Abstract
Cofactor F420 plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent. It mediates a variety of important redox transformations involved in bacterial persistence, antibiotic biosynthesis, pro-drug activation and methanogenesis. However, the biosynthetic pathway for F420 has not been fully eluci-dated: neither the enzyme that generates the putative intermediate 2-phospho-L-lactate, nor the function of the FMN-binding C-terminal domain of the γ-glutamyl ligase (FbiB) in bacteria are known. Here we show that the guanylyltransferases FbiD and CofC accept phosphoenolpyruvate, rather than 2-phospho-L-lactate, as their substrate, leading to the formation of the previously uncharacterized intermediate, dehydro-F420-0. The C-terminal domain of FbiB then utilizes FMNH2 to reduce dehydro-F420-0, which produces mature F420 species when combined with the γ-glutamyl ligase activity of the N-terminal domain. This new insight has allowed the heterologous expression F420 from a recombinant F420 biosynthetic pathway in Escherichia coli.
Cofactor F420 is a deazaflavin that acts as a hydride carrier in diverse redox reactions in both bacteria and archaea1,2. While F420 structurally resembles the flavins FMN and FAD, it func-tions as an obligate two-electron hydride carrier and hence is functionally analogous to the nicotinamides NAD+ and NADP+3. The lower reduction potential of the F420, relative to the flavins, results from the substitution of N5 of the isoalloxazine ring in the flavins for a carbon in F4204,5. Originally characterized from methanogenic archaea in 19724,5, F420 is an important catabolic cofactor in methanogens and mediates key one-carbon transformations of methano-genesis6. F420 has since been shown to be synthesized in a range of archaea and bacteria1,2,7,8. In Mycobacterium tuberculosis, the causative agent of tuberculosis, F420 has been shown to contribute to persistence9,10 and to activate the new clinical antitubercular prodrugs delamanid and pretomanid11. There are also growing numbers of natural products that have been shown to be synthesized through F420-dependent pathways, including tetracyclines12, lincosamides13, and thiopeptides14. F420-dependent enzymes have also been explored for bioremediation and biocatalytic applications15,16.
The currently accepted F420 biosynthetic pathway consists of two branches2. In the first branch, tyrosine is condensed with 5-amino-6-ribitylamino-2,4[1H,3H]-pyrimidinedione from the flavin biosynthetic pathway to generate the deazaflavin chromophore Fo (7,8-dide-methyl-8-hydroxy-5-deazariboflavin) via the activity of the two-domain Fo synthase FbiC, or the CofG/H pair (where ‘Fbi’ refers to mycobacterial proteins and ‘Cof’ refers to archaeal homologs). In the second branch, it has been hypothesized that a 2-phospho-L-lactate guan-ylyltransferase (CofC in archaea and the putative enzyme FbiD in bacteria) catalyzes the guanylylation of 2-phospho-L-lactate (2-PL) using guanosine-5’-triphosphate (GTP), yielding L-lactyl-2-diphospho-5’-guanosine (LPPG)17. The two branches then merge at the reaction catalyzed by the transferase FbiA/CofD, where the 2-phospho-L-lactyl moiety of LPPG is transferred to Fo, forming F420-018,19. Finally, the γ-glutamyl ligase (FbiB/CofE) catalyzes the poly-glutamylation of F420 to generate mature F420, with poly-γ-glutamate tail lengths of ∼2-8, depending on species20,21.
There are three aspects of the F420 biosynthetic pathway that require clarification. First, the metabolic origin of 2-PL, the proposed substrate for CofC, is unclear. It has been assumed that a hypothetical kinase (designated CofB) phosphorylates L-lactate to produce 2-PL22. However, no such enzyme for the production of 2-PL has been identified in bacteria or archaea, and our genomic analysis of F420 biosynthesis operons has failed to identify any can-didate enzymes with putative L-lactate kinase activity2. Second, the existence of FbiD has only been inferred through bioinformatics and genetic knockout studies and the enzyme has not been formally characterized23,24. Finally, the bacterial γ-glutamyl ligase FbiB is a two-do-main protein20, in which the N-terminal domain is homologous to other F420-γ-glutamyl ligases (including the archaeal equivalent, CofE) and the C-terminal domain adopts an FMN-binding nitroreductase (NTR) fold20. Although both domains are required for full γ-glutamyl ligase activity, no function has been associated with either the C-terminal domain or the FMN cofactor, given no redox reactions are known to be involved in F420 biosynthesis.
Here we demonstrate that 2-PL is not required for F420 biosynthesis in bacteria and instead phosphoenolpyruvate (PEP), an abundant intermediate of glycolysis and gluconeogenesis, is incorporated into F420. We show that PEP guanylylation is catalyzed by the CofC and FbiD enzymes that were previously thought to act upon 2-PL. In bacteria, the incorporation of PEP in the pathway results in the production of the previously undetected intermediate dehydro-F420-0, which is then reduced by the C-terminal domain of FbiB alongside poly-glu-tamylation. These findings result in a substantially revised pathway for F420 biosynthesis and have allowed us to heterologously produce functional F420 in engineered Escherichia coli, an organism that does not normally produce F420, at levels comparable to some native F420-producing organisms.
Results
FbiD/CofC use phosphoenolpyruvate, rather than 2-phospho-L-lactate, as a substrate
The archaeal enzyme CofC has previously been suggested to catalyze the guanylylation of 2-PL to produce LPPG during F420 biosynthesis (Fig. 1a)25. Another study, using transposon mutagenesis, has shown that MSMEG_2392 of Mycobacterium smegmatis is essential in the biosynthesis of F420 from Fo23. We have recently shown that homologs of this gene have sequence homology to CofC and belong to operons with other validated F420 biosynthetic genes in a wide variety of bacteria2. In keeping with the bacterial naming system, we refer to this enzyme as FbiD. To test the function of this putative bacterial FbiD, we cloned the Rv2983 gene from M. tuberculosis26 into a mycobacterial expression vector and purified heterologously expressed Mtb-FbiD from M. smegmatis mc24517 host cells. We also expressed and purified Mtb-FbiA (the enzyme thought to transfer the 2-phospho-L-lactyl moiety of LPPG to Fo to produce F420-0)18 to use in coupled HPLC-MS enzymatic assays with Mtb-FbiD. Surprisingly, we found that when Mtb-FbiD and Mtb-FbiA were included in an assay with 2-PL, GTP (or ATP) and Fo, no product was formed (Fig. 1b). We then tested whether Mtb-FbiA and CofC from Methanocaldococcus jannaschii (Mj-CofC) could catalyze F420-0 formation under the same conditions, which again yielded no product (Fig. 1b).
Although 2-PL is hypothesized to be an intermediate in F420 biosynthesis, this has never been experimentally confirmed in bacteria. Additionally, no enzyme capable of phosphorylating L-lactate to 2-PL has been identified in F420 producing organisms, despite considerable investigation22. 2-PL has been little studied as a metabolite and is only known to occur as a by-product of pyruvate kinase activity27. 2-PL has not been implicated as a substrate in any metabolic pathway outside the proposed role in F420 biosynthesis; rather it has been shown in vitro to inhibit several enzymes involved in glycolysis and amino acid biosynthesis28-30. Our inability to detect activity with 2-PL led us to consider alternative metabolites that could potentially substitute for 2-PL, namely the structurally analogous and comparatively abundant molecule phosphoenolpyruvate (PEP)31 (Fig. 1a).
While there was no activity when 2-PL was used in the FbiD/CofC:FbiA coupled assays, when these enzymes were incubated with PEP, GTP (or ATP) and Fo, a previously un-reported intermediate in the F420 biosynthesis pathway, which we term ‘dehydro-F420-0’, was produced (Fig. 1b). The identity of this compound, which is identical to F420-0 except for a methylene group in place of the terminal methyl group, was verified by tandem mass spec-trometry (Fig. 1c). The only difference that we observed between Mtb-FbiD or Mj-CofC was that while Mtb-FbiD exclusively utilizes GTP to produce dehydro-F420-0, Mj-CofC can also catalyze the reaction with ATP, albeit to a lesser extent (Fig. 1b). Interestingly, in our experi-ments the FbiD/CofC enzymes were only active in the presence of FbiA. This was not unexpected given that the inferred intermediate (enolpyruvyl-diphospho-5’-guanosine; EGGP) is expected to be unstable22 (Fig. 1a).
To understand the molecular basis of PEP recognition by Mtb-FbiD, we crystallized the protein and solved the structure by selenium single-wavelength anomalous diffraction (Se-SAD), and then used this selenomethionine-substituted structure to obtain the native FbiD structure by molecular replacement. The latter was then refined at 1.99 Å resolution (R/Rfree=0.19/0.22) (SI Table 2). As expected, Mtb-FbiD adopts the same MobA-like nucleoside triphosphate (NTP) transferase family protein fold as CofC: a central 7-stranded β-sheet (six parallel strands and one antiparallel), with α-helices packed on either side (Fig. 2a). Superposition of CofC from Methanosarcina mazei (PDB code 2I5E) on to Mtb-FbiD gives a root mean square difference (rmsd) of 1.85 Å over 181 Cα atoms, with 25.4% sequence identity, establishing clear structural homology.
We also soaked PEP into pre-formed FbiD crystals to obtain an FbiD-PEP complex (2.18 Å resolution, R/Rfree=0.22/0.26). FbiD has a cone-shaped binding cleft with a groove running across the base of the cone, formed by the C-terminal end of the central β-sheet (Fig. 2a). PEP binds in the cleft with its phosphate group anchored through two Mg2+ ions to three acidic side chains (D116, D188 and D190) (Fig. 2b). The PEP carboxylate group is hydrogen bonded to the hydroxyl group of S166 and the main chain NH groups of T148 and G163. All PEP binding residues are conserved in the CofC protein of M. mazei (PDB code 2I5E) (Fig. 2c and Fig. S2), consistent with the enzymatic assays that showed PEP is the substrate for ar-chaeal CofC, as well as FbiD. In the GTP-bound structure of E. coli MobA32 (PDB code 1FRW), GTP binds in a characteristic surface groove, providing a structural framework for substrate binding and catalysis. In our enzyme assays, neither FbiD nor CofC showed activity in the absence of FbiA. Moreover, we did not observe GTP binding in either our co-crystalli-zation or differential scanning fluorimetry experiments. We speculate that the GTP binding site is not formed until FbiD/CofC interacts with FbiA/CofD, enabling catalysis to proceed through to formation of dehydro-F420-0. This may provide an advantage by producing EPPG/EPPA only when both proteins are available, thereby overcoming the issue of interme-diate instability.
The C-terminal domain of FbiB catalyzes reduction of dehydro-F420-0 to F420-0
Dehydro-F420-0 would yield F420-0 upon reduction of the terminal methylene double bond. However, no masses corresponding to F420-0 were identified in any of the LC-MS traces from the FbiD:FbiA coupled assays, suggesting that an enzyme other than FbiD or FbiA catalyzes dehydro-F420-0 reduction. We have previously shown that full-length FbiB consists of two domains: an N-terminal domain that is homologous to the archaeal γ-glutamyl ligase CofE21,33, and a C-terminal domain of the NTR fold34 that binds to FMN and has no known function20, but is essential for extending the poly-γ-glutamate tail.
We tested whether FbiB could use dehydro-F420-0 as a substrate with a three enzyme assay in which FbiB and L-glutamate were added to the FbiD:FbiA coupled assay. Mtb-FbiB was observed to catalyze the addition of L-glutamate residues to dehydro-F420-0, forming de-hydro-F420 species with one ([M+H]+, m/z of 643.40) and two ([M+H]+, m/z of 772.40) gluta-mate residues (Fig. S1). We then tested the hypothesis that the orphan function of the FMN-binding C-terminal domain could in fact be a dehydro-F420-0 reductase. We used a four-en-zyme in vitro assay where E. coli NAD(P)H:flavin oxidoreductase35 (Fre), FMN, NADH and 10 mM dithiothreitol (to maintain reducing conditions and generate reduced FMNH2) were added to the FbiD:FbiA:FbiB assay and the reaction was performed in anaerobic conditions (to prevent reaction of FMNH2 with oxygen). We found that F420-1, i.e. the fully reduced and mature glutamylated cofactor, was produced; but only in the presence of both FbiB and Fre/FMNH2 (Fig. 3a). Thus, dehydro-F420-0 is a bona fide metabolic intermediate that can be converted to mature F420 by FbiB in an FMNH2-dependent fashion. These results demonstrate that bacterial FbiB is a bifunctional enzyme, functioning as a dehydro-F420-0 reductase and as a γ-glutamyl ligase (Fig. 3c).
When the previously published crystal structures of Mtb-FbiB are analyzed in the context of these results, the molecular basis for this activity becomes clear. Crystal structures with both F420 and FMN bound have been separately solved and when these are overlaid, it is apparent that the FMN molecule is ideally situated to transfer a hydride to the terminal methylene of dehydro-F420-0 (assuming deydro-F420-0 binds in a similar fashion to F420). Interestingly, the phospholactyl group of F420 appears to be disordered in these crystal structures, suggesting it may adopt multiple conformations. To test this, we docked and minimized dehydro-F420-0 and FMNH2 simultaneously using the OPSL3a forcefield. The results show that in this ternary complex the two molecules can adopt ideal positions and orientations for the reduction of dehydro-F420-0 (Fig. 3b). The methylene group of dehydro-F420-0 is accommo-dated by a small hydrophobic pocket mostly comprised of P289 and M372 allowing it to be positioned above the N5 atom of FMNH2, in a plausible Michaelis complex for hydride transfer. We therefore suggest that the phosphoenolpyruvyl (analogous to the phospholactyl) group of dehydro-F420-0 most likely samples conformations within this pocket where it can be reduced.
Interestingly, the γ-glutamyl ligase CofE from archaea is a single domain enzyme; there is no homology to the C-terminal NTR-fold domain of FbiB. In an analogous situation, Fo synthesis is performed by two single domain enzymes in archaea, CofH and CofG2, whereas in bacteria this reaction is catalyzed by a two-domain protein, FbiC (with N-and C-terminal domains homologous to CofH and CofG, respectively). Previous analysis of archaeal genomes revealed that cofH and cofG are closely associated in genomic context36. We there-fore investigated the genomic context of archaeal cofE genes to investigate whether genes with homology to the C-terminal NTR domain of FbiB were located nearby. From over 1000 archaeal genomes, we only detected 16 open reading frames (ORFs) in the neighboring con-text of cofE that could encode proteins with an NTR fold, although none of these shared sub-stantial (>34% sequence identity) homology and all lacked the key F420 binding residues ob-served in FbiB. There was one interesting exception: the unusual archaea Lokiarchaeum sp.37 are unique among all sequenced archaea in that they alone encode an FbiB-like γ-glutamyl ligase:NTR fusion protein.
A recombinant F420 biosynthesis operon can be heterologously expressed in Escherichia coli to produce mature F420
Cofactor F420 is only produced by certain bacterial species; the majority of bacteria, including Escherichia coli, lack the genes required for F420 biosynthesis. Our in vitro assay results suggest that 2-PL, and by extension, the hypothesized L-lactate kinase CofB, are not required for heterologous production of F420 in a non-native organism. To test our hypothesis, we generated a plasmid expressing the Mycobacterium smegmatis F420 biosynthesis genes fbiB/C/D along with the fbiA homolog cofD from Methanosarcina mazei19, which was substituted as Ms-FbiA was found to express poorly in E. coli.
We generated two versions of a plasmid-encoded recombinant F420 biosynthetic operon, both of which encode codon-optimized genes for expression in E. coli: one encodes the native enzymes (pF420), and the second encodes C-terminal FLAG-tagged versions of the enzymes to allow their detection in a western blot (pF420-FLAG) (Fig. 4a). Plasmids were designed to allow induction of F420 biosynthesis in the presence of anhydrotetracycline. A western blot using anti-FLAG antibodies was used to detect whether the proteins were expressed in soluble form in E. coli, and confirmed that all were expressed to varying degrees (Fig. S3). We then tested, using HPLC with fluorescence detection, whether F420 was heterologously produced in the cell lysate of our recombinant E. coli strain expressing this operon. As shown in Fig. 4b, E. coli expressing both the FLAG-tagged, and untagged, plasmids produced traces consistent with mature, poly-glutamylated F420, although they differed slightly to the M. smegmatis produced F420 standard in terms of the distribution of poly-γ-glutamate tail lengths.
To confirm that this was indeed mature F420, and not dehydro-F420 species, we purified the compound from E. coli lysate and performed high-resolution MS/MS analysis. Mass frag-mentation did indeed show that the compound was reduced, and not dehydro-F420 (Fig. 4d). No dehydro-F420 species were detected. The yield of purified F420 was approximately 27 nmol/L of culture, which is comparable to physiological levels of several F420-producing species38. UV-Vis and fluorescence spectra of the purified F420 matched literature values (Fig. S4)4,5. Finally, we confirmed that the purified cofactor was functional by measuring enzyme kinetic parameters with F420-dependent glucose-6-phosphate dehydrogenase (FGD) from M. smegmatis (Fig. 4c). The apparent Michaelis constant was within error of that observed with FGD and F420 produced from M. smegmatis, while the kcat was approximately half that of the M. smegmatis F420 (Fig. 4c), which could result from slight differences in the distribution of tail lengths, as previously reported (Table S3)39. These results confirm that the recombinant production of F420 was achieved with a biosynthetic pathway containing only CofD (equivalent to FbiA)/FbiB/FbiC/FbiD.
Discussion
It has become widely accepted within the field that one of the essential initial steps in F420 bi-osynthesis involves a hypothetical L-lactate kinase that produces 2-PL, which is subsequently incorporated into F420 through the activities of CofC and CofD. However, neither bioinformatics nor genetic knockout studies have identified plausible candidate genes for a L-lactate kinase2,18,23. Furthermore, 2-PL has been shown to inhibit several enzymes involved in central metabolism28-30. In terms of pathway flux, this makes 2-PL an unusual starting point for biosynthesis of an abundant metabolite such as F420, which can exceed 1 μM in some species38. The results presented in this paper unequivocally demonstrate that PEP, rather than 2-PL, is the authentic starting metabolite in bacteria. These results reconcile the previously problematic assumptions that are required to include 2-PL within the biosynthetic pathway and establish a revised pathway (Fig. 5) that is directly linked to central carbon metabolism (via PEP) through the glycolysis and gluconeogenesis pathways.
Our observation that CofC accepts PEP (and not 2-PL) in vitro, appear to contradict previous studies in which Mj-CofC was reported to use 2-PL as substrate19,33. However, this discrepancy is most likely due to the supplementation of the coupled CofC/CofD reaction in these studies with pyruvate kinase and 2 mM PEP, a strategy that was used to overcome apparent product inhibition by GMP19,33. Regardless, we cannot explain how a pathway starting from PEP can produce mature (i.e. not dehydro-F420) F420 in archaea given that their equivalent of FbiB (CofE) lacks the C-terminal domain dehydro-F420-0 reductase domain seen in FbiB. One possibility is that an unknown dehydro-F420-0 reductase exists elsewhere in the genome (remote from CofE). Further studies are required to resolve this step in archaeal F420 biosynthesis.
The discovery of dehydro-F420-0 as the product of FbiD:FbiA activity in mycobacteria indicated that another enzyme must be required to reduce dehydro-F420-0 and produce F420-0. This allowed us to define the function of the orphan C-terminal domain of mycobacterial FbiB20. The N-terminal domain is homologous with the archaeal γ-glutamyl ligase CofE33, but is only capable of catalyzing glutamylation of F420-120. The C-terminal domain, which binds both F420 and FMN, is essential for extending the poly-γ-glutamate tail of F420-0 20, but we could not explain the possible function of the FMN cofactor. Here, we show that FbiB can catalyze both polyglutamylation and reduction of dehydro-F420-0, via the activities of the N-and C-terminal domains, respectively.
The increasing recognition of the importance of F420 in a variety of biotechnological, medical and ecological contexts underlines the need for making the compound more widely accessible to researchers; however our inability to produce F420 recombinantly in common laboratory organisms has been a major barrier to wider study. Here, we confirm the results of our in vitro experiments by showing that recombinant expression of the four characterized F420 biosynthesis genes allows production of F420 in E. coli. This should now facilitate the use of F420 in a variety of processes with recombinant organisms, such as biocatalysis using a bio-orthogonal cofactor, directed evolution of F420-dependent enzymes, recombinant production of antibiotics for which F420 is a required cofactor, and metabolic engineering.
Methods
Bacterial strains and growth conditions
Protein expression utilized either M. smegmatis mc2451740, E. coli BL21(DE3) or LOBSTR-BL21(DE3)41 cells. For growth of M. smegmatis, media were supplemented to 0.05% (v/v) Tween80. M. smegmatis cells were grown in ZYM-505242 or modified auto-induction media TB 2.0 (2.0% tryptone, 0.5% yeast extract, 0.5% NaCl, 22 mM KH2PO4, 42 mM Na2HPO4, 0.6% glycerol, 0.05% glucose, 0.2% lactose)43. For selenomethionine labelling, cells were grown in PASM-5052 media42. E. coli expressions were conducted in either the above modified auto-induction media, or Terrific Broth (TB) medium modified for auto-induction of protein expression (1.2% tryptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4, 2 mM MgSO4, 0.8% glycerol, 0.015% glucose, 0.5% lactose, 0.375% aspartic acid), grown for 4 h at 37° C followed by overnight incubation at 18° C.
Protein expression and purification
Mtb-FbiD
The open reading frame encoding FbiD (Rv2983) was obtained by PCR from M. tuberculosis genomic DNA (Table S1). The pYUBDuet-fbiABD co-expression construct was then prepared by cloning fbiD into pYUBDuet44 using BamHI and HindIII restriction sites, followed by cloning the fbiAB operon using NdeI and PacI restriction sites. This construct expresses FbiD with an N-terminal His6-tag, whereas the FbiA and FbiB proteins are expressed without any tags.
The pYUBDuet-fbiABD vector was transformed into M. smegmatis mc24517 strain40 for expression. The cells were grown in a fermenter (BioFlo®415, New Brunswick Scientific) for 4 days before harvesting. The cells were lysed in 20 mM HEPES, pH 7.5, 150 mM NaCl, 20 mM imidazole, 1 mM β-mercaptoethanol using a cell disruptor (Microfluidizer M-110P) in the presence of Complete protease inhibitor mixture mini EDTA-free tablets (Roche Applied Science). The lysate was centrifuged at 20,000 ×g to separate the insoluble material. Re-combinant FbiD was separated from other proteins by immobilized metal affinity chromatog-raphy (IMAC) on a HisTrap FF 5-ml Ni-NTA column (GE Healthcare), eluted with imidaz-ole, and further purified by Size Exclusion Chromatography (SEC) on a Superdex 75 10/300 column (GE Healthcare) pre-equilibrated in 20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM β-mercaptoethanol.
Mtb-FbiA
The open reading frame encoding M. tuberculosis FbiA was commercially synthesized and cloned into pRSET-A (Invitrogen). The pYUB28b-fbiA construct used for expression in M. smegmatis mc24517 was prepared by subcloning fbiA into pYUB28b44 using an NdeI site introduced by overhang PCR utilizing the HindIII restriction site present on both vectors amplified with the T7 reverse primer (Table S1). The resulting pYUB28b-fbiA construct expresses FbiA with an N-terminal His6-tag. The protein was expressed in M. smegmatis mc24517 in ZYM-5052 media auto-induction media42,45 in a fermenter (BioFlo®415, New Brunswick Scientific) for 4 days. The protein was then purified using Ni-NTA and size-exclusion chromatography steps, as described above, in 20 mM HEPES, pH 7.5, 200 mM NaCl, 5% glycerol, 1 mM β-mercaptoethanol.
Mj-CofC
The open reading frame encoding Methanocaldococcus jannaschii CofC (MJ0887)25 was synthesized (GenScript) and cloned into pYUB28b vector44 using NdeI and HindIII restriction sites. The protein was expressed in TB auto-induction media and purified using Ni-NTA and size-exclusion chromatography steps, as described above, in 20 mM HEPES, pH 7.5, 200 mM NaCl, 5% glycerol, 1 mM β-mercaptoethanol.
Ec-Fre
The E. coli flavin reductase35 was cloned into pProEX-HTb using KasI and HindIII restriction sites (Table S1). Protein expression and purification was performed similar to Mj-CofC.
Ms-FbiD
The open reading frame encoding M. smegmatis FbiD (MSMEG_2392) was synthesized (Integrated DNA Technologies) and cloned into pETMCSIII by Gibson Assembly as outlined previously43. The protein was expressed overnight ay 30 °C in auto-induction media and purified similarly to Mj-CofC.
Construction of synthetic F420 biosynthesis operon
Ribosome binding sites were individually optimized for each of the codon-optimized F420 biosynthesis genes using the Ribosome Binding Site Calculator server36,46. Multiple operon designs were analyzed using the server’s operon calculator and modified to remove unwanted translation products and RNA instability elements while maintaining predicted translation initiation rates for all coding sequences within an order of magnitude. The final design placed the operon under the control of the tetracycline-inducible promoter BBa_R0040 and the artificial terminator BBa_B1006. For subsequent assembly the operon was flanked by BioBrick prefix and suffix sequences. The operon was synthesized by GenScript and cloned into pSB1C3 containing the constitutive tetracycline repressor cassette BBa_K145201 using the standard BioBrick assembly protocol with EcoRI and XbaI/SpeI restriction enzymes47.This construct, hereafter referred to as pF420, allowed for production of F420 to be induced by the addition of anhydrotetracycline. For western blot analysis as second version of the operon with single C-terminal FLAG tags on all four genes was likewise synthesized.
To test for in vivo F420-depended reductase/oxidase activity the open reading frame of M. smegmatis FGD (MSMEG_0777) was codon optimized and commercially synthesized by GenScript and cloned into multiple cloning site 1 of pCOLADuet-1 (Novagen) using NcoI and HindIII sites. Subsequently MSMEG_2027 was subcloned from pETMCSIII-MSMEG_202743 into multiple cloning site 2 using NdeI and KpnI to give pFGD2027.
Mtb-FbiD crystallization and structure determination
Crystallization. Apo-FbiD (20 mg/mL in 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM β-mercaptoethanol) was crystallized using the sitting drop vapor diffusion method in 30% PEG 1500, 3% MPD, 0.2 M MgSO4, 0.1 M sodium acetate pH 5.0. For experimental phasing, selenomethionine-substituted FbiD crystals were grown using protein produced in M. smegmatis host cells45. Se-SAD anomalous diffraction data sets were collected at the Australian Synchrotron. Data collection statistics are summarized in Table S2. Crystals of Mtb-FbiD in complex with PEP were obtained by soaking pre-formed apo crystals in precipitant solutions containing 10 mM PEP for 30 min.
Structure determination. All data sets were indexed and processed using XDS48, and scaled with AIMLESS49 from the CCP4 program suite50. The structure was solved using the SAD protocol of Auto-Rickshaw51, the EMBL-Hamburg automated crystal structure determi-nation platform. Based on an initial analysis of the data, the maximum resolution for sub-structure determination and initial phase calculation was set to 2.83 Å. All three of the expected heavy atoms were found using the program SHELXD52. The initial phases were im-proved using density modification and phase extension to 2.33 Å resolution using the pro-gram RESOLVE53. Cycles of automatic model building by ARP/wARP54 and phenix.au-tobuild55 resulted in a protein model that was completed manually using COOT56. Water molecules were identified by their spherical electron density and appropriate hydrogen bond geometry with the surrounding structure. Following each round of manual model building, the model was refined using REFMAC557, against the data to 1.99 Å resolution. The PDB_redo program58 was used in the final stages of refinement. Full refinement statistics are shown in Table S2.
The structure of Mtb-FbiD in complex with PEP was solved by molecular replacement using PHASER59 with the apo-FbiD structure as a search model. The structure was refined by cycles of manual building using COOT56 and refinement using REFMAC557, against the data to 2.18 Å resolution. Full refinement statistics are shown in Table S2.
HPLC assays
FbiD/CofC-FbiA coupled activity was monitored in a reaction mixture containing 100 mM HEPES pH 7.5, 2 mM GTP, 0.1 mM Fo, 5 mM MgCl2, 1 mM 2-PL or PEP, 1 μM FbiD and 5μM FbiA. The reactions were incubated at 37 °C and stopped using 20 mM EDTA at various time points. Separation of F420 species was performed on an Agilent HP 1100 HPLC system equipped with photodiode array and fluorescence detectors (Agilent Technologies). Samples were kept at 4°C, and the injection volume was 20 μL. Samples were separated on a Phenomenex Luna C18 column (150 × 3 mm, 5 μm) with a 0.2 μm in-line filter that was maintained at 30 °C. The mobile phase consisted of 100% methanol (A) and 25 mM sodium acetate buffer, pH 6.0 (B), with a gradient elution at a flow rate of 0.5 ml/min and a run time of 30 min. The gradient profile was performed as follows: 0–25 min 95–80% B, 25–26 min 80% B, 26–27 min 95% B, 27–30 min 95% B, and a post-run of 2 min. The wavelengths used for photodiode array were 280 and 420 nm (bandwidth 20 nm) using a reference of 550 nm (bandwidth 50 nm). The wavelengths used for the fluorescence detector were 420 nm (excita-tion) and 480 nm (emission).
LC-MS characterization of dehydro-F420 species
Enzymatic reactions were set up as described above. 10 uL aliquots were injected onto a C18 trap cartridge (LC Packings, Amsterdam, Netherlands) for desalting prior to chromatographic separation on a 0.3 × 100 mm 3.5 μm Zorbax 300SB C18 Stablebond column (Agilent Tech-nologies, Santa Clara, CA, USA) using the following gradient at 6 uL/min: 0-3 min 10% B, 24 min 50% B, 26 min 97% B, 29 min 97% B, 30.5 min 10% B, 35 min 10% B, where A was 0.1% formic acid in water and B was 0.1% formic acid in acetonitrile. The column eluate was ionised in the electrospray source of a QSTAR-XL Quadrupole Time-of-Flight mass spectrometer (Applied Biosystems, Foster City, CA, USA). For IDA (Information Dependent Analysis) analyses, a TOF-MS scan from 330-1000 m/z was performed, followed by three rounds of MS/MS on the most intense singly or doubly charged precursors in each cycle. For targeted work, defined Product Ion Scans were created to isolate and fragment specific ions of interest with various collision energies (10-60 kV). Both positive and negative modes of ionisation were used as appropriate.
MS/MS confirmation of reduction of dehydro-F420 species
To confirm the reduction of the PEP moiety in vitro assays were prepared in 50 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, 2 mM GTP, 0.1 mM Fo, 1 mM PEP, 5 µM Mtb-FbiA, 6.5 µM Msg-FbiD, 10 mM DTT, 20 µM FMN, 0.2 mM NADH, 0.1 µM Ec-Fre, 2 µM Mtb-FbiB and 1 mM L-glutamate. To minimize futile oxidation of FMNH2 by oxygen the reaction mixture was repeatedly evacuated and purged with nitrogen and maintained under a nitrogen atmosphere. Samples were incubated at ambient temperature for up to 36 hours and stopped by addition of 20 mM EDTA. Samples were desalted using Bond Elut C18 tips (Agilent Technologies) and eluted in 0.1 % formic acid in acetonitrile. Samples were injected on to a Q Exactive Plus (ThermoFischer Scientific) at 100 µl/min with isocratic 50% B where A was 10 mM ammonium acetate pH 6.0 and B was 0.02% ammonia in methanol. Scans from 150-2250 m/z were performed and data-dependent MS/MS on targeted metabolites was done in both positive and negative mode.
Production of F420 in E. coli
The pF420 vector was transformed into E. coli BL21(DE3) for expression. Cells were culti-vated in M9 minimal media supplemented with chloramphenicol (25 µg/mL) and tyrosine (18 µg/mL) in 1 L shake flasks with 500 ml working volume at 28 °C. At O.D. of 0.60, 200 ng/mL of tetracycline was added to induce the expression of F420 biosynthesis pathway. After induction, cells were cultivated for at least 16 h. To test expression of the F420 biosynthetic genes, cell pellets were lysed by resuspending to O.D. of 0.8 in buffer containing BugBuster (Novagen). Following centrifugation at 16,000 ×g proteins were resolved on a gel (BoltTM 4-12% Bis-Tris Plus, Invitrogen) for 1 h and visualized using Coomassie Brilliant Blue. For im-munoblotting, proteins were transferred to a membrane (iBlot® 2 NC Regular Stacks, Invitrogen) using iBlot Invitrogen (25 V, 6 mins). After staining and de-staining, the membrane was blocked with 3 % skim milk solution and blotted with anti-FLAG antibodies conjugated with HRP (DYKDDDDK Tag Monoclonal Antibody, Thermofisher Scientific).
For detection of F420 in E. coli lysate by HPLC-FLD, cells were grown overnight in media containing 2.0% tryptone, 0.5% yeast extract, 0.5% NaCl, 22 mM KH2PO4, 42 mM Na2HPO4, 100 ng/mL anhydrotetracycline, 34 µg/mL chloramphenicol at 30 °C. Cells from 500 µL of culture were pelleted by centrifugation at 16,000 ×g. Cells were resuspended in 500 µL of 50 mM Na2HPO4 pH 7.0 and lysed by boiling at 95 °C for 10 minutes. Cell debris was pelleted by centrifugation at 16,000 ×g and filtered through a 0.22 µm PVDF filter. Anal-ysis was conducted as described previously2. pSB1C3 containing only BBa_K145201 was used as a control.
Purification and analysis of E. coli-produced F420
For F420 extraction, 1 L of cell culture was centrifuged at 5,000 ×g for 15 minutes. The cell pellet was re-suspended in 30 ml of 75% ethanol and boiled at 90 °C in a water bath for 6 min for cell lysis. The cell extract was again centrifuged at 5,000 ×g for 15 minutes to remove cell debris and the supernatant was lyophilized. The lyophilized cell extract was re-dissolved in 10 ml milli Q water centrifuged at 5,000 ×g for 15 minutes and the supernatant passed through 0.45 µm syringe filter (Millex-HV). The filtered cell extract was purified for F420 using a 5 ml HiTrap QFF column (GE Healthcare) as previously described44. The purified F420 solution was further desalted by passing it through C18 extract column (6 ml, HyperSep C18 Cartridges, ThermoFischer Scientific). The C18 extract column was first equilibrated by passing through 10 ml of 100% methanol and 10 ml of milli Q. Afterwards the F420 solution was passed through the C18 extract column, F420 was eluted in 2 ml fraction of 20% methanol.The purified F420 solution was further concentrated in GeneVac RapidVap and re-dissolved in 500 µl of milliQ for further analysis and assays.
UV and fluorescence spectra were collected on a Varian Cary 60 and a Varian Cary Eclipse, respectively, in a 10 mm QS quartz cell (Hellma Analytics). Samples were buffered to pH 7.5 with 50 mM HEPES and scanned from 250-600 nm. For fluorescence, the excitation wavelength was 420 nm and the emission scanned from 435 to 600 nm.
Activity assays with M. smegmatis and E. coli-derived F420 were conducted with M. smegmatis FGD1 expressed and purified as described previously15. Assays were performed in 50 mM Tris pH 7.5, 300 mM NaCl, 50 nM FGD1, 5 µM F420 and 0-1000 µM glucose-6-phosphate using a SpectraMAX e2 plate reader. Activity was measured by following loss of F420 fluorescence at 470 nm. Apparent kcat and Km values were calculated using GraphPad Prism 7.04 (GraphPad Software, La Jolla California).
2-PL synthesis
In the absence of a commercial source, 2-PL was chemically synthesized by a slight modifi-cation of the method of Kirsch60. Briefly, benzyl lactate was condensed with chlorodiphenyl phosphate in pyridine, with cooling, to give benzyldiphenylphosphoryl lactate. Hydrogenolysis of this material in 70% aqueous tetrahydrofuran over 10% Pd-C gave phospholactic acid as a colorless, viscous oil, which was characterized by proton, carbon and phosphorus NMR spectroscopy, and by mass spectrometry. 1H NMR (DMSO-d6) δ 11.68 (br, 3H), 4.53 (m, 1H), 1.36 (d, J=6.8 Hz, 3H). 13C NMR (DMSO-d6) δ 172.50 (d, JP-C=0.05 Hz), 69.56 (d, JP-C=0.04 Hz), 19.27 (d, JP-C=0.04 Hz). 31P NMR (DMSO-d6) δ −1.64. APCI-MS found: [M+H]+=171.1, [M-H]-=169.1.
Fo purification
Fo was purified from M. smegmatis culture medium over-expressing Mtb-FbiC as described previously44.
Genomic context analysis
A non-redundant CofE dataset of 4,813 sequences was collected using the Pfam identifier PF01996 and the InterPro classifications IPR008225, IPR002847, and IPR023659. Archaeal CofE sequences were extracted from this dataset, resulting in a set of 1,060 sequences that included representatives across twelve phyla: Crenarchaeota, Euryarchaeota, Thaumarchaeota, Candidatus Bathyarchaeota, Candidatus Diapherotrites, Candidatus Heimdallarchaeota, Candidatus Korarchaeota, Candidatus Lokiarchaeota, Candidatus Marsarchaeota, Candidatus Micrarchaeota, Candidatus Odinarchaeota, and Candidatus Thorarchaeota. The genomic context (10 upstream and 10 downstream genes) of each archaeal CofE was analyzed for the presence of a neighboring PF00881 domain with homology to the C-terminal domain of FbiB using the Enzyme Function Initiative-Genome Neighborhood Tool61.
Author contribution
JA performed experiments, analysed results and cowrote the manuscript; ENMJ, MH, JC, SMS, SS, BP, MM performed experiments and analyzed results; BN conceived project, per-formed experiments and analyzed results; CG, ENB conceived project, designed experiments, analyzed results and cowrote the manuscript; CS designed experiments, analyzed results and cowrote the manuscript; GB, CJJ conceived project, designed experiments, performed experiments, analyzed results and cowrote the manuscript. All authors provided feedback on the manuscript.
Declaration of interests
The authors declare no competing financial interest.
Acknowledgment
We thank Assoc. Prof. Chris Squire, Dr Carol Hartley and Dr Andrew Warden, and Dr Mat-thew Taylor for helpful discussions, Dr Adam Carroll and Dr Thy Truong for technical assis-tance. This research is supported by a Sir Charles Hercus Fellowship (GB), an NHMRC New Investigator Grant (CG; 1142699), an ARC DECRA Fellowship (CG; DE170100310), the Health Research Council of New Zealand (ENB), an AGRTP Scholarship (JA), the Australian Research Council, and the National Health and Medical Research Council (CJ). Crystal data collection was undertaken on the MX1 beamline at the Australian Synchrotron (Victoria, Australia). Access to the Australian Synchrotron was supported by the New Zealand Synchrotron Group Ltd.