Within the family, while the residues involved in binding the difficile. We therefore set out to characterize the substrate + Ca2 ion and the positioning of the ribose moiety are conserved, specificity of predicted Q precursor transporters in this organism. the residues interacting with the nucleobase are variable. Initially, several nucleoside hydrolase subclasses were defined on the basis of Substrate Specificity Analysis of the Q Precursor Transporters C. difficile their preferred substrate, i.e., the purine-specific inosine-adenosine- Suggested Salvages Not Only preQ1 but Also Queuine guanosine–preferring nucleoside hydrolases (IAGNHs), the 6- and Queuosine. The strain 630 of C. difficile does not encode a oxopurine–specific inosine-guanosine–preferring nucleoside hydro- homolog of YhhQ but encodes 3 homologs of QrtT/QueT (31) lases (IGNHs), and the base-aspecific inosine-uridine–preferring (CD1683, CD2097, and CD3073; UniProt IDs Q186P1, Q188G5, nucleoside hydrolases (IUNHs). However, with the increasing and Q184R1, respectively; Fig. 3A). These ECF S components number of sequences becoming available, it appears that this clas- interact with the core components of ECF transporter encoded in sification is not in accordance with what has been identified at the C. difficile by CD0100, CD0101,andCD0102 (46). We showed sequence level (44). here earlier that deletion of the operon containing CD1683 In order to place the CD1682 protein within the nucleoside hy- abolished Q presence in this organism (Fig. 3B). To further drolase family (IPR036452), we performed SSN analyses (39, 40). In characterize these transporters, we constructed 3 pBAD33 deriv- the UniRef90 SSN generated at a low alignment score threshold of atives each expressing the 3 core components genes in 1 operon 58, CD1682 (UniProt ID Q186N9) falls into cluster 1 (the largest), with 1 of the 3 predicted ECF S components and transformed the containing SwissProt-annotated sequences RihA, B, and C, IUNH, plasmids in the E. coli ΔqueD ΔyhhQ strain that expresses the C. and uridine nucleosidase (SI Appendix,Fig.S4A). This confirmed that trachomatis tgt gene (tgtCt). This strain can salvage both provided q CD1682 is related to sequences known to hydrolyze nucleobases. and preQ0/preQ1 only if an appropriate transporter gene is When subjected to a higher alignment score threshold of 75, CD1682 expressed in trans (Fig. 5A). The presence of Q in tRNA was

and highly homologous sequences are segregated into their own monitored in that strain after feeding with 10 nM preQ0,preQ1,or MICROBIOLOGY cluster (SI Appendix,Fig.S4B). This suggests that sequences consti- q(Fig.5B). We found that preQ1 but not preQ0 was transported tuting the CD1682 cluster are different enough from the rest of the both by CD1683 and CD2097 and that CD1683 is able to trans- family that they could be active against a different substrate, namely Q. port q, albeit with a lower efficiency. No transport of any precursor Genome Neighborhood Network (GNN) analysis (39, 40) of a was observed when expressing CD3073. local high-resolution SSN generated around CD1682 identified a The same strains were used to test if these ECF transport pro- set of closely related nucleoside hydrolases that are highly likely teins as well as the previously characterized YhhQ proteins could dedicated for Q salvage (SI Appendix,Fig.S5). A multiple se- be involved in Q import (Fig. 5C). Q-tRNA was detected when quence alignment for UniRef90 representative sequences from CD1683 (S component) and genes of ECF core components were the CD1682 cluster (156 sequences) showed the absolute con- coexpressed with CD1682 (Fig. 5D) in the presence of Q (10 nM), + servation of residues involved in Ca2 chelation (SI Appendix, Fig. confirming the nucleoside hydrolase activity of CD1682 in vivo. We S6). However, the typical conserved residues known to be involved propose to name members from this subgroup of the nucleoside in the coordination of the pyrimidine or purine moieties in the hydrolase family: queuosine hydrolase or QueK. It is worth noting characterized enzymes among the family are not present (45). As that a small amount of Q was detected in tRNA in the presence of queuosine is a 7-deazaguanine carrying a cyclopentenediol ring high Q concentrations (500 nM) when yhhQCt or CD1683 was linked at position 7 through an aminomethyl linkage, a more expressed while CD1682 is absent (SI Appendix,Fig.S9). This sizeable substrate pocket to accommodate the queuine moiety is observation could result from a nonspecific Q nucleoside hydrolase required. The divergence detected at the sequence level for the activity in E. coli, potentially carried out by an endogenous member nucleobase recognition is thus consistent with the proposed di- of the nucleoside hydrolase family. vergence in activity carried: Q hydrolysis. Bioinformatic evidence associates CD1682 (and closely re- The C. difficile q Salvage Pathway Uses a Queuine Lyase of the Radical lated sequences) with Q salvage. To directly test the hypothesis SAM Enzyme Family. In combination, the experiments described that CD1682 catalyzes the hydrolysis of Q to q, CD1682 was here so far suggest that Q can be transported in C. difficile by an expressed in E. coli with a C-terminal hexahistidine tag, purified ECF transporter using CD1683 as a specificity component and ++ by Ni -affinity chromatography followed by size-exclusion hydrolyzed to q by CD1682 (Fig. 3A). As the substrate of TGTCd chromatography (SI Appendix, Fig. S7), and assayed for activ- (CD2802) is preQ1 (SI Appendix, Fig. S2), the conversion of q to ity. Analytical size-exclusion chromatography performed on the preQ1, a reaction never described previously to our knowledge, is purified recombinant CD1682 reveals a molecular weight of needed to complete the pathway. The third gene of the CD1682 118 kDa, while the molecular weight predicted from the coding operon encodes for a protein of the radical-SAM (RS) enzyme sequence is 37.1 kDa, suggesting that a trimeric complex forms in family, known for its remarkable and versatile enzymology (47– solution. Other nucleoside hydrolases have been reported to 50). We hypothesized that CD1684 (UniProt ID Q186P0) could exist as dimers or tetramers in solution (44). Activity was eval- act as a lyase, breaking a C-N bond to generate preQ1 from q. uated quantitatively by following the time-dependent elution of The hypothesis that CD1684 is a q lyase was tested by using the E. substrate and product by LC-MS. As visualized with absorption coli ΔqueD ΔqueF pBAD33:yhhQCt strain, which is able to import q at 260 nm (Fig. 4A) and confirmed with MS for their specific but cannot catalyze its insertion in tRNAs (Fig. 6A). Remarkably, masses (Fig. 4 B and C), over time, Q is consumed concomitantly when the CD1684 gene was expressed in this strain, q could be with q being produced, providing in vitro support for the pro- salvaged (Fig. 6B). TGTEc is preQ1-specific, and the q salvage ac- posed hypothesis. The hydrolysis capacity of the purified hy- tivity is abolished by deletion of the downstream gene queA (Fig. 6 B, drolase against other purines nucleosides was also evaluated (SI Right). Thus, the insertion of Q in tRNAs when q is provided ex- Appendix, Fig. S8). In identical conditions regarding substrate ternally required the production of the preQ1 intermediate and is concentration, enzyme amount, and time units, Q appears con- not the result of a direct q incorporation. These results taken

Yuan et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 27, 2021 Fig. 4. CD1682 is a queuosine hydrolase (QueK). (A) The queuosine hydrolysis activity of purified recombinant CD1682 was assayed and analyzed at different time points by injection of the reaction mixture into an HPLC system and measurement of the absorbance at 260 nm. The assay was performed with 100 μM queuosine and 100 nM CD1682. A control incubated without enzyme is included. (B and C) The identities of the substrate and product, with corresponding + + retention times, were verified by extracting the ion counts for the expected masses [M H ] = m/z 278 for queuine and 410 for queuosine, respectively.

together suggest that, indeed, CD1684 is an enzyme capable of protein was part of a set of closely related radical SAM enzymes producing preQ1 from q. that form a separate group that is likely to be involved in Q salvage SNNs were built to place CD1684 within the RS superfamily based on gene neighborhood information (SI Appendix,Fig.S11). (composed of overlapping IPR006638 and IPR007197). As de- To directly test the hypothesis that CD1684 could act as a tailed in SI Appendix,Fig.S10, SSNs analyses showed that this lyase, breaking a C-N bond to generate preQ1 from q, CD1684,

Fig. 5. The ECF substrate specificity component CD1683 transports preQ1, queuine, and queuosine. (A) Scheme of Q metabolism in the E. coli derivatives used to test substrate specificity (queuine, preQ1, and preQ0) of the ECF transporter genes. Genes encoding C. difficile ECF core components (CD100, CD101, CD102)with those encoding different substrate-specific components (CD1683, CD2097,andCD3073) were expressed in E. coli ΔqueD ΔyhhQ pBAD24::tgtCt strain. Dashed Asp arrows represent reactions being tested. (B) Q-tRNA levels were detected by the ABP assay in tRNA GUC extracted 60 min after supplementing with different precursors. (C) Scheme of Q metabolism in the E. coli strains used to test queuosine transport and hydrolysis activity. Genes coding different transporters, including

YhhQCt and ECF complex with S component (CD1683, CD2097, or CD3073), were coexpressed with the predicted queuosine hydrolase (CD1682)inE. coli ΔqueD ΔyhhQ strains expressing both tgtEc and tgtCt. Red dashed arrows represent reactions being tested. Precursors in gray are not synthesized de novo in these strains. Asp (D) Detection of Q-tRNA by the APB assay in tRNA GUC extracted 60 min after supplementing with exogenous queuosine (Q; 10 or 500 nM).

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1909604116 Yuan et al. Downloaded by guest on September 27, 2021 Fig. 6. CD1684 generates preQ1 from queuine in vivo. (A) Scheme of Q metabolism in the E. coli derivatives used to test the activity of CD1684. Red dashed arrows represent reactions being tested. Precursors in gray are not synthesized de novo in these strains. (B) Detection of Q-tRNA by the APB assay in Asp tRNA GUC extracted 60 min after supplementing with different precursors. The C. difficile CD1684 gene was expressed in E. coli ΔqueD ΔqueF pBAD33::yhhQCt strain and ΔqueD ΔqueF ΔqueA pBAD33::yhhQCt strain.

with a C-terminal hexahistidine tag, was expressed in E. coli, bonding with protein backbone functionalities, as is the case for ++ purified in aerobic conditions by Ni -affinity chromatography most RS enzymes (54). The only sequence-specific interactions (SI Appendix, Fig. S7), followed by reconstitution of its Fe/S coming from QueLCs are Glu123 and Asn184, which directly cluster and then by size-exclusion chromatography in anaerobic hydrogen-bond with the dihydroxycyclopentene moiety (Fig. 7G). conditions. UV/Vis absorption spectra of CD1684 as purified and Interestingly, QueLCs contains a highly conserved active-site cys after reconstitution reveal characteristics of proteins possessing a residue (C154) within ∼4 Å of the SAM methyl group. When this Fe/S center (SI Appendix,Fig.S12). Estimation of the molar con- residue was modeled as cysteine, extra electron density was appar- tent of Fe and S before and after reconstitution reveals 2.17 ± 0.27 ent, which was best filled by a model that included a methyl-cys Fe and 3.19 ± 0.05 S for the as-purified protein and 1.92 ± 0.15 Fe (mCys) residue (SI Appendix,Fig.S13C), similar to mCys355 and 4.45 ± 0.09 S for the reconstituted protein. The reconstituted found in the RS methyltransferase RlmN (55). Currently, the im- enzyme was assayed for activity under anaerobic conditions. Ac- portance of this modification is unclear, even if it does form part of tivity was evaluated at the qualitative level in the presence of so- the SAM binding pocket. Studies are under way to determine how

dium dithionite (chemical electron donor) by following the this modification forms and its importance for the catalyzed re- MICROBIOLOGY evolution of substrates and products by LC-MS over time. As vi- action. Unlike SAM, q is coordinatedbyanextensivenetworkof sualized with absorption at 260 nm (Fig. 7A) and confirmed with hydrogen bonds provided by several conserved amino acids, in- MS (Fig. 7 B–E), q and SAM were consumed concomitantly with cluding Ser67, Glu96, Lys119, and Asp229 (SI Appendix,Fig.S14). production of preQ1 and 5′dA, validating in vitro the proposed Interestingly, the side chain of Asp229 does not actually participate hypothesis, and confirming that CD1684 is indeed a radical SAM in the hydrogen bonding; instead, it is the C-terminal carboxylate of enzyme, because of its utilization of SAM to produce 5′dA (47). Asp229 that H-bonds to the N9 of deazaguanine in q (Fig. 7G). This Because we have not identified the coproduct produced by interaction is stabilized by Arg187, which plays dual roles by also CD1684 during preQ1 production, we cannot unambiguously assign providing H-bonds to the adenosine moiety of SAM with its amide the type of reaction catalyzed. However, based on our genetic and and carbonyl backbone atoms. In addition to H-bonds, q is also biochemical characterization, we preliminarily named this enzyme further stabilized by a pi-stacking interaction with Tyr33. H-bonding queuine lyase or QueL. reinforces this interaction between the Tyr33 carbonyl atom and the exocyclic N in q, with additional binding interactions provided by an Crystal Structure of Queuine Lyase from Clostridium spiroforme DSM ordered water molecule that is positioned between the hydroxyl of 1552. To gain insight into the reaction catalyzed by QueL, we Tyr33 and the bridging amine in q. The dihydroxycyclopentene cloned several homologous members from the CD1684 SSN moiety of queuine has 2 primary interactions: Glu96 forms H-bonds cluster, heterologously expressed and purified them as previously with the 4′-and5′-position hydroxyl groups, and Lys119 H-bonds to described (51), and subjected them to sparse matrix screening the 4′-position hydroxyl (Fig. 7G). These interactions orient the crystallization trials by using the sitting-drop vapor diffusion dihydroxycyclopentene to place the 5′-position hydroxyl proton in method. The QueL from Clostridium spiroforme DSM 1552 direct alignment and distance (3.5 Å) from the 5′ carbon of SAM, (QueLCs; UniProt ID B1C2R2) formed hexagonal rod-like which indicates that this hydrogen atom is likely abstracted by the crystals in the presence of SAM and q, which exhibited strong 5′deoxyadenosyl-radical (5′dA•) to start catalysis, which is consistent diffraction at a synchrotron X-ray source. We determined the with the distances found in other RS enzymes crystalized in the structure by using single-wavelength anomalous dispersion by presence of SAM and their requisite substrate (54, 56). The re- collecting data at a wavelength of 1.378 Å, which takes advantage mainder of the binding interactions with q involve van der Waals of Fe absorption from the native [4Fe-4S] cluster. The resulting interactions with several Val, Leu, and Ile residues. 1.73-Å–resolution structure of QueLCs (deposited in the PDB, ID 6P78) contains all 229 amino acids from the native sequence, Discussion as well as a [4Fe-4S] cluster, SAM, and q (SI Appendix, Fig. S13 This work illustrates that bacteria adapt their Q salvage strate- A and B), in a single molecule in the asymmetric unit. A search gies to their environments. Bacteria that are located inside a of the PDB with the Dali server (52) found that QueLCs has human cell such as C. trachomatis have streamlined their metabo- structural similarly with pyruvate formate-lyase activating en- lism and rely on import for many nutrients (57–60). Only q or Q are zyme (PFL-AE; RMSD of 3.9 Å for 192 Cα atoms; PDB ID code supposedly available in the intracellular environment, and we show 3CB8), even though these enzymes share only 12% sequence here that Chlamydiae species have evolved to salvage the q base: a identity. Overall, QueLCs adopts the same (β/α)6 partial TIM YhhQ family member (CT140) imports q, and a bacterial TGT barrel as PFL-AE (Fig. 7F), but unlike the “splayed” core of homolog (CT193) catalyzes the base exchange between q and the PFL-AE, its core is compressed by ∼2 Å, resulting in a more target guanine in tRNAs. Those transport and base-exchange ac- complete active site (53). This difference is likely because, while tivities are not the historically identified and canonical ones but are the substrate for PFL-AE is a large folded protein (PFL) that related. Clearly, depending on the organism, the actual substrates contributes to the active site, QueLCs acts on the much smaller q for YhhQ and bacterial TGT homologs vary. This illustrates the molecule. SAM binds to QueLCs mostly through hydrogen difficulty in defining the boundaries between functions and thus

Yuan et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 27, 2021 Fig. 7. Queuine lyase (QueL) activity and structure. (A) The queuine lyase activity of purified recombinant CD1684 was analyzed by separation of quenched reaction mixture by an HPLC system with monitoring at 260 nm absorbance. Data from a representative assay are presented from an assay performed under anaerobic

conditions with 100 μM queuine, 200 μM sodium dithionite, 66.67 μM S-adenosyl-L-methionine (SAM limited to allow for visualization of preQ1; otherwise, a high concentration of SAM was used), and 10 μM of purified CD1684. The control is a reaction lacking enzyme. (B–E) The identities of the substrate and product, with

corresponding retention times, were verified by extracting the ion counts for the expected masses [M+H+] = m/z 278, 180, 399, and 252 for queuine, preQ1,SAM,and 5′dA (5′deoxyadenosine), respectively. The UV signal and mass corresponding to queuine and SAM are reduced over time, while signal and mass corresponding to

preQ1 and 5′dA are increased, demonstrating that CD1684 is an RS enzyme with queuine lyase activity. (F) Overall view of QueLCs with secondary structural elements assigned numerically. The α6 extension (blue) caps the active site terminating in Asp229. SAM (light gray) and queuine (dark gray) are depicted as ball-and-stick il- lustrations (oxygen, red; nitrogen, blue). The RS cluster is depicted as spheres (iron, burnt orange; sulfur, yellow). (G) A top-down view of the active site showing highly

conserved amino acids (depicted as ball and sticks in pink) and hydrogen bonds (dashed lines) formed between substrates (coloring as in A) and the QueLCs active site. The red dashed line denotes the distance (3.5 Å) between the 5′-carbon of SAM and the 5′-oxygen of queuine. (H) Mechanistic proposal for catalysis by QueL.

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1909604116 Yuan et al. Downloaded by guest on September 27, 2021 annotations within protein families. Defining accurate annotation ring closure would produce 7, which would be followed by a bulk therefore requires context information and/or signature motifs (61). solvent-catalyzed tautomerization to yield cyclopentenone 8, For example, in order to differentiate the subfamilies of bacterial which is reminiscent of the Nazarov cyclization (67) (Fig. 7H). TGT that incorporate q from those that incorporate preQ1, one can combine the absence of QueA with the difference in residues in the Methods binding pocket for the substrate’s 7-substituent moiety (Fig. 2A and Bioinformatics. For sequence analyses, the BLAST tools (68) and resources at SI Appendix, Supplementary Text). Similarly, analysis of the speci- NCBI (69) (https://www.ncbi.nlm.nih.gov/), UniProt (70) (https://www.uniprot.org), ficity of YhhQ subfamilies requires combination of the presence of and PATRICBRC (71) (https://patricbrc.org/) were routinely used. Further details on Q biosynthetic genes from the organism in the same cluster with the all bioinformatic analyses are provided in SI Appendix. specificity of their TGT enzymes (SI Appendix,Fig.S15). Gut microbiota represent a complex ecosystem that develops Strains, Media, and Growth Conditions. Strains and plasmids used in this study are listed in SI Appendix, Table S1. Oligonucleotides used for mutant con- in close interaction with the host. In terms of Q metabolism, this struction and plasmid construction are listed in SI Appendix, Table S2.Fur- environment is particularly complex, as specific microbes can be ther details are provided in SI Appendix. sources but also sinks of q, Q, preQ0, or preQ1 (62) with the

additional competition for the q precursor by the human host. Chemicals. PreQ0 was purchased from ArkPharm (AK-32535). PreQ1 was Indeed, a recent analysis of Q metabolism genes in 2,216 repre- purchased from Sigma-Aldrich (SML0807-5MG). Queuine was purchased sentative bacteria of the gut microbiome found that ∼50% of from Toronto Research Chemicals (Q525000). Queuosine was synthesized as these bacteria must salvage a Q precursor (63). The final num- previously described (72) and detailed in SI Appendix. bers are given in SI Appendix, Fig. S16 and Dataset S1.To summarize, 51% of the bacteria analyzed are predicted to syn- Exogenous q Precursors Feeding. As previously described (73), cells were cul- thesize Q de novo. While a few strains such as Streptococcus turedinM9definedmediawithglycerol as carbon source and arabinose for pneumoniae are predicted to import preQ (2%), a large pro- induction. After supplementing with queuosine precursors, the transport reaction 0 was stopped, followed by tRNA extraction. Details are provided in SI Appendix. portion (29%) must salvage preQ1 (based on the absence of queF and the presence of tgt and queA), while 13% salvage q. Finally, tRNA Extraction. As previously described (28), small RNAs of E. coli cells were only 5% of the genomes analyzed do not encode TGT homologs extracted by using a PureLink miRNA Isolation kit (Life Technologies) accord- and are hence predicted not to modify their tRNA with queuosine. ing to manufacturer protocol. For C. difficile, small RNAs were extracted by The characterization of the Q salvage pathway in C. difficile using a FastRNA Pro Blue Kit (MP Biomedicals) and FastPrep instrument

now allows the study of the physiological role of this tRNA according to manufacturer protocol. Details are provided in SI Appendix. MICROBIOLOGY modification in the important enteropathogen. The absence of Q does not seem to affect growth rate, but we found that WT Detection of Queuosine in Bulk tRNA. Detection of the presence of Q in tRNA is tRNAs are only partially modified in BHI, presumably because based on a method originally developed by Igloi and Kössel (42) and later the Q source is limiting (Fig. 3B). Further studies are required in improved (73), as detailed in SI Appendix. more relevant physiological conditions, as Q might be important for fitness under stress conditions and during colonization. In- Enzyme Expression, Purification, and Assays. C-terminal hexahistidine-tagged CD1682 (QueK, Q186N9) and CD1684 (QueL, Q186P0) were overexpressed in E. deed, the expression of genes from the CD1682 operon was coli Rosetta DE3 pLysS cells and E. coli BL21 DE3 cells, respectively, followed by reported to be up-regulated in biofilm compared with planktonic purification and enzyme assay, as detailed in SI Appendix. The CD1684 homolog

cells (64), when iron is low (65), during exponential growth in C. spiroforme DSM 1552, QueLCs (UniProt ID code B1C2R2), with a N-ter compared with stationary phase, and when the sigma factor of hexahistidine tag, was overexpressed in E. coli BL21 DE3 cells, followed by the stationary phase, SigH, is deleted (66). purification and structure determination. Details are provided in SI Appendix. Finally, this study is another example of the power of comparative genomics approaches to discover novel enzymatic re- X-Ray Diffraction and Structure Determination. QueLCs was crystallized fol- actions. The q lyase activity had never been described in the lowed by diffraction and structure determination as detailed in SI Appendix. literature to our knowledge, and the discovery that a subgroup of the radical-SAM enzyme superfamily catalyzes this reaction is ACKNOWLEDGMENTS. This work was funded by the National Institutes of Health (R01 GM70641 to V.d.C.-L.; P01 GM118303 to J.A.G. and S.C.A.; U54- unexpected. The crystal structure of QueLCs bound to its sub- GM093342 to J.A.G. and S.C.A.; R21-AI133329 to T.L.G. and S.C.A.; U54- strates allows us to propose how preQ1 is generated from q. As GM094662 to S.C.A.; GM110588 to M.A.S.), the Price Family Foundation proposed in Fig. 7H, the reaction starts by abstraction of the 5- (S.C.A.), and the California Metabolic Research Foundation (M.A.S.). We position hydroxyl H-atom by 5′dA•, producing intermediate 1. acknowledge the Albert Einstein Anaerobic Structural and Functional Genomics Resource (http://www.nysgxrc.org/psi3/anaerobic.html). We thank This is the most likely outcome because all other hydrogen atoms Martin I. H. McLauglin and Wilfred A. van der Donk for providing the guid- in q are greater than 5 Å away and would require large move- ance and the plasmid isc-pBADCDF for in vivo maturation of metalloen- ments in the active site to place them in resonance with the zymes. The LC-MS analyses were performed by Furong Sun at the Mass 5′dA•. The presence of the 5-position hydroxyl radical would allow Spectrometry Laboratory, a Service Facility from the School of Chemical ′ ′ Sciences at University of Illinois at Urbana–Champaign. The Einstein fission of the 4 -5 C-C bond of the dihydroxycyclopentene ring, Crystallographic Core X-Ray diffraction facility is supported by NIH Shared generating a vinylic-stabilized radical intermediate 2 (Fig. 7H). Instrumentation Grant S10 OD020068. This research used resources of the The redox potential of this radical would be sufficiently oxidizing Advanced Photon Source, a US Department of Energy (DOE) Office of to accept an electron from a reduced RS cluster to yield anion 3 Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of the (Fig. 7H). The anion could then isomerize, in the process elim- Lilly Research Laboratories Collaborative Access Team (LRL-CAT) beamline inating preQ1 to produce the divinyl ketone 5 (Fig. 7H). With the at Sector 31 of the Advanced Photon Source was provided by Eli Lilly assistance of a proton from Glu96, a cationic 4π-electrocyclic- Company, which operates the facility.

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