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. Author manuscript; available in PMC: 2022 Jun 9.
Published in final edited form as: Methods Enzymol. 2022 Feb 3;669:71–90. doi: 10.1016/bs.mie.2021.12.014

Studies of GenK and OxsB, two B12-dependent radical SAM enzymes involved in natural product biosynthesis

Yu-Hsuan Lee a, Hung-wen Liu a,b,?
PMCID: PMC9178707  NIHMSID: NIHMS1788471  PMID: 35644181

Abstract

The B12-dependent radical SAM enzymes are an emerging subgroup of biological catalysts that bind a cobalamin cofactor in addition to the canonical [Fe4S4] cluster characteristic of radical SAM enzymes. Most of the B12-dependent radical SAM enzymes that have been characterized are mediated methyltransfer reactions; however, a small number are known to catalyze more diverse reactions such as ring contractions. Thus, Genk is a methyltransferase from the gentamicin C biosynthetic pathway, whereas Ox catalyzes the oxidative ring contraction of 2′-deoxyadenosine 5′-phosphates to generate an oxeae aldehyde during the biosynthesis of oxetanocin A. The preparation and in vitro characterization of such enzymes is complicated by the presence of two redox sensitive cofactors in addition to challenges in obtaining soluble protein for study. This chapter describes expression, purification and assay methodologies for GenK and OxsB highlighting the use denaturation/refolding protocols for solubilizing inclusion bodies as well as the use of cluster assembly and cobalamin uptake machinery during in vivo expression.

1. Introduction

Radical S-adenosyl-L-methionine (SAM) enzymes catalyze transformations involving radical intermediates. They are characterized by the presence of a low potential [Fe4S4]1+ cluster typically bound to the highly conserved CX3CX2C motif in the active site (Broderick, Duffus, Duschene, & Shepard, 2014; Frey, Hegeman, & Ruzicka, 2008; Sofia, Chen, Hetzler, Reyes-Spindola, & Miller, 2001). This cluster serves to induce reductive homolysis of the C5′–S bond of SAM (1). The resulting 5′-deoxyadenosyl radical (2) or its equivalent then abstracts a hydrogen atom from the substrate (3) to form 5′-deoxyadenosine (4), methionine (5), and a substrate radical (6), which can undergo a large number of possible transformations leading to a wide array of potential products depending on the constraints of the active site (see Fig. 1).

Fig. 1.

Fig. 1

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The initial steps of reactions catalyzed by radical SAM enzymes.

A number of radical SAM enzymes are also known to bind cobalamin (coenzyme B12) in their active sites and are referred to as B12-dependent radical SAM enzymes (Wang, 2018). Cobalamin (Cbl) is a cobalt-containing tetrapyrrole and can participate in both one- and two-electron processes since the cobalt center can accommodate oxidation states from 1 + to 3 +. While more than 9700 UniRef50 sequence clusters have been annotated as B12-dependent radical SAM enzymes (Oberg, Precord, Mitchell, & Gerlt, 2021), only a handful have been characterized (Allen & Wang, 2014a; Bridwell-Rabb, Zhong, Sun, Drennan, & Liu, 2017; Huang et al., 2015; Kim, Liu, McCarty, & Liu, 2017; Kim et al., 2013; Marous et al., 2015; Maruyama et al., 2020; Parent et al., 2016; Pierre et al., 2012; Radle, Miller, Laremore, & Booker, 2019; Sinner, Lichstrahl, Li, Marous, & Townsend, 2019; Wang, Schnell, Baumann, Müller, & Begley, 2017; Werner et al., 2011). Moreover, only three B12-dependent radical SAM enzymes, OxsB (Bridwell-Rabb et al., 2017), TsrM (Knox et al., 2021) and TokK (Knox, Sinner, Townsend, Boal, & Booker, 2021), have been structurally characterized to date.

The majority of B12-dependent radical SAM enzymes that have been characterized in vitro are limited to catalyzing methylation reactions with methylcobalamin (7) serving as the immediate methyl donor (McLaughlin & van der Donk, 2018; Sato, Kudo, Kim, Kuzuyama, & Eguchi, 2017; Werner et al., 2011). GenK represents one such example that catalyzes C6′ methylation of gentamicin X2 (9, GenX2) to G418 (11) during the biosynthesis of gentamicin C1 (13) (Kim et al., 2017, 2013; Unwin et al., 2004). GenK catalysis proceeds via abstraction of the C6′ pro-R H-atom of 9 by the 5′-deoxyadenosyl radical equivalent (2) to give the substrate radical intermediate 10 (Fig. 2). Transfer of a methyl radical from Cbl(III)-Me (7) generates G418 (11) and Cbl(II) (8). Two additional reducing equivalents are then required to both reduce the [Fe4S4]2+ cluster back to the [Fe4S4]1+ state and regenerate Me-Cbl(III) from Cbl(II) and an additional molecule of SAM yielding SAH (12) as a byproduct.

Fig. 2.

Fig. 2

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Reaction catalyzed by GenK.

OxsB is the only B12-dependent radical SAM enzyme characterized in vitro to catalyze an oxidative ring contraction, which takes place during the biosynthesis of oxetanocin-A (OXT-A, 18) (Bridwell-Rabb et al., 2017). Oxetanocin-A is a nucleoside analog having potent antiviral, anti-tumor, and antimicrobial activities due to its ability to inhibit DNA polymerases (Shimada et al., 1986; Ueda, Tsurimoto, Nagahata, Chisaka, & Matsubara, 1989). Its biosynthesis has been shown to be catalyzed by the cobalamin (Cbl)-dependent radical SAM enzyme OxsB (Bridwell-Rabb et al., 2017). The transformation catalyzed by OxsB involves a sequence of 14 → 15 → 16; however, the functional form of the cobalamin cofactor as well as its catalytic role remain unclear (Fig. 3).

Fig. 3.

Fig. 3

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Reaction catalyzed by OxsB/OxsA.

A major hurdle in the study of B12-dependent radical SAM enzymes is acquiring sufficient protein in soluble form. Manipulation of buffer salt concentration can be utilized to solubilize inclusion bodies and then refold the denatured proteins (Allen & Wang, 2014a, 2014b; Kim et al., 2013). Another approach is co-expression of the radical SAM enzyme with [Fe4S4] cluster biogenesis enzymes in order to facilitate in vivo reconstitution of the clusters (Anton et al., 2008; Chatterjee et al., 2008; Grove, Lee, St Clair, Krebs, & Booker, 2008; Hänzelmann et al., 2004; Kriek, Peters, Takahashi, & Roach, 2003; Wecksler et al., 2009). This can lead to improved solubility and two plasmid systems have been frequently utilized. These include pDB1282, which encodes the isc operon from Azotobacter vinelandii (Zheng, Cash, Flint, & Dean, 1998), and pRKSUF017, which encodes the suf operon from Escherichia coli (Takahashi & Tokumoto, 2002). Moreover, Lanz et al. have also reported that co-expression of B12-dependent radical SAM enzymes with the pBAD42-BtuCEDFB plasmid, which encodes the Btu system for cobalamin uptake in E. coli, can also improve the yield of soluble protein (Lanz et al., 2018).

The following sections provide methodologies currently in practice for the preparation and assay of GenK and OxsB. GenK typically expresses as inclusion bodies necessitating a refolding protocol in order to obtain soluble protein. In contrast, OxsB can be isolated as a soluble protein without the need for refolding. However, the preparation and purification of OxsB can be improved by codon-optimization and co-expression with [Fe4S4] cluster biogenesis enzymes. Therefore, this chapter provides details for each of these two different approaches to the in vitro study of B12-dependent radical SAM enzymes.

2. GenK catalyzes 6′-C methylation in gentamicin biosynthesis

2.1. Cloning, overexpression, purification and chemical reconstitution of GenK

Either plasmid pDB1282 or pRKSUF017 can be used for co-expression of [Fe4S4] biogenesis machinery (Takahashi & Tokumoto, 2002; Zheng et al., 1998), whereas BtuCEDFB-pBAD42 encodes proteins for the cobalamin uptake system (Lanz et al., 2018). In the absence of these plasmids, expression of GenK lacking an affinity tag is primarily in the form of inclusion bodies. While coexpression of native GenK with pDB1282 or pRKSUF017/BtuCEDFB-pBAD42 does appear to improve the yield of soluble enzyme it may also make purification of native protein more challenging. Consequently, this approach may be inferior to the expression of GenK alone followed by denaturation and refolding.

2.1.1. Cloning

The gene encoding GenK is polymerase chain reaction (PCR)-amplified from the genomic DNA of the gentamicin producing strain Micromonospora echinospora (NRRL 2953) using KOD polymerase (Novagen). The forward primer (5′-CAACATATGAACGCGCTGGTGGCAGC-3′) and reverse primer (5′-TAACGAATTCAGTGGGAAACCGCCTCGG-3′) for the PCR reaction are engineered with an NdeI and an EcoRI digestion site, respectively underlined. The resulting DNA fragment is then subcloned into a pCR:blunt vector (Invitrogen) followed by excision with NdeI and EcoRI (New England Biolabs, NEB) and ligated into pET-24b + (Novagen). The resulting plasmid is designated GenK:pET-24 and encodes a construct of native GenK that lacks an affinity tag.

2.1.2. Overexpression

GenK is susceptible to proteolytic degradation especially when expressed at low temperatures (e.g., 18 °C) where inclusion bodies are less stable (De Groot & Ventura, 2006). Yields appear to be improved, however, when expression is carried out at a more elevated temperature (e.g., 25C) with proteinase inhibitors included in the lysis buffer.

  1. Competent E. coli BL21 star (DE3) cells are transformed with the GenK:pET-24 plasmid and selected by plating on Luria-Bertani (LB) agar plates with 50 μg/mL kanamycin at 37 °C for 16 h

  2. A single colony is used to inoculate 10 mL LB broth containing 50 μg/mL kanamycin. This starter culture is incubated at 37 °C with 200 rpm shaking for 12 h

  3. The starter culture is added to 1 L LB broth containing 50 μg/mL kanamycin. The resulting culture is incubated at 37 °C with 200 rpm shaking until the optical density at 600 nm (OD600) reaches 0.6

  4. The culture is then cooled to 25 °C, and 40 mg ammonium iron(II) sulfate is added along with isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM

  5. The culture is incubated at 25 °C for another 20 h with 120 rpm shaking

  6. Cells are harvested by centrifugation at 4000 g and 4 °C for 12 min, and the cell pellet can be stored at − 80 °C

2.1.3. Purification

GenK is isolated in soluble form via a denaturation/refolding process. The entire purification process may take several days to complete and can be performed aerobically. The purity of isolated GenK is examined by SDS-PAGE. Refolded GenK can then be flash frozen and stored at − 80 °C. Prior to assay, GenK is thawed, and the [Fe4S4] clusters are reconstituted under anaerobic conditions. The following purification procedure is designed for a 3 L expression culture as described above. All steps are carried out at 4 °C under aerobic conditions.

Buffers.

The purification and reconstitution are carried out using the following buffer solutions. All buffers are chilled at 4 °C and β-mercaptoethanol is freshly added before use. Phenylmethylsulfonyl fluoride (PMSF) can be stored in isopropanol as a 100 mM stock solution and is freshly added before use.

Lysis buffer:

50 mM Tris·HCl (pH 8.0), 1 mM EDTA, 1 mM (PMSF), 1 mM β-mercaptoethanol and 10% (v/v) glycerol.

Denaturation buffer:

50 mM Tris·HCl (pH 8.0), 8 M urea, 1 mM β-mercaptoethanol and 10% (v/v) glycerol.

Refolding buffer:

50 mM Tris·HCl (pH 8.0), 1 mM MgSO4, 400 mM L-arginine, 5 mM glutathione (reduced), 0.5 mM glutathione (oxidized), 5 mM DTT and 10% (v/v) glycerol.

Reconstitution buffer:

50 mM Tris·HCl (pH 8.0), 0.15 M NaCl and 20% (v/v) glycerol.

  1. The cell pellet from the 3 L culture is suspended in 75 mL lysis buffer

  2. The suspended cell pellet is sonicated with a Fisher Scientific 550 Dismembrator at power 9 for 300 s total on-time with each 10 s burst followed by a 25 s pause

  3. The cell lysate is pelleted by centrifugation at 18000 g for 25 min at 4 °C

  4. The lysate pellet is suspended in 75 mL chilled lysis buffer, centrifuged at 18000 g for 25 min at 4 °C and the supernatant is discarded. This step is repeated at least twice

  5. The lysate pellet is suspended in 15 mL denaturation buffer using sonication for 40 s total on-time with each 5 s burst followed by a 20 s pause

  6. The suspension from step 5 is centrifuged at 18000 g for 25 min. The dark yellow supernatant (ca. 15 mL) can be either flash frozen with liquid N2 for storage at − 80 °C or immediately refolded

  7. The urea buffer containing denatured GenK from step 6 (10 mL) is combined with 200 mL refolding buffer slowly over 2 days. This is done by adding ca. 1 mL of the denatured GenK dropwise over 1 min to the refolding buffer approximately every 3 h

  8. The refolded GenK is concentrated to ca. 5 mg/mL protein, aliquoted, flash frozen and stored at − 80 °C or immediately reconstituted. The purity of GenK can be examined by SDS-PAGE (Fig. 4).

Fig. 4.

Fig. 4

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SDS-PAGE analysis of GenK (69 kDa). Lanes 1 and 8 are molecular weight markers; lanes 2–4 are whole cell lysate, pellet and supernatant; lanes 5–6 are pellet and supernatant in denaturation buffer and lane 7 is GenK from refolding buffer.

2.1.4. Reconstitution of the [Fe4S4]

Reconstitution is carried out in an anaerobic chamber with buffers that have been equilibrated with the anaerobic atmosphere.

  1. An aliquot of 2.85 mL containing ca. 5 mg refolded GenK (diluted with reconstitution buffer) is equilibrated with the atmosphere of the anaerobic chamber by stirring gently for 2.5 h in an uncapped vial at 4 °C

  2. To the reconstitution solution is added 30 μL of 1 M dithiothreitol (DTT), and the mixture is gently stirred for another 30 min

  3. A total of 60 μL 100 mM Fe(NH4)2(SO4)2 in water is added to the reconstitution mixture over 10 min and then stirred for another 10 min

  4. A total of 60 μL 100 mM Na2S in reconstitution buffer is added to the reconstitution mixture over 20 min and stirred for another 3 h. The resulting dark brown mixture contains ca. 70 μM GenK, 5 mM DTT, 2 mM iron and 2 mM sulfide in a volume of ca. 3 mL

  5. The reconstitution mixture is desalted using an Econo-Pac 10DG desalting column from Bio-Rad Laboratories pre-equilibrated with anaerobic reconstitution buffer. The enzyme elutes in olive-brown colored aliquots, which are combined and concentrated by centrifugal filtration to yield ca. 70 μM GenK monomer

2.2. In vitro functional characterization

GenK activity can be assessed using the GenX2 substrate (9). Under anaerobic conditions, a 50 μL solution containing 5 μM reconstituted GenK, 10 mM DTT, 1 mM methyl viologen, 4 mM NADPH, 4 mM SAM (1) and 1 mM methylcobalamin (Me-Cbl, 7) is prepared in 50 mM Tris·HCl buffer (pH 8.0). The reaction is initiated with the addition of 1 mM GenX2 followed by incubation at room temperature over 20 h and halted by filtration to remove the protein. The deproteinized solution can then be analyzed by electrospray ionization mass spectrometry (ESI-MS). Under positive ion mode, the [M + H]+ ions of substrate GenX2 (9) and product G418 (11) are detected at m/z 483.27 and 497.28, respectively.

Reduction and demethylation of SAM can also be followed by HPLC without the need for mass spectrometry. Thus, a 27 μL assay solution reaction can be quenched by adding 3 μL 30% (v/v) aqueous trichloroacetic acid and centrifuging to remove the precipitated protein. The resulting supernatant can then be analyzed by HPLC using UV absorbance detection at 260 nm to follow the formation and consumption of the adenine nucleosides SAM (1), 5′-deoxyadenosine (4) and SAH (12). Chromatographic separation is achieved with a Varian Microsorb-MV 100–5 C18 (4.6 mm × 250 mm) column. A two-solvent system is used with 0.1% trifluoroacetic acid (TFA) in ddH2O as mobile phase A and 0.1% TFA in acetonitrile as mobile phase B. The column is eluted at a flow rate of 1 mL/min with a linear gradient from 0% to 20% B over 30 min.

Despite the lack of a chromophore in GenX2 (9) and G418 (11), these aminoglycosides can also be detected by UV–vis absorption following derivatization with 1-fluoro-2,4-dinitrobenzene (19, DNFB) (Ryan, 1984). Mass spectrometry demonstrates complete modification of all three primary amino groups in 9 or 11 with the 2,4-dinitrophenyl (DNP) moiety (Fig. 5). Thus, a 30 μL aliquot of the GenK reaction solution described above is combined with 120 μL of a mixture prepared from 112 μL MeOH, 1.8 μL 8.4 M DNFB and 6 μL of 0.5 M sodium hydroxide. The resulting solution is heated at 80 °C for 5 min and then centrifuged at 15000 g for 5 min to pellet any particulates and protein precipitate. The supernatant can be analyzed by HPLC with UV–vis absorbance detection at 340 nm using a Varian Microsorb-MV 100–5 C18 (4.6 mm × 250 mm) column. The column is eluted with ddH2O as mobile phase A and acetonitrile as mobile phase B at a flow rate of 1 mL/min and a linear gradient from 40% to 50% B over 20 min.

Fig. 5.

Fig. 5

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DNFB derivatization of substrate GenX2 (9) and product G418 (11). The derivatization installs three DNP moieties and yields 9-DNP3 and 11-DNP3.

3. OxsB and OxsA catalyzes ring contraction in oxetanocin A biosynthesis

The oxetanocin A (OXT-A, 18) biosynthetic gene cluster along with potential resistance determinants is plasmid borne (pOXT1) in the producing strain Bacillus megaterium strain NK84-0128 and encoded on a single 6.8 kb fragment (BglII-D) obtained via the enzymatic digestion of pOXT1 with the restriction enzyme BglII (Morita et al., 1999). Only two genes encoded by this cluster are strictly required for OXT-A biosynthesis, namely oxsA and oxsB (Bridwell-Rabb et al., 2017; Zhong, Lee, Liu, & Liu, 2021). OxsB is the B12-dependent radical SAM enzyme directly responsible for the oxidative ring contraction 14 → 16. OxsA is an HD (His-Asp) domain phosphohydrolase capable of dephosphorylating oxetanocin A 5′-triphosphate (17) to OXT-A (Bridwell-Rabb, Kang, Zhong, Liu, & Drennan, 2016). However, in vivo and in vitro assays have shown that the presence of OxsA is required for OxsB activity suggesting a direct interaction between the two proteins (Bridwell-Rabb et al., 2017; Zhong et al., 2021).

3.1. Cloning, overexpression, purification and chemical reconstitution of OxsB

3.1.1. Cloning

The oxsB gene in the original studies (Bridwell-Rabb et al., 2017) was synthesized by the Mr. Gene service according to the published primary sequence of oxsB (NCBI, accession No. AB005787) following codon optimization. Once obtained, the oxsB gene can be PCR-amplified and subcloned into the NdeI and HindIII sites of pET-28b. This modifies oxsB to encode the N-His6 tagged OxsB protein, and the resulting plasmid is designated OxsB:pET-28b.

An expression construct can also be generated for the isolation of untagged OxsB. This is accomplished by subcloning the PCR-amplified oxsB gene fragment into a MalE-pET vector at the NdeI and HindIII cut sites. This plasmid is designated as OxsB:MalE-pET. The MalE-pET vector allows for expression of OxsB as a fusion protein with C-His10 tagged maltose binding protein (MBP) linked to the N-terminus of OxsB via a 7-residue (ENLYFQG) linker that can be cleaved by the tobacco etch virus (TEV) protease (Kapust et al., 2001).

3.1.2. Overexpression

Two E. coli strains have been investigated for the expression of both OxsB constructs. The first is E. coli BL21 star (DE3), which harbors the pDB1282 plasmid for in vivo reconstitution of [Fe4S4] clusters (Zheng et al., 1998). The second is E. coli BL21 star (DE3) containing both the pDB1282 and pBAD42-BtuCEDFB plasmids to allow for reconstitution of both the [Fe4S4] cluster as well as the cobalamin cofactor (Lanz et al., 2018).

Overexpression from E. coli BL21 star (DE3)/pDB1282.

This protocol is applicable to both the OxsB:pET-28b and OxsB:MalE-pET plasmids. The culture broth will assume a pale pink color upon addition of cyanocobalamin; however, the harvested cell pellet generally does not have a pink color.

  1. Competent E. coli BL21 star (DE3) cells are sequentially transformed with pDB1282, which confers ampicillin resistance, and the OxsB containing plasmid, which confers kanamycin resistance. The transformants containing both plasmids are selected by plating on LB agar containing 50 μg/mL kanamycin and 100 μg/mL ampicillin at 37 °C for 16 h

  2. A single colony is selected and used to inoculate 10 mL LB broth containing 50 μg/mL kanamycin and 100 μg/mL ampicillin. The resulting starter culture is incubated at 37 °C with 200 rpm shaking for 12 h

  3. The 10 mL starter culture is then used to inoculate 1 L LB broth containing 30 μg/mL kanamycin, 50 μg/mL ampicillin, 0.1% (v/v) ethanolamine and 3 mg/L cyanocobalamin (CN-Cbl). The inoculated culture is incubated at 37 °C with 200 rpm shaking until the OD600 reaches 0.2

  4. Once the OD600 reaches 0.2, expression of the pDB1282 plasmid is induced by the addition of 0.5 g L-arabinose along with 40 mg Fe(NH4)2(SO4)2 and 20 mg L-cysteine. Incubation is continued at 37 °C and 200 rpm shaking

  5. Once the OD600 reaches 0.6, the culture is chilled on ice for 10 min, and overexpression of the OxsB construct is induced by adding IPTG to a final concentration of 0.1 mM. The resulting culture is further incubated at 18 °C for 20 h with 150 rpm shaking

  6. The cell pellet is then harvested by centrifugation at 4000 g and 4 °C for 12 min and can be immediately purified or stored at − 80 °C

Overexpression from BL21 star (DE3)/pDB1282/pBAD42-BtuCEDFB.

This protocol is applicable to both the OxsB:pET-28b and OxsB:MalE-pET plasmids. The procedure is the same as that from E. coli BL21 star (DE3)/pDB1282; however, 100 μg/mL ampicillin, 50 μg/mL kanamycin and 50 μg/mL streptomycin are added to the selection agar, starter culture and expression culture following co-transformation of the E. coli BL21 star (DE3) cells with the pDB1282, pBAD42-BtuCEDFB and OxsB containing plasmids. In contrast to coexpression with the pDB1282 plasmid alone, the harvested cell pellet is also pink in color consistent with coexpression of the cobalamin uptake system.

3.1.3. Purification

Purifications are carried out using the following buffers, which are prepared with fresh β-mercaptoethanol. All procedures are carried out aerobically at 4 °C.

Lysis buffer:

50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES, pH 8.0), 300 mM NaCl, 10 mM imidazole, 2 mM β-mercaptoethanol and 10% (v/v) glycerol.

Wash buffer:

50 mM HEPES (pH 8.0), 500 mM NaCl, 25 mM imidazole, 2 mM β-mercaptoethanol and 10% (v/v) glycerol.

Elution buffer:

50 mM HEPES (pH 8.0), 300 mM NaCl, 200 mM imidazole, 2 mM β-mercaptoethanol and 10% (v/v) glycerol.

Dialysis buffer:

50 mM HEPES (pH 8.0), 100 mM NaCl, 2 mM β-mercaptoethanol and 10% (v/v) glycerol.

Reconstitution buffer:

25 mM HEPES (pH 7.5).

Purification of N-His6-tagged OxsB from plasmid OxsB:pET-28b.

The quantities are quoted for a 6 L culture. The typical yield is 120–200 mg protein per 6 L culture.

  • 1

    The harvested cell pellet is suspended in 180 mL lysis buffer on ice

  • 2

    The suspended cell pellet is sonicated for 180 s total on-time with each 10 s burst followed by a 30 s pause

  • 3

    The cell debris is pelleted by centrifugation at 18000 g and 4 °C for 25 min. The supernatant is then decanted onto a chilled column prepacked with 10 mL Ni-NTA resin pre-equilibrated with lysis buffer. The initial flow through is poured back through the column at least two times

  • 4

    The column is washed with wash buffer until Bradford assay shows no further elution of non-specific binding proteins (ca. 500 mL).

  • 5

    N-His6-tagged OxsB is then eluted with the elution buffer, and the pooled fractions are dialyzed (12–14 kDa MWCO) three times against 1 L dialysis buffer for 1 h each at 4 °C. The dialyzed protein is then concentrated to ca. 0.1 mM, at which point it can be flash frozen and stored at − 80 °C. The final protein solution from overexpression in the presence of pBAD42-BtuCEDFB is typically clear and bright pink in color. The overall cobalamin occupancy is estimated to be ca. 32% (Zhong et al., 2021).

Purification of untagged OxsB from plasmid OxsB:MalE-pET.

Steps 1–5 are the same as in the protocol for the purification of N-His6-tagged OxsB. The typical yield is 120–200 mg protein per 6 L culture. A His6-tagged TEV protease with the S219V point mutation is used in this experiment which has been reported to show improved efficiency in this methodology (Kapust et al., 2001).

  • 6

    Approximately 1 mg TEV protease per 10 mg MBP-OxsB is added to the OxsB preparation, and the mixture is incubated at 4 °C for 12 h. If a significant level of MBP-tagged OxsB (131 kDa) remains, additional TEV protease can be added and the incubation extended for another 12 h

  • 7

    The MBP tag (44 kDa) and TEV protease (29 kDa) are then bound by adding 10 mL Ni-NTA resin. The mixture is then gently agitated at 4 °C for 1 h

  • 8

    The slurry is then poured into an empty column, and the flow through is poured back to the column at least three times to maximize binding between Ni-NTA and the tagged impurities

  • 9

    After the flow-through is collected, the Ni-NTA column is washed with another 10 mL dialysis buffer to elute any bound OxsB. The purity of OxsB (87 kDa) can be checked by SDS-PAGE (Fig. 6).

  • 10

    The purified protein is concentrated to ca. 0.1 mM OxsB monomer and can be aliquoted, flash frozen and stored at − 80 °C or immediately reconstituted

Fig. 6.

Fig. 6

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SDS-PAGE analysis of MBP-OxsB (131 kDa), OxsB (87 kDa), MBP (44 kDa) and TEV (29 kDa). Lane 1 is molecular weight marker; lanes 2–4 are whole cell lysate, pellet and supernatant; lane 5 is the flow through; lane 6 is after incubation with TEV for 24 h; lanes 7–10 are sequential flow through showing increasing purity of the untagged OxsB.

3.1.4. Reconstitution of OxsB

Iron sulfur reconstitution of OxsB follows essentially the same procedure as that for GenK (see Section 2.1.4).

3.1.5. Cobalamin reconstitution of OxsB

This step is unnecessary if cobalamin is to be added to the assays. If performed, however, it must follow iron-sulfur reconstitution.

  1. To a 1.2 mL solution of 30 μM [Fe4S4]-reconstituted OxsB is added 36 μL of 10 mM hydroxocobalamin (HO-Cbl) prepared in reconstitution buffer

  2. The mixture is stirred at 4 °C ice for at least 3 h. Although HO-Cbl is not as light sensitive as the organocobalamin cofactors (i.e., Ad-Cbl & Me-Cbl), the mixture is nevertheless covered with foil to minimize exposure to ambient light

  3. Centrifugal filtration (MWCO 10 kDa) is used to remove the unbound cobalamin via buffer exchange

  4. The reconstituted enzyme can be stored anaerobically in an amber vial at ca. 10 °C

3.2. Cloning, overexpression and purification of OxsA

3.2.1. Cloning

The OxsA expression construct in the original studies was synthesized according to the published primary sequence using the Mr. Gene service after codon optimization (Bridwell-Rabb et al., 2016). The oxsA gene can then be PCR-amplified and subcloned into the NdeI and HindIII sites of the pET-28b and pET-24 plasmids, which are designated OxsA:pET-28b and OxsA:pET-24, respectively. The former construct encodes the OxsA protein with an N-His6 affinity tag. The latter construct yields the OxsA protein in its native form with no modifications.

3.2.2. Overexpression

Overexpression of OxsA from E. coli BL21 star (DE3).

The following protocol is applicable to both the OxsA:pET-24 and OxsA:pET-28b plasmids.

  1. Competent E. coli BL21 star (DE3) cells are transformed with the relevant OxsA plasmid. The correct transformant can be selected after plating on LB agar containing 50 μg/mL kanamycin at 37 °C for 16 h

  2. A single colony is selected to inoculate 10 mL LB broth containing 50 μg/mL kanamycin. The starter culture is incubated at 37 °C with 200 rpm shaking for 12 h

  3. The starter culture is used to inoculate 1 L LB broth containing 30 μg/mL kanamycin. The resulting culture is incubated at 37 °C with 200 rpm shaking until the OD600 reaches 0.6

  4. The turbid culture is chilled on ice for 10 min, and OxsA overexpression is induced by the addition of IPTG to a final concentration of 0.1 mM

  5. The culture is incubated at 18 °C for another 20 h with 150 rpm shaking

  6. The cell pellet is harvested at 4000 g and 4 °C for 12 min and can be immediately purified or stored at − 80 °C

3.2.3. Purification

Purification of N-His6 tagged OxsA follows the standard procedure for His6-tagged proteins. Due to the high yield of OxsA expression, ammonium sulfate precipitation of the untagged OxsA followed by anion exchange chromatography yields OxsA as the major component on SDS-PAGE analysis. However, OxsB catalysis is sluggish under the assay conditions currently identified such that trace enzyme contaminants can lead to side reactions when the OxsB/OxsA assay is conducted over long time intervals (Zhong et al., 2021). Therefore, size-exclusion chromatography can be used to further purify the untagged OxsA.

Purification of N-His6-tagged OxsA (from OxsA:pET-28b).

The purification of N-His6-tagged OxsA follows the same procedure as that described for N-His6-tagged OxsB (Section 3.2.3). The typical yield of N-His6-tagged OxsA from a 2 L culture is 10 mg.

Purification of untagged OxsA (from OxsA:pET-24).

The purification of untagged OxsA uses Tris·HCl buffers during ammonium sulfate precipitation and (diethylaminoethyl-cellulose) DEAE column chromatography. The purified protein is then dialyzed against HEPES buffer for storage.

Lysis buffer:

50 mM Tris·HCl (pH 8.0), 300 mM NaCl and 10% (v/v) glycerol.

Dialysis buffer:

50 mM Tris·HCl (pH 8.0) and 10% (v/v) glycerol.

Elution buffer:

50 mM Tris·HCl (pH 8.0), 1 M NaCl and 10% (v/v) glycerol.

Storage buffer:

50 mM HEPES (pH 8.0), 100 mM NaCl and 10% (v/v) glycerol.

  1. The OxsA cell pellet from a 1 L culture is suspended in 35 mL lysis buffer on ice

  2. The suspended cell pellet is sonicated for 100 s total on-time with each 10 s burst followed by a 30 s pause

  3. The cell debris is pelleted by centrifugation at 18000 g and 4 °C for 25 min

  4. The isolated supernatant is stirred slowly at 4 °C, and solid ammonium sulfate (47.2 g/100 mL supernatant) is added over 4 h. The solution will become opaque

  5. The precipitated OxsA solution is centrifuged at 18000 g and 4 °C for 25 min. The precipitated OxsA pellet is then dissolved in a minimum volume (ca. 2 mL) of the dialysis buffer and dialyzed (3.5 kDa MWCO membrane) against 1 L dialysis buffer three times over 3 h

  6. A column packed with 250 mL DEAE cellulose is pre-equilibrated with dialysis buffer

  7. A two-chamber gradient mixer is prepared with 300 mL dialysis buffer in the first chamber and 300 mL elution buffer in the second. The dialyzed OxsA isolate is applied to the DEAE column. The first chamber is then connected to the column and 50 mL of dialysis buffer is run through the column before opening the second chamber to begin the gradient elution

  8. Eluted fractions containing OxsA by SDS-PAGE are pooled and concentrated to 30 mL before dialysis

  9. The concentrated OxsA is dialyzed for 1 h against 1 L storage buffer three times

  10. The resulting OxsA can be aliquoted, flash frozen and stored at − 80 °C

  11. If further purification is necessary, FPLC size exclusion chromatography using a Superdex 75 10/300 GL column (GE healthcare) can be used. A 0.8 mL sample containing around 150 μM OxsA is eluted isocratically with storage buffer at a flow rate of 0.5 mL/min

3.3. In vitro assay

Activity of reconstituted OxsB can be assayed anaerobically using 2′-dAMP, 2′-dADP or 2′-dATP as the substrate (Bridwell-Rabb et al., 2017). A 50 μL solution containing 1 mM substrate, 1 mM SAM, 1 mM DTT, 10 mM MgCl2, 0.2 mM HO-Cbl, 1 mM NADPH, 0.25 mM methyl viologen is prepared in 25 mM HEPES buffer (pH 7.5). The reaction is initiated by adding OxsA and reconstituted OxsB each to a final concentration of 20 μM, and the reaction is allowed to incubate for 12 h. The reaction is then deproteinized by centrifugal filtration and can be analyzed by HPLC using a Dionex column (CarboPac PA1, Thermo Scientific, 250 mm × 4 mm). The elution is monitored by UV absorbance detection at 260 nm. Separation can be achieved by running ddH2O as mobile phase A and 1.5 M NH4OAc as mobile phase B at a flow rate of 1 mL/min with the following linear gradient: 0–5 min 10% B, 5–34 min 10–30% B, 34–35 min 30% B, 35–37 min 30–10% B, 37–41 min 10% B.

Improved separation of the reaction products using reversed phase HPLC can be obtained following dephosphorylation and reduction of the product aldehyde using NaBH4 to yield OXT-A (18). A 50 μL aliquot of the reaction mixture is incubated with 5 μL CutSmart buffer (NEB) and 0.5 μL calf intestinal phosphatase (NEB) for 30 min at 37 °C before adding 55 μL acetonitrile and centrifuging to remove the precipitates. To 90 μL of the supernatant is then added 9 μL of 100 mM aqueous NaBH4 prepared fresh. The reaction is incubated at room temperature for 30 min before adding 1 μL glacial acetic acid. The quenched reaction is then evaporated and dissolved with ddH2O before HPLC analysis using a C18 column (Agilent Microsorb-MV 100–5 C18 250 × 4.6 mm) with UV absorbance at 260 nm. Separation can be achieved by running 1% (w/v) NH4OAc as mobile phase A and acetonitrile as mobile phase B at a flow rate of 1 mL/min with the following linear gradient: 0–5 min 2% B, 5–23 min 2–10% B, 23–32 min 10–35% B, 32–35 min 35% B, 35–37 min 35–2% B, 37–47 min 2% B. OXT-A (18) elutes at ca. 18 min.

4. Conclusion

As more and more radical SAM enzymes are discovered and investigated, significant new insights are made into the chemistry of these metalloenzymes and the roles played by radicals in enzymological systems. At the same time, however, just as many new questions and unusual mechanisms of catalysis are becoming apparent and in need of further study. This is especially true of the B12-dependent radical SAM subgroup, which often poses challenges with respect to both obtaining soluble protein for study and the identification of suitable assay conditions for in vitro characterization. While much work is left to be done, progress is nevertheless being made. The methodologies provided in this chapter thus afford a starting point for the enzymologist interested in the study of these complex enzyme systems.

Acknowledgment

We are grateful to all coworkers who have made contributions to our studies of GenK and OxsB over the years. We would also like to thank Dr. Mark Ruszczycky for his valuable comments of this paper. The plasmid pDB1282 was a generous gift from Professor Dennis Dean at the Virginia Polytechnic Institute and State University. The plasmid pBAD42-BtuCEDFB was a kind gift from Professor Squire Booker at Pennsylvania State University. This work is supported by grants from the National Institutes of Health (GM035906 and GM040541) and the Welch Foundation (F-1511).

References

  1. Allen KD, & Wang SC (2014a). Initial characterization of Fom3 from Streptomyces wedmorensis: The methyltransferase in fosfomycin biosynthesis. Archives of Biochemistry and Biophysics, 543, 67–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allen KD, & Wang SC (2014b). Spectroscopic characterization and mechanistic investigation of P-methyl transfer by a radical SAM enzyme from the marine bacterium Shewanella denitrificans OS217. Biochimica et Biophysica Acta, 1844(12), 2135–2144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anton BP, Saleh L, Benner JS, Raleigh EA, Kasif S, & Roberts RJ (2008). RimO, a MiaB-like enzyme, methylthiolates the universally conserved Asp88 residue of ribosomal protein S12 in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 105(6), 1826–1831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bridwell-Rabb J, Kang G, Zhong A, Liu H. w., & Drennan CL (2016). An HD domain phosphohydrolase active site tailored for oxetanocin-A biosynthesis. Proceedings of the National Academy of Sciences of the United States of America, 113(48), 13750–13755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bridwell-Rabb J, Zhong A, Sun HG, Drennan CL, & Liu H. w. (2017). A B12-dependent radical SAM enzyme involved in oxetanocin A biosynthesis. Nature, 544(7650), 322–326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Broderick JB, Duffus BR, Duschene KS, & Shepard EM (2014). Radical S-adenosylmethionine enzymes. Chemical Reviews, 114(8), 4229–4317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chatterjee A, Li Y, Zhang Y, Grove TL, Lee M, & Krebs C, et al. (2008). Reconstitution of ThiC in thiamine pyrimidine biosynthesis expands the radical SAM superfamily. Nature Chemical Biology, 4(12), 758–765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. De Groot NS, & Ventura S (2006). Effect of temperature on protein quality in bacterial inclusion bodies. FEBS Letters, 580(27), 6471–6476. [DOI] [PubMed] [Google Scholar]
  9. Frey PA, Hegeman AD, & Ruzicka FJ (2008). The radical SAM superfamily. Critical Reviews in Biochemistry and Molecular Biology, 43(1), 63–88. [DOI] [PubMed] [Google Scholar]
  10. Grove TL, Lee KH, St Clair J, Krebs C, & Booker SJ (2008). In vitro characterization of AtsB, a radical SAM formylglycine-generating enzyme that contains three [4Fe-4S] clusters. Biochemistry, 47(28), 7523–7538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hänzelmann P, Hernández HL, Menzel C, García-Serres R, Huynh BH, & Johnson MK, et al. (2004). Characterization of MOCS1A, an oxygen-sensitive iron-sulfur protein involved in human molybdenum cofactor biosynthesis. The Journal of Biological Chemistry, 279(33), 34721–34732. [DOI] [PubMed] [Google Scholar]
  12. Huang C, Huang F, Moison E, Guo J, Jian X, & Duan X, et al. (2015). Delineating the biosynthesis of gentamicin x2, the common precursor of the gentamicin C antibiotic complex. Chemistry & Biology, 22(2), 251–261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kapust RB, Tözsér J, Fox JD, Anderson DE, Cherry S, & Copeland TD, et al. (2001). Tobacco etch virus protease: Mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Engineering, 14(12), 993–1000. [DOI] [PubMed] [Google Scholar]
  14. Kim HJ, Liu YN, McCarty RM, & Liu H. w. (2017). Reaction catalyzed by GenK, a cobalamin-dependent radical S-adenosyl-L-methionine methyltransferase in the biosynthetic pathway of gentamicin, proceeds with retention of configuration. Journal of the American Chemical Society, 139(45), 16084–16087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kim HJ, McCarty RM, Ogasawara Y, Liu YN, Mansoorabadi SO, & LeVieux J, et al. (2013). GenK-catalyzed C-6′ methylation in the biosynthesis of gentamicin: Isolation and characterization of a cobalamin-dependent radical SAM enzyme. Journal of the American Chemical Society, 135(22), 8093–8096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Knox HL, Chen PY, Blaszczyk AJ, Mukherjee A, Grove TL, & Schwalm EL, et al. (2021). Structural basis for non-radical catalysis by TsrM, a radical SAM methylase. Nature Chemical Biology, 17(4), 485–491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Knox HL, Sinner EK, Townsend CA, Boal AK, & Booker SJ (2021). Structural basis for carbapenam C-alkylation by TokK, a B12-dependent radical SAM enzyme. BioRxiv. doi: 10.1101/2021.07.10.451909.2021.07.10.451909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kriek M, Peters L, Takahashi Y, & Roach PL (2003). Effect of iron-sulfur cluster assembly proteins on the expression of Escherichia coli lipoic acid synthase. Protein Expression and Purification, 28(2), 241–245. [DOI] [PubMed] [Google Scholar]
  19. Lanz ND, Blaszczyk AJ, McCarthy EL, Wang B, Wang RX, & Jones BS, et al. (2018). Enhanced solubilization of class B radical S-adenosylmethionine methylases by improved cobalamin uptake in Escherichia coli. Biochemistry, 57(9), 1475–1490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Marous DR, Lloyd EP, Buller AR, Moshos KA, Grove TL, & Blaszczyk AJ, et al. (2015). Consecutive radical S-adenosylmethionine methylations form the ethyl side chain in thienamycin biosynthesis. Proceedings of the National Academy of Sciences of the United States of America, 112(33), 10354–10358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maruyama C, Chinone Y, Sato S, Kudo F, Ohsawa K, & Kubota J, et al. (2020). C-methylation of S-adenosyl-L-methionine occurs prior to cyclopropanation in the biosynthesis of 1-amino-2-methylcyclopropanecarboxylic acid (Norcoronamic acid) in a bacterium. Biomolecules, 10(5), 775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McLaughlin MI, & van der Donk WA (2018). Stereospecific radical-mediated B12-dependent methyl transfer by the fosfomycin biosynthesis enzyme Fom3. Biochemistry, 57(33), 4967–4971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Morita M, Tomita K, Ishizawa M, Takagi K, Kawamura F, & Takahashi H, et al. (1999). Cloning of oxetanocin A biosynthetic and resistance genes that reside on a plasmid of Bacillus megaterium strain NK84–0128. Bioscience, Biotechnology, and Biochemistry, 63(3), 563–566. [DOI] [PubMed] [Google Scholar]
  24. Oberg N, Precord TW, Mitchell DA, & Gerlt JA (2021). RadicalSAM.org: A resource to interpret sequence-function space and discover new radical SAM enzyme chemistry. ACS Bio & Med Chem Au. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Parent A, Guillot A, Benjdia A, Chartier G, Leprince J, & Berteau O (2016). The B12-rdical SAM enzyme PoyC catalyzes valine Cβ-methylation during polytheonamide biosynthesis. Journal of the American Chemical Society, 138(48), 15515–15518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pierre S, Guillot A, Benjdia A, Sandström C, Langella P, & Berteau O (2012). Thiostrepton tryptophan methyltransferase expands the chemistry of radical SAM enzymes. Nature Chemical Biology, 8(12), 957–959. [DOI] [PubMed] [Google Scholar]
  27. Radle MI, Miller DV, Laremore TN, & Booker SJ (2019). Methanogenesis marker protein 10 (Mmp10) from Methanosarcina acetivorans is a radical S-adenosylmethionine methylase that unexpectedly requires cobalamin. The Journal of Biological Chemistry, 294(31), 11712–11725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ryan JA (1984). Colorimetric determination of gentamicin, kanamycin, tobramycin, and amikacin aminoglycosides with 2,4-dinitrofluorobenzene. Journal of Pharmaceutical Sciences, 73(9), 1301–1302. [DOI] [PubMed] [Google Scholar]
  29. Sato S, Kudo F, Kim SY, Kuzuyama T, & Eguchi T (2017). Methylcobalamin-dependent radical SAM C-methyltransferase Fom3 recognizes cytidylyl-2-hydroxyethylphosphonate and catalyzes the nonstereoselective C-methylation in fosfomycin biosynthesis. Biochemistry, 56(28), 3519–3522. [DOI] [PubMed] [Google Scholar]
  30. Shimada N, Hasegawa S, Harada T, Tomisawa T, Fujii A, & Takita T (1986). Oxetanocin, a novel nucleoside from bacteria. The Journal of Antibiotics, 39(11), 1623–1625. [DOI] [PubMed] [Google Scholar]
  31. Sinner EK, Lichstrahl MS, Li R, Marous DR, & Townsend CA (2019). Methylations in complex carbapenem biosynthesis are catalyzed by a single cobalamin-dependent radical S-adenosylmethionine enzyme. Chemical Communications (Cambridge, England), 55(99), 14934–14937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sofia HJ, Chen G, Hetzler BG, Reyes-Spindola JF, & Miller NE (2001). Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: Functional characterization using new analysis and information visualization methods. Nucleic Acids Research, 29(5), 1097–1106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Takahashi Y, & Tokumoto U (2002). A third bacterial system for the assembly of iron-sulfur clusters with homologs in archaea and plastids. The Journal of Biological Chemistry, 277(32), 28380–28383. [DOI] [PubMed] [Google Scholar]
  34. Ueda K, Tsurimoto T, Nagahata T, Chisaka O, & Matsubara K (1989). An in vitro system for screening anti-hepatitis B virus drugs. Virology, 169(1), 213–216. [DOI] [PubMed] [Google Scholar]
  35. Unwin J, Standage S, Alexander D, Hosted T Jr., Horan AC, & Wellington EM (2004). Gene cluster in Micromonospora echinospora ATCC15835 for the biosynthesis of the gentamicin C complex. The Journal of Antibiotics, 57(7), 436–445. [DOI] [PubMed] [Google Scholar]
  36. Wang SC (2018). Cobalamin-dependent radical S-adenosyl-L-methionine enzymes in natural product biosynthesis. Natural Product Reports, 35(8), 707–720. [DOI] [PubMed] [Google Scholar]
  37. Wang Y, Schnell B, Baumann S, Müller R, & Begley TP (2017). Biosynthesis of branched alkoxy groups: Iterative methyl group alkylation by a cobalamin-dependent radical SAM enzyme. Journal of the American Chemical Society, 139(5), 1742–1745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wecksler SR, Stoll S, Tran H, Magnusson OT, Wu SP, & King D, et al. (2009). Pyrroloquinoline quinone biogenesis: Demonstration that PqqE from Klebsiella pneumoniae is a radical S-adenosyl-L-methionine enzyme. Biochemistry, 48(42), 10151–10161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Werner WJ, Allen KD, Hu K, Helms GL, Chen BS, & Wang SC (2011). In vitro phosphinate methylation by PhpK from Kitasatospora phosalacinea. Biochemistry, 50(42), 8986–8988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zheng L, Cash VL, Flint DH, & Dean DR (1998). Assembly of iron-sulfur clusters. Identification of an iscSUA-hscBA-fdx gene cluster from Azotobacter vinelandii. The Journal of Biological Chemistry, 273(21), 13264–13272. [DOI] [PubMed] [Google Scholar]
  41. Zhong A, Lee YH, Liu YN, & Liu H. w. (2021). Biosynthesis of oxetanocin-a includes a B12-dependent radical SAM enzyme that can catalyze both oxidative ring contraction and the demethylation of SAM. Biochemistry, 60(7), 537–546. [DOI] [PMC free article] [PubMed] [Google Scholar]

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