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. Author manuscript; available in PMC: 2019 Jul 13.
Published in final edited form as: Methods Enzymol. 2018 Jul 13;606:95–118. doi: 10.1016/bs.mie.2018.05.001

QueE: A radical SAM enzyme involved in the biosynthesis of 7-deazapurine containing natural products

Julia K Lewis *, Nathan A Bruender †, Vahe Bandarian *
PMCID: PMC6484087  NIHMSID: NIHMS1013991  PMID: 30097106

Abstract

7-Carboxy-7-deazaguanine (CDG) is a common intermediate in the biosynthesis of 7-deazapurine-contianing natural products. The biosynthesis of CDG from GTP requires three enzymes: GTP cyclohydrolase I, 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) synthase, and CDG synthase (QueE). QueE is a member of the radical S-adenosyl-l-methionine (SAM) superfamily and catalyzes the SAM-dependent radical-mediated ring contraction of CPH4 to generate CDG. This chapter focuses on methods to reconstitute the activity of QueE in vitro.

1. Introduction

Pyrrolopyrimidine moieties are components of structurally diverse group of natural products that share a 7-deazapurine core. These compounds are found in all kingdoms of life and their function(s) range from secondary metabolites produced by Actinomyces, to the modified bases, queuosine and archaeosine, in tRNA [see (McCarty & Bandarian, 2012) for review]. The first example of a deazapurine, toyocamycin, was isolated on the basis of its biological activity in 1956 (Nishimura, Katagiri, Sato, Mayama, & Shimaoka, 1956). To date, >30 compounds with deazapurine cores have been observed in Nature (McCarty et al, 2012).

Prior to elucidation of the biosynthetic pathway to deazapurines, radiotracer isotope experiments had demonstrated that deazapurines were derived from purines in a pathway that retains some of the carbons of the sugar, and C-2 from the base, but removes C-8 (Buff & Dairman, 1975; Burg & Brown, 1968; Elstner & Suhadolnik, 1971; Elstner, Suhadolnik, Suh, 1975; Guroff & Strenkoski, 1966; Kuchino, Kasai, Nihei, & Nishimura, 1976; Reynolds & Brown, 1962; 1964; Smulson & Suhadolnik, 1967; Suhadolnik & Uematsu, 1970; Uematsu & Suhadolnik, 1970). Despite the structural diversity, it now appears the deazapurine moiety is biosynthesized in three steps from guanosine triphosphate (GTP) (McCarty et al, 2012; McCarty, Krebs, & Bandarian, 2012; McCarty, Somogyi, & Bandarian, 2009a; McCarty, Somogyi, Lin, Jacobsen, & Bandarian, 2009b; Miles, Roberts, McCarty, & Bandarian, 2014; Reader, Metzgar, Schimmel, & de Crécy-Lagard, 2004), and modified as appropriate by additional species-specific tailoring enzymes (McCarty et al, 2012; Bai, Fox, Lacy, Van Lanen, & Iwata-Reuyl, 2000; Dowling et al., 2016; Frey, McCloskey, Kersten, & Kersten, 1988; Lee, Van Lanen, & Iwata-Reuyl, 2007; Miles, McCarty, Molnar, & Bandarian, 2011; Miles, Myers, Kincannon, Britt, & Bandarian, 2015; Nelp & Bandarian, 2015; Nelp, Astashkin, Breci, McCarty, & Bandarian, 2014; Nelp, Song, Wysocki, & Bandarian, 2016; Okada, Harada, & Nishimura, 1976; Okada et al., 1979; Phillips et al., 2008; Reuter, Slany, Ullrich, & Kersten, 1991; Shindo-Okada, Okada, Ohgi, Goto, & Nishimura, 1980; Song, Nelp, Bandarian, & Wysocki, 2015; Van Lanen et al., 2005; Watanabe et al., 1997) (Fig. 1). In the first step of the reaction GTP cyclohydrolase I (GCH I) converts GTP to 7,8-dihydroneopterin triphosphate (H2NTP), which is subsequently modified to 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) by CPH4 synthase (QueD), prior to undergoing a ring contraction to form 7-carboxy-7-deazaguanine (CDG) catalyzed by CDG synthase (McCarty et al, 2012; McCarty et al, 2009a; McCarty et al, 2009b; Phillips et al., 2008). CDG likely serves as the starting point for all known deazapurines.

Figure 1. Biosynthesis of the 7-deazapurine core.

Figure 1.

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The 7-deazapurine core is obtained in three steps from GTP in Nature.

QueE catalyzes the radical-mediated ring contraction of CPH4 to form CDG (McCarty et al., 2009a). It is a member of the radical S-adenosyl-L-methionine (SAM) enzymes, which were initially identified on the basis of a conserved CxxxCxxC motif (Sofia, 2001; Frey, Hegeman, & Ruzicka, 2008). The Cys sidechains in the conserved motif coordinate three iron atoms of a site-differentiated 4Fe-4S cluster. The fourth iron to binds SAM through its amino and carboxylate moieties (Berkovitch et al., 2003; Walsby, Ortillo, Broderick, Broderick, & Hoffman, 2002; Walsby et al., 2005). In the +1 oxidation state, the iron-sulfur cluster reductively cleaves SAM to generate 5’-deoxyadenosyl radical (5’-dAdo•), which initiates chemistry by H-atom abstraction in most radical SAM enzymes (Moss & Frey, 2001). Recent bioinformatic studies suggest that radical SAM enzymes constitute a wide superfamily of >100,000 members (S. Brown & Babbitt, 2015).

In QueE, the reversible cleavage of SAM generates 5’-dAdo• which initiates ring contraction in CPH4 is by abstracting a H-atom from C-6 of the substrate (Fig. 3) (McCarty et al., 2012). The substrate radical is thought to rearrange to a product radical that is quenched by H-atom abstraction from dAdo. Stereospecific abstraction of the C-7 proR hydrogen and elimination of the amino group complete the catalytic cycle and reforming SAM. In addition to detailed biochemical studies, QueE has been quite amenable to structural studies. Multiple high resolution crystal structures of the protein provide detailed snapshots of the active site of the protein in the ± various ligands, including the cofactor, substrate, and substrate analogs (Bruender et al., 2017; Bruender, Young, & Bandarian, 2015; McCarty et al., 2012; McCarty et al, 2009b; Wilcoxen, Bruender, Bandarian, & Britt, 2018) (Fig. 4).

Figure 3. Radical mediated ring contraction mechanism of QueE.

Figure 3.

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The dAdo• abstracts a hydrogen atom from the C-6 position of CPH4 which is thought to undergo rearrangement to a gem-amino carboxylate product. The product maintains the C-7 proS proton of the substrate.

Figure 4. Crystal Structure of QueE.

Figure 4.

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The overall structure of QueE with SAM, CPH4, and 4Fe-4S cluster (A), and close up of the active site (B). SAM is bound to the cluster through its α-amino and α-carboxylate moieties.

Studies with QueE require access to a number of reagents. As with other radical SAM enzymes, QueE activity requires the presence of a means reducing the 4Fe-4S cluster to the catalytically active +1 state (Fig. 2 and Fig. 5). Dithionite is often used in the lab, but it has been demonstrated that higher activities are achieved with biological reducing systems, such as flavodoxin (Fld)/flavodoxin reductase (FPR)/NADPH (Bruender et al., 2015). However, it is also possible to bypass the FPR/NADPH and use dithionite-reduced Fld to achieve efficient reduction of the enzyme. In addition to a source of reductant, the substrate, CPH4, is not commercially available and must be obtained by enzymatic synthesis from GTP using the biosynthetic enzymes. This chapter will focus on the methods to obtain each of these components to reconstitute in vitro activity of recombinant QueE. We note that while the foregoing discussion will focus on the B. subtilis enzyme, the methods have also been applied successfully to the B. multivorans protein as well (Dowling et al., 2013) and are therefore general to QueE proteins.

Figure 2. Reduction cleavage of SAM.

Figure 2.

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The [4Fe-4S] cluster in RS enzymes reductively cleaves SAM to 5’-dAdo•, which initiates the catalytic cycle.

Figure 5. Biological Reduction Pathways.

Figure 5.

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The biological reduction pathway from NADPH or the chemical reduction via dithionite. Reprinted with permission from American Chemical Society (Bruender et al., 2015).

2. Expression and Purification of QueE

The 4Fe-4S cluster that is required in all RS enzymes is often oxygen sensitive, requiring the enzymes to be purified and studied under strictly anaerobic conditions. Since QueE is highly overexpressed in E. coli hosts, a single step purification under anoxic conditions, using the His6 affinity tag step suffices to purify sufficient protein for biochemical, structural, and spectroscopic studies.

2.1. Expression of QueE

The gene of queE was amplified from genomic DNA of B. subtilis (ATCC 23857D) and ligated into pET28a using the NdeI and BamHIII restriction sites to make the plasmid pRM78 (McCarty et al., 2009b). The pET28a plasmid encodes kanamycin (Kan) resistance and expression is induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). We find that it is necessary to express queE with pDB1282, which contains the genes iscSUA-hscB-hscA-fdx-orf3 from Azobacter vinelandii that are involved in assembly of the iron-sulfur clusters in nitrogenase, to enhance cluster content (Frazzon, Fick, & Dean, 2009). The pDB1282 plasmid contains ampicillin (Amp) resistance and the expression of the isc operon is induced with arabinose.

Protocol for Expression of queE gene

  1. Co-transform BL21(DE3) E. coli cells with plasmids pRM78 and pDB1282. Plate cells on LB agar plates containing 100 μg/mL Amp and 34 μg/mL Kan.

  2. Prepare the LB media in 2.8 L Fernbach flasks by dissolving 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl in 1 L of deionized water. Autoclave 30 min and cool to room temperature before use.

  3. Select a single colony from the transformation plate and inoculate 100 mL LB media containing both 100 μg/mL Amp and 34 μg/mL Kan (see step 1). Grow the starter culture at 37 °C overnight with shaking at 200 rpm.

  4. Use the overnight culture to inoculate the expression cultures. Add 10 mL of overnight culture to the 1 L of fresh LB in 2.8 L Fernbach flasks supplemented with 100 μg/mL Amp and 34 μg/mL Kan. Allow the cells to grow at 37 °C with shaking at 175 rpm to an optical density at 600 nm (OD600) of ~0.3 and inudce expression of the isc genes from the pDB1282 plasmid by addition of 0.5 g/L of solid arabinose. Also add 50 μM (final concentration) FeCl3 to ensure that enough iron is present for reconstitution of the cluster. Incubate the cultures with continued shaking at 37 °C to OD600 of ~0.5 prior to inducing expression of QueE with IPTG (100 µM final concentration). Shake the cultures at 120 rpm for 6 h after inducing expression of QueE.

  5. Harvest the cells at 5,000 xg, flash freeze in liquid nitrogen, and store at −80 °C until use.

2.2. Purification of QueE

Purification of QueE should be performed in an anaerobic chamber under a nitrogen atmosphere. We use a Coy chamber, which typically has an atmosphere composed of 97% nitrogen and 3% hydrogen. Anaerobicity is achieved by allowing H2 to react with O2 on the surface of the palladium catalyst within the chamber to generate water. Excess moisture in the chamber is removed by both dessicant packs and a Coy dehumidifier. The anaerobicity of the chamber is monitored by a Coy cam-12 detector. The chamber is in a laboratory which is maintained at 22 °C. Materials to be introduced into the chamber pass through an antechamber and made anaerobic by repeated cycling between vacuum and either N2 or a 95%N2/5%H2 gas mix. We have modified the default cycle program for the antechamber so that it is vacuumed to 19.9 in Hg and refilled with N2 four times prior to final round of evacuation and filling with mixed gas (95%N2/5%H2). To facilitate purifications steps, we have placed an ÄKTAprime plus inside the chamber. All tubes, tips, and other plasticware are placed in the glovebox at least 24 h prior to use. Water and concentrated buffer stocks are prepared aerobically and stirred in the glovebox for 12 h to exchange the gas. Cell disruption is carried out on ice and the centrifugation steps are at 4 °C. Cells are disrupted by a Branson sonifier as indicated below. We maintain the horn of the sonifier in the glove box and attach it to a controller outside the glove box.

Protocol for Purification of QueE

  1. Place 5–10 g of frozen cell pellet in a stainless steel beaker with a stir bar and 1 mM phenylmethane sulfonyl fluoride (PMSF) in the middle of a crystallization dish filled with ice and cycle into the glove box.

  2. Add deoxygenated lysis solution (4 mL/g of wet cell paste) containing 0.05 M piperazine N,N’-bis(2-ethanesulfonic acid) (PIPES-NaOH) (pH 7.4), 0.5 M KCl, 0.05 M imidazole, and 10 mM dithiothreitol (DTT).

  3. While the suspension is stirred, lyse cells a total of 10 min, with 15 sec bursts at 50% power that are followed by 45 sec rests to allow the solution to chill in between.

  4. Transfer the lysate into an Oakridge centrifuge tube with O-rings, tightly seal the tube inside the glove box, and remove from the chamber.

  5. Centrifuge at 18,500 xg for 35 min at 4 °C to pellet cell debris.

  6. Charge a 5 mL HisTrapHP column (GE Healthcare) with NiSO4 according to manufacturer’s directions and equilibrate with lysis solution (step 2).

  7. Transfer the centrifuge tube into the anaerobic chamber and carefully decant the supernatant.

  8. Using a 50 mL SuperLoop, load the protein onto the column. Wash with lysis solution (step 2) until the UV trace returns to baseline (~50 mL). Elute the protein with a 50 mL gradient from 0.05 M – 0.5 M imidazole in lysis buffer.

  9. Pool the dark brown fractions containing QueE and exchange into a solution containing 0.05 M PIPES-NaOH (pH 7.4) and 10 mM DTT using a BioRad 10DG desalting column.

  10. Concentrate to a volume of ~5–10 mL using an Amicon concentrator under N2 with a YM-10 membrane (Millipore). N2 can be brought into the glove box through a tube that is fed through a side nipple on the Coy chamber. Freeze the resulting protein in small aliquots in liquid nitrogen and store at −80 °C. A typical SDS-PAGE of purified QueE is shown in Fig. 6.

  11. Quantify the QueE stock by a Bradford assay (BioRad) with BSA as standard. We have empirically determined a Bradford concentration correction factor of 0.67 for QueE, which is based on the absolute amino acid content (McCarty et al., 2012).

Figure 6. SDS-PAGE of purified proteins.

Figure 6.

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The gel shows (1) ladder, (2) QueE, (3) FPR, and (4) YkuN.

2.3. Chemical Reconstitution of QueE

While as purified QueE has some intact 4Fe-4S cluster, it is necessary to carry out a chemical reconstitution of QueE under anaerobic conditions to obtain cofactor-replete enzyme. We have empirically determined that efficient reconstitution can be achieved when QueE is incubated with 8 molar excess of iron (FeCl3) and sulfide (Na2S) as described below.

  1. Weigh out ~20 mg of Na2S and FeCl3 into separate glass scintillation vials. Cap, and cycle into the anaerobic chamber.

  2. Dissolve the Na2S in water and the FeCl3 in saturated sodium bicarbonate to a final concentration of 100 mM.

  3. Add 8 molar equivalents of FeCl3 and Na2S to the protein based on its corrected concentration determined by the Bradford assay (Section 2.2, step 11). Dropwise add the FeCl3 and allow it to stir for about 3 min before adding the Na2S.

  4. Allow the reconstitution to proceed at room temperature for 4 h after Na2S has been added.

  5. At the end of the 4 h, the solution often has some precipitate. Remove the precipitate by centrifugation prior to desalting with a BioRad 10DG desalting column into solution of 0.05 M PIPES-NaOH (pH 7.4) containing 10 mM DTT.

  6. Concentrate the desalted QueE using an Amicon concentrator under N2 with a YM-10 membrane (Millipore) to a minimal volume (~1 mL).

  7. Freeze the protein in liquid nitrogen as small aliquots and store at −80 °C. Typical yield from this procedure is ~150 mg of QueE from 5 g of wet cell paste.

  8. As needed, QueE can be further purified by gel filtration on a HiPrep 16/60 Sephacryl S-200 high resolution column (GE Healthcare). Equilibrate the column with solution containing 0.05 M PIPES-NaOH (pH 7.4), 0.15 M NaCl and 10 mM DTT. We usually calibrate the column with chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), bovine serum albumin (BSA) (67 kDa), and aldolase (158 kDa).

  9. Inject 1 mL of concentrated QueE onto the equilibrated column and collect fractions while washing with the loading buffer.

  10. Analyze the fractions with an SDS-PAGE gel and compare to standards to determine the oligomeric state. We only elute fractions that correspond to dimeric QueE.

  11. The fractions containing QueE are pooled and concentrated in the same manner as in step 6. The concentrated QueE is then aliquoted, flash frozen in liquid nitrogen, and stored at −80 °C until use.

3. Expression and Purification of YkuN

The reduction of the FeS cluster in QueE to initiate the catalytic cycle can be accomplished by a number of methods (see Fig. 5). The simplest route is to use a strong reductant, such as sodium dithionite. However, we have shown that the biological reducing system, such as flavodoxin/flavodoxin reductase is a better choice for QueE, as significantly improves the activity (Bruender et al., 2015). We generally use the B. subtilis flavodoxin homolog, YkuN, in our assays to pass electrons to QueE either from dithionite, or flavodoxin reductase and NADPH.

3.1. Expression of B. subtilis flavodoxin YkuN

The gene ykuN was amplified by PCR from B. subtilis subsp. subtilis str. 168. The gene was A-tailed and ligated into the pGEM Easy-T vector (Promega) for blue and white screening. The gene was then moved to the pET29a expression vector to generate pNB466 (Bruender et al., 2015).

Protocol for expression of B. subtilis flavodoxin YkuN

  1. BL-21 (DE3) E. coli cells were transformed with pNB466 and plated onto LB agar plates containing 34 μg/mL Kan. Incubate at 37 °C overnight.

  2. Pick a colony from the LB agar plate to inoculate a 100 mL overnight starter culture containing 34 µg/mL Kan. Incubate at 37 °C overnight with shaking at 200 rpm.

  3. Add 10 mL of the overnight culture to 1 L of fresh LB broth in 2.8 L Fernbach flasks containing 1 L LB each and supplemented with 34 µg/mL Kan. Incubate at 37 °C with shaking at 175 rpm until they reach OD600~0.5.

  4. Induce expression of ykuN with 0.1 mM IPTG (final concentration) and add riboflavin to a final concentration of 0.1 mM at the time of induction to ensure that cofactor replete protein is obtained. Allow cells to express the protein at 37ºC overnight while shaking at 175 rpm.

  5. Collect the cells after ~ 18 h by centrifugation at 5,000 xg for 15 min at 4 °C.

  6. Flash freeze wet cell pastes in liquid nitrogen and store at −80 °C until use.

3.2. Purification of YkuN

The expression vector for YkuN produces native protein, which is readily purified by successive anion exchange and hydrophobic interactions chromatographic steps as described below.

Protocol for the purification of YkuN

  1. In a stainless steel beaker with a stir bar, add 40 g of frozen cell paste and solution (4 mL/g of wet cell paste) containing 0.05 M Tris-HCl (pH 7.2), 1 mM EDTA, 2 mM DTT, 1 mM PMSF. Place the beaker in the middle of a crystallization dish filled with ice.

  2. While the suspension is stirred, lyse cells with a Branson digital sonifier for total of 10 min, with 15 sec bursts at 50% power that are followed by 45 sec rests to allow the solution to chill in between.

  3. Centrifuge at 18,000 xg for 35 min to remove cell debris.

  4. Equilibrate a DEAE-Sepharose column (GE Healthcare) (11 × 3 cm) with 3 column volumes of the lysis solution (0.05 M Tris-HCl (pH 7.2), 1 mM EDTA, 2 mM DTT) on an ÄKTAprime FPLC.

  5. Load the cleared lysate via a 50 mL SuperLoop onto the column. Wash the column with the loading buffer until the UV absorbance is at the baseline. Elute YkuN with a 0.85 L linear gradient 0 −1 M KCl in lysis solution.

  6. Pool fractions containing YkuN on the basis of SDS-PAGE analysis of the yellow fractions (Flavodoxin (YkuN) binds FMN which is a yellow chromophore).

  7. Slowly add an equal volume of 50 mM Tris-HCl (pH 7.0) containing 3 M ammonium sulfate and 2 mM DTT to the pooled YkuN fractions over 30 min.

  8. Equilibrate a butyl-Sepharose column (GE Healthcare) (11 × 3 cm) with solution containing 0.05 M Tris-HCl (pH 7.0), 1.5 M ammonium sulfate, and 2 mM DTT on an ÄKTAprime FPLC.

  9. Using a 50 mL SuperLoop load YkuN onto the column and wash with the loading buffer until the UV absorbance returns to baseline. Elute the protein with a 0.7 L linear gradient from the 1.5M −0 M ammonium sulfate in loading solution.

  10. Identify YkuN-containing fractions by SDS-PAGE gel of yellow fractions.

  11. Concentrate the protein to ~ 1 mL using Amicon concentrator under N2 with a YM-10 membrane (Millipore). Freeze YkuN in small aliquots in liquid nitrogen and store at −80 °C until use. See Fig. 6 for representative SDS-PAGE of purified YkuN.

4. Expression and Purification of flavodoxin reductase (FPR)

FPR is used in tandem with YkuN to biologically reduce the [4Fe-4S] cluster of QueE by shuttling electrons that are ultimately derived from NADPH to QueE. This section describes expression and purification of FPR.

4.1. Expression of the fpr gene

The gene for E. coli fpr was synthesized in codon optimized form (Genscript), and cloned into HindIII and NdeI restriction sites of pET28a for expression of His6 tagged protein. The sequence for the synthetic gene and the corresponding protein is shown in Fig. 6.

Protocol for fpr gene expression

  1. Transform HMS174(DE3) E. coli cells with the fpr expression plasmid and plate onto an LB agar plate containing 34 µg/mL kanamycin. Put the LB agar plate at 37 °C overnight.

  2. Select a colony from plate to inoculate a 0.1 L starter culture containing 34 µg/mL kanamycin and incubate at 37 °C overnight with shaking at 200 rpm.

  3. Inoculate six 2.8 L Fernbach flasks containing 1 L LB each with 34 µg/mL Kan with 10 mL of overnight culture. Shake the cultures at 175 rpm at 37 °C.

  4. At an OD600~0.5 add 0.1 mM (final concentration) IPTG to induce expression. As with YkuN, add 0.1 mM riboflavin to ensure that cofactor replete protein is obtained.

  5. Harvest the cells after ~ 6 h by centrifugation at 5,000 xg for 15 min at 4 °C.

  6. Flash freeze the cell paste in liquid nitrogen and store at −80 °C until use.

4.2. Purification of FPR

FPR is produced as a His6 tagged protein, which simplifies purification using Ni-NTA affinity chromatography. FPR is a flavoprotein and fractions containing the enzyme are readily identified by the bright yellow color due to the presence of the flavin chromophore and by SDS-PAGE.

Protocol for FPR purification

  1. Place ~5 g of cell paste and a stir bar in a stainless steel beaker. Add lysis buffer containing 0.02 M KPi (pH 7.2) and 0.5 M NaCl at a ratio of ~4 mL buffer per g of cell paste. Add solid PMSF to obtain 1 mM final concentration. Put the beaker in the center of a recrystallization dish filled with ice.

  2. Sonicate the cells with 48 bursts of 15 sec each at 50% power for 12 min with 45 sec rest to minimize heating of the sample.

  3. Transfer the lysate to Oakridge tubes and centrifuge at 18,000 xg for 35 min.

  4. Charge a 1 mL His trap column (GE Healthcare) with NiSO4 according to manufacturer’s protocol and equilibrate a 1 mL His-trap (GE Healthcare) with lysis buffer on an ÄKTAprime FPLC.

  5. Load the clarified lysate onto the column using a 50 mL SuperLoop.

  6. Wash the column with ~ 5 mL of lysis buffer until the UV absorbance is near baseline and stable.

  7. Elute FPR with a 5 mL linear gradient in lysis buffer from 0.05 to 0.5 M imidazole. Pool the yellow fractions containing FPR.

  8. Dialyze the pooled fraction against 4 L of 0.02 M HEPES-NaOH (pH 7.4) at 4 ºC for ~16 h. Change the dialysis buffer twice.

  9. Concentrate the protein with Amicon concentrator with a YM-10 membrane (Millipore) under N2. Use liquid nitrogen to flash freeze the protein aliquots and store at −80 °C until use. See Fig. 6 for representative SDS-PAGE of purified YkuN.

5. Enzymatic synthesis of CPH4 with GCH I and QueD from GTP

The substrate for QueE, CPH4, is not commercially available but can be synthesized enzymatically using GCH I and QueD (see Fig. 1). In this section we describe protocols for preparation of GCH I and QueD, and the enzymatic preparation of CPH4 from GTP. These methods can be adapted to synthesis of deuterated isotopomers of CPH4 with label at C7proR, or C7proS (McCarty et al., 2012) (Fig. 8). The [7R-D] isotope of CPH4 is generated by completing the first step of the reaction of GTP with GCH I in D2O, followed by QueD in H2O. The [7S-D] isotope of CPH4 is made using 97% [U-D] GTP (Cambridge Isotope Laboratories, Inc.) as the starting material with the GCH I and QueD reactions run in H2O. The C6 labeled isotope is obtained by reacting sepiapterin (Sigma), with QueD in D2O. Fig. 8 summarizes the strategies for enzymatic synthesis of isotopologs of CPH4.

Figure 8. Enzymatic synthesis of isotopologs of CPH4.

Figure 8.

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(a) Isotopologs of CPH4 can be prepared enzymatically by carrying out the GCH I and QueD reactions in H2O or D2O. (b) Mass spectra of the synthetic isotopologs. Reprinted with permission from American Chemical Society (McCarty et al, 2012)

5.1. Expression and purification of GCH I

The gene, for GCH I (folE) was amplified from the E. coli W3110 genomic DNA and cloned into the HindIII and NdeI site of pET28a to generate pJB163. GCH I is purified via anion exchange chromatography and hydrophobic interaction chromatography.

Protocol for expression of GCH I

  1. Transform BL21 (DE3) E. coli cells with pJB163 and plate onto an LB agar plate containing 34 µg/mL Kan. Incubate the plate at 37 °C overnight.

  2. Select a colony from the LB agar plate and use the colony to inoculate a 0.1 L starter culture containing 34 µg/mL Kan. Allow the culture to grow at 37 °C overnight.

  3. Use 10 mL of starter culture to inoculate six 2.8 L Fernbach flasks, each having 1 L of LB with 34 µg/mL Kan and 100 µM ZnSO4. Grow at 37 °C and 200 rpm.

  4. At an OD600~0.7 induce the cells with 100 µM IPTG.

  5. Harvest the cells after 4 h of growth by centrifugation at 5,000 xg at 4 °C. Flash freeze the cell paste in liquid nitrogen and store at −80 °C until use.

Protocol for purification of GCH I

  1. GCH I is purified at 4 °C under aerobic conditions using an ÄKTA FPLC.

  2. Weigh out ~10 g of cell paste and place in a stainless steel beaker with a stir bar. Dissolve in lysis buffer (4 mL/g) containing 0.02 M Tris-HCl (pH 8.0), 10 mM DTT and supplemented with 1 mM PMSF. Place beaker in the middle of a recrystallization dish packed with ice.

  3. While stirring the cells, sonicate using a Branson digital sonifier with 40 bursts at 50% power for 15 sec, followed by 45 sec of rest to avoid heating the sample.

  4. Pour the cell lysate into Oakridge tubes and centrifuge for 1 h at 18,000 xg and 4 °C.

  5. Equilibrate a Q-Sepharose column (GE Healthcare) (2.6×14.5 cm) with ~ 3 column volumes of the lysis buffer.

  6. Use a SuperLoop to inject the clarified lysate onto the column.

  7. Wash the column with ~2 column volumes of loading buffer until the UV trace returns to near baseline.

  8. Elute the protein with 0.85 L linear gradient from 0 −0.5 M KCl in loading buffer.

  9. Identify the fractions containing GCH I by SDS-PAGE.

  10. Pool the fractions containing GCH I and concentrate to ~50 mL using an Amicon concentrator with a YM-10 membrane (Millipore) under N2.

  11. Equilibrate a butyl-Sepharose column (GE Healthcare) (11 × 3 cm) with loading buffer containing 0.02 M Tris-HCl (pH 8.0), 1 M ammonium sulfate, and 10 mM DTT.

  12. To the pooled fractions of GCH I from step 9 add an equal volume of buffer containing 20 mM Tris-HCl (pH 8.0), 2 M ammonium sulfate, and 10 mM DTT. This solution should be added slowly over a period of 30 min while the pooled fractions are stirring on a stir plate.

  13. Use a SuperLoop to inject the fractions of GCH I onto the column. After loading, wash the column with the loading buffer until the UV trace has returned to baseline.

  14. Elute the protein over a 0.7 L linear from 1 to 0 M ammonium sulfate in loading solution.

  15. Identify and pool fractions containing GCH I by SDS-PAGE.

  16. Dialyze the GCH I fractions against 4 L of buffer containing 20 mM Tris-HCl (pH 8.0) and 10 mM DTT at 4 ºC for ~16 h. Dialyze against fresh buffer a second time.

  17. Concentrate the GCH I to a minimal volume using an Amicon concentrator under N2 gas with a YM-10 membrane (Millipore).

  18. Aliquot the protein and flash freeze with liquid nitrogen. Store at –80°C until use.

5.2. Expression and purification of QueD

Native QueD from E. coli is expressed from a pET29 based plasmid with ygcM (b2765) cloned between HindIII and NdeI sites (McCarty et al, 2009a). The gene, ygcM, was PCR amplified from E. coli W3110 chromosomal DNA.

Protocol for expression of queD gene

  1. Transform BL21(DE3) with vector containing E. coli QueD gene and incubate on a LB plate containing 34 µg/mL Kan at 37 °C overnight.

  2. Inoculate 6 L of LB media containing 34 µg/mL Kan with 10 mL from a starter culture prepared from a single colony picked from the transformation plate.

  3. Grow at 37 °C and 200 rpm to OD600~0.5. Induce the cells with 0.1 mM IPTG. Since QueD requires Zn2+ for activity, supplement the cultures with 100 µL of 1 M ZnSO4 (dissolved in water) to give a final concentration of 100 µM.

  4. Continue to allow the cells to grow at 37 °C for 4 h after induction.

  5. Collect cells by centrifugation at 5,000 xg at 4 °C. Use liquid nitrogen to flash freeze the cell paste and store at −80ºC until use.

Purification of QueD

QueD is purified by a combination of anion exchange and hydrophobic interaction chromatographic steps as follows. All purification steps are carried out 4 °C under aerobic conditions.

  1. In a stainless steel beaker containing a stir bar suspend ~10 g of cell paste in 40 mL of loading buffer containing 0.02 M Tris-HCl (pH 8) and 10 mM DTT, and supplemented with 1 mM PMSF. Place the beaker in a recrystallization dish packed with ice.

  2. Lyse the cells by sonication using a Branson digital sonifier at with 48 bursts at 50% amplitude 45 sec rests to allow the mixture to remain cold.

  3. Clear the lysate by centrifugation at 18,000 xg for 35 min at 4 °C.

  4. Equilibrate a Q-Sepharose column (GE Healthcare) (2.6×14.5 cm) with loading buffer. Load the cleared lysate onto the column, wash with ~3 column volumes of the loading buffer and elute with a 0.8 L linear gradient from 0 −0.5 M NaCl in the loading buffer.

  5. Identify fractions containing QueD by SDS-PAGE analysis. Pool the fractions and slowly add solid ammonium sulfate to 1 M saturation over 20 min while stirring.

  6. Equilibrate a butyl sepharose CL-4B column (GE Healthcare) (2.6×14.5 cm) with a butyl loading buffer containing 0.02 M Tris•HCl (pH 8), 1 M ammonium sulfate, and 10 mM DTT.

  7. Load the QueD onto the column, wash with ~2 column volumes of the loading buffer and elute with a 0.7 L linear gradient from 1M-0 ammonium sulfate in loading buffer.

  8. Identify fractions containing QueD by SDS-PAGE analysis, pool, and concentrate in an Amicon pressure concentration device fitted with a YM-10 membrane (Millipore).

  9. Dialyze the protein against 4 L of 0.02 M HEPES-NaOH (pH 7.5) at 4ºC for ~16 h with one buffer change.

  10. The protein can be concentrated as necessary by Amicon filtration as before.

  11. Freeze the protein in small aliquots in liquid nitrogen and store at −80 °C.

5.4. Enzymatic synthesis of CPH4

CPH4 is synthesized by the combined action of GCH I and QueD from GTP. The resulting CPH4 is readily purified by anion exchange chromatography on DEAE-Sepharose. CPH4 is light and oxygen sensitive. Therefore, the synthesis and purification of CPH4 are carried out inside a Coy anaerobic chamber (5%H2/95%N2) and in the dark.

  1. Set up a synthesis mixture that contains 0.05 M Tris-HCl (pH 8.0), 10 mM DTT, 0.5 mM GTP, 20 μM GCH I, and 20 μM QueD in a total volume of 20 mL. Stir the reaction 12 h under anaerobic conditions. The flask is kept dark by covering in foil.

  2. Equilibrate a DEAE-Sepharose (11 × 3 cm) column (GE Healthcare) with 10 mM ammonium bicarbonate. The column and the fractions are also kept dark by covering in foil.

  3. Load the reaction mixture onto the column and elute the CPH4 isocratically with 10 mM ammonium bicarbonate.

  4. CPH4 typically elutes ~0.45 L and can be detected by UV absorbance at 280 nm of the eluate.

  5. Fractions containing CPH4 are pooled and lyophilized. The lyophilization vessel is also kept dark by covering with foil.

  6. The lyophilized CPH4 is resuspended in a minimal volume of 0.05 M Tris-HCl (pH 8.0) and flash frozen small aliquots in liquid nitrogen and stored at −80ºC until use.

  7. CPH4 is quantified using the extinction coefficient of tetrahydrobiopterin at 297 nm at pH 8.0 (ε297=8.71×103 M−1 cm−1).

6. Typical assay conditions for QueE

QueE, is assayed under anaerobic conditions using YkuN/Fpr/NADH to reduce the protein. The conversion of CPH4 to CDG is followed by LC-MS analysis of the reaction. CDG is quantified by comparison of the peaks in the UV chromatograms to standards.

Protocol for assaying QueE in vitro

QueE is assayed in the anaerobic chamber using solutions that are prepared inside the chamber with anaerobic water. Assays are typically carried out in 0.05 M PIPES-KOH (pH 7.4) containing 10 mM DTT, 2 mM MgSO4 and 2 mM SAM. The reduction of QueE is achieved by including 2 mM NADPH, 4 µM FPR and 10 µM YkuN. Alternatively, dithionite (10 mM) and YkuN can also be used to bypass FPR/NADPH. The reactions at various time points can be quenched with addition of 30% (w/v) trichloroacetic acid (TCA), to a final concentration of 3% (w/v) TCA. The reaction mixtures are then analyzed by LC-MS.

7. LC-MS analysis of turnover by CDG

CDG is readily detected and quantified by HPLC methods. We use the UV trace to quantify the product and the MS detector to ensure the identity of the peak. Reverse phase chromatography at 0.2 mL/min on a C18 column is carried out on a Vanquish UPLC that is interfaced with a LTQ Orbitrap XL instrument (ThermoFisher) operated in positive ion mode, scanning m/z 75–1000 at a resolution of 100,000.

Protocol for LC-MS detection of CDG

  1. Centrifuge the quenched reaction mixtures to remove any precipitated protein.

  2. Equilibrate a ThermoFisher Hypersil GOLD C18 column (2.1×150 mm, particle size of 1.9 µm) in Optima water (ThermoFisher) containing 0.1 % Optima TFA (ThermoFisher).

  3. Inject an aliquot (60 µL) of the reaction mixture and elute with a gradient to 100% Optima acetonitrile (ThermoFisher) containing 0.1 % TFA with the following program: 0% acetonitrile from 0 −3 min; 0–10% acetonitrile from 3–38 min; 100% acetonitrile from 38.1–40.1 min; and 0% acetonitrile from 40.1–43.1 min to re-equilibrate the column.

  4. A representative chromatogram is shown in Fig. 9. Under these conditions, CDG elutes at 13 min. The UV-visible spectrum of the product has distinct features at 230 and 300 nm. Identity of the peak as CDG is confirmed by the extracted ion chromatogram at m/z 195.05. Note that the small difference in the retention times observed in the UV-visible and extracted ion chromatographic traces results from the short distance between the UV-visible detector and the electrospray.

  5. Quantify the CDG by comparison to known standards using the area under the UV-visible trace corresponding to CDG.

Figure 9. LC-MS analysis of turnover by QueE.

Figure 9.

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Production of QueE is monitored at 254 nm using a diode array detector (A). CDG has characteristic spectral features at 229 and 299 nm (B). The in-line mass spectrometer allows confirmation of the mass of the CDG (C).

11. Conclusions

The wealth of structural and biochemical data available on QueE makes this an ideal system for exploring structure/function relationships in a radical SAM enzyme. In addition to cofactor-replete enzyme, studies of QueE require access to a source of reductant and substrate. In this chapter we have described methods to all other components that are necessary for in vitro studies of the enzyme.

Figure 7. Sequence of codon optimized FPR.

Figure 7.

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The clonging sites are shown in italic.

Acknowledgements

This work was supported by the National Institutes of Health (GM072623 and GM120638).

Abbreviations:

AmSO4

ammonium sulfate

Amp

ampicillin

CDG.

7-carboxy-7-deazaguanine

CPH4

6-carboxy-5,6,7,8-tetrahydropterin

dAdo•

5’-deoxyadenosyl radical

DTT

dithiothrietol

IPTG

isopropyl β-D-1-thiogalactopyranoside

Kan

kanamycin

KPi

potassium phosphate

LB

lysogeny broth

PIPES

piperazine-N,N’-bis(2-ethanesulfonic acid)

PMSF

phenylmethane sulfonyl fluoride

SAM

S-adenosyl-l-methionine

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