Development of an in vitro method for activation of X-succinate synthases for fumarate hydroalkylation

Summary Anaerobic microbial degradation of hydrocarbons is often initiated through addition of the hydrocarbon to fumarate by enzymes known as X-succinate synthases (XSSs). XSSs use a glycyl radical cofactor, which is installed by an activating enzyme (XSS-AE), to catalyze this carbon-carbon coupling reaction. The activation step, although crucial for catalysis, has not previously been possible in vitro because of insolubility of XSS-AEs. Here, we take a genome mining approach to find an XSS-AE, a 4-isopropylbenzylsuccinate synthase (IBSS)-AE (IbsAE) that can be solubly expressed in Escherichia coli. This soluble XSS-AE can activate both IBSS and the well-studied benzylsuccinate synthase (BSS) in vitro, allowing us to explore XSSs biochemically. To start, we examine the role of BSS subunits and find that the beta subunit accelerates the rate of hydrocarbon addition. Looking forward, the methodology and insight gathered here can be used more broadly to understand and engineer XSSs as synthetically useful biocatalysts.


INTRODUCTION
Hydrocarbons are abundant within both natural (e.g., marine hydrocarbon seeps) and artificial (e.g., oil pipelines) environments. Aerobic microbial degradation of hydrocarbons has been well characterized and used in bioremediation of crude-oil-polluted environments; however, hydrocarbons inevitably end up in marine and terrestrial anoxic environments as well. 1,2 Even within oxic zones, intensive respiration of facultative microbes creates anoxic microenvironments. Further understanding of how hydrocarbons are anaerobically degraded by microbes is necessary to create better tools for bioremediation and to inhibit microbial corrosion 3 within crude-oil-containing facilities. Characterization of microbial communities within these anaerobic environments remains an important, active area of research, 2 and as more anaerobic degraders are discovered, it is also critical that we understand the underlying molecular mechanisms that allow these microbes to accomplish hydrocarbon degradation in the absence of molecular oxygen. 1 One of the key reactions catalyzed by these organisms involves metabolism of hydrocarbon substrates via addition to fumarate. This hydroalkylation reaction proceeds via homolytic cleavage of a C-H bond on the hydrocarbon substrate, addition of the hydrocarbyl radical to the C=C bond of fumarate, and addition of hydrogen to the resulting succinyl radical. Beyond environmental significance, this type of reaction is a synthetically attractive method for forming C-C bonds, as it has the potential to rapidly build structural complexity within small molecules without byproducts or pre-functionalized substrates. The growing class of enzymes that catalyze this impressive reaction is known as the X-succinate synthases (XSSs).
In oxic environments, hydrocarbons are activated by enzymes that use iron, copper, and flavin cofactors to oxygenate using O 2 . 4 When molecular oxygen is not present, other cofactors must be used to initiate radical chemistry for hydrocarbon activation. The XSS enzymes use a simple glycyl radical cofactor to initiate radical-based catalysis, which makes them members of the large glycyl radical enzyme (GRE) superfamily. 5 Members of the GRE superfamily are structurally comprised of a 10-stranded b/a barrel with Gly and Cys loops located within the barrel ( Figure 1A). The only structurally characterized XSS to date is benzylsuccinate synthase (BSS), which catalyzes the formation of benzylsuccinate from fumarate and toluene. 6,7 Once a radical is formed on the Gly residue within the Gly loop of BSS, it can form a transient thiyl radical on Figure 1. Overview of BSS mechanism X-ray crystallography of benzylsuccinate synthase (BSS) has provided insight into the molecular mechanisms involved in fumarate addition to toluene. 6,7 (A) Snapshots of the active site of BSS (PDB: 5BWE) 7 show how substrates are positioned for radical catalysis. The glycyl radical cofactor is harbored within the Gly loop (yellow), which is proximal to the Cys loop (purple). (B) The glycyl radical is proposed to be in equilibrium with a thiyl radical formed on the Cys residue within the Cys loop. (C) The thiyl radical is proposed to abstract a hydrogen atom from the methyl group of toluene. The resulting benzylic group can add to the alkene of fumarate, resulting in a succinate radical which can abstract a hydrogen atom from Cys to ultimately form R-benzylsuccinate and regenerate the thiyl radical. [8][9][10] (D) Working model for glycyl radical installation based on X-ray crystal structures. Note that PDB: 5BWE is a structure with glycine, not with the glycyl radical. 1 depicts the crystal structure of BSSag (PDB: 5BWD), which contains a partially open, though still buried, glycyl radical domain (GRD, dark gray) as compared to the crystal structure of BSSabg (4). It is proposed that this slightly open conformation of BSSag (1) could be in equilibrium with a fully open conformation of the GRD (2). The fully open conformation of BSSag allows binding of the activating enzyme (AE, green) to the GRD. Once bound, the AE can install the glycyl radical cofactor, thus activating BSS for catalysis (2-> 3). The AE can dissociate and BSSb can bind to the BSSag complex to stabilize the closed conformation (4). It is proposed that a closed conformation of the enzyme is required for toluene binding and fumarate addition. iScience Article active site ( Figures 1D, 1D2, and 1D3). 11,14 Once glycyl radical is installed, it must move back into the GRE barrel, where it can catalyze multiple rounds of turnover.
Though most characterized GREs consist of a single protein subunit, there are a few exceptions that contain additional smaller subunits, including 4-hydroxyphenylacetate decarboxylase (HPAD) 20 and BSS. 21 HPAD contains one additional 9.5 kDa subunit, which harbors two [4Fe-4S] clusters. 22 BSS contains two extra subunits -BSSg, which is 7 kDa, and BSSb, which is 9 kDa, in addition to the 98 kDa large catalytic subunit BSSa. 23 Like the extra subunit of HPAD, both BSSg and BSSb contain [4Fe-4S] clusters. 24 BSSa cannot be solubly expressed in Escherichia coli without BSSg. 23 The structure of BSSag shows that part of BSSg fits into a hydrophobic surface pocket of BSSa, which is most likely the reason for increased solubility when the two subunits are coexpressed. 6 In vivo, it is known that BSSg is necessary for organism survival on toluene, 25 but the native function of this subunit and its [4Fe-4S] cluster is unknown. When comparing the structures of BSSag and BSSabg, it was observed that the Gly loop of BSSag moves out of the active site by 2 Å and the protein begins to partially open in a clam-shell like motion ( Figure S2). 6 It is proposed that the BSSag structure has captured a ''partially open'' snapshot of BSS, where the Gly loop is starting to move out of the GRE active site ( Figure 1D1). Based on these movements, BSSb is proposed to play a role in the conformational changes needed to move between the ''closed'' state where the Gly loop is near the Cys loop ( Figures 1D-1D4) and the ''open'' state where the Gly loop moves out of the active site completely so it can bind to the GRE-AE for cofactor installation ( Figures 1D2 and 1D3). 6,7 Although this working model for BSSb 0 s role in activation fits the structural data ( Figure 1D), it has not been explored biochemically because of the inability to install the glycyl radical within BSS in vitro. Multiple attempts have been made to express BSS-AE from Thauera aromatica (BSS-AE Ta ) as soluble protein in E. coli, but only very small amounts of folded protein could be obtained, even when expressed as fusion proteins. 7,26 Activation experiments have shown that these small amounts of BSS-AE are not able to install glycyl radical on BSS. 26 This problem with in vitro activation has not only hampered our understanding of XSS mechanism, which is unique among the GREs in that it includes multiple subunits, but it has also severely limited the amount of mutagenesis experiments and engineering efforts using these enzymes.
A robust system for glycyl radical cofactor installation would open the door to exploring members of this environmentally and synthetically important enzyme class to gain fundamental insight into molecular mechanism and to engineering green, selective biocatalysts for organic synthesis.
Ever since the discovery of BSS in the 1990's, 27,28 the radical chemistry used to carry out this challenging olefin hydroalkylation reaction has been the topic of multiple reports; however, the inability to install the radical cofactor in vitro limited the types of experiments that could be accomplished with this system. Here, we have found an XSS-AE that can be recombinantly produced in E. coli and used to form glycyl radical on its native XSS in vitro. Moreover, we have shown that it has cross-reactivity with BSS. This cross-reactivity is atypical for the GRE superfamily, as GRE-AEs are typically observed to be highly specific for their native GRE partner. 12,29 We have also gleaned important insights about the roles of XSS subunits in glycyl radical formation subsequent fumarate hydroalkylation. These studies not only complement previous structural data in determining the function for BSSb, but also serve as a starting point for future biochemical investigations of XSS enzymes and directed evolution campaigns. This is enabling technology that will allow the larger community to more rapidly mutate XSS enzymes to both understand how hydrocarbons are anaerobically degraded and to engineer this class for asymmetric C-C bond formation.

RESULTS
Finding an XSS-AE that can be produced in E. coli Many gene clusters containing putative XSSs have been discovered and sequenced from anaerobic organisms that are capable of degrading aromatic hydrocarbons, such as toluene. Given the reported insolubility of BSS-AE Ta , 7,26 we wondered whether recalcitrance to heterologous expression in E. coli was a hallmark of XSS-AEs, or whether unexplored homologs could be more easily obtained as pure enzyme. We performed a BLAST search using BSS-AE Ta as a query sequence and chose 6 putative XSS-AEs with different sequences (see Percent Identity Matrix in Table S1) from different organisms ( Figure 2, Table S2 and Figure S1). These 6 XSS-AEs, along with BSS-AE Ta , were cloned into a pET28a vector that included a C-terminal His 6 -tag. Like all AdoMet-dependent enzymes, the XSS-AEs were predicted to have 3 Cys residues that coordinate an active site [4Fe-4S] cluster ( Figure S3). In addition, 8  iScience Article clusters ( Figure S3). The domain harboring these auxiliary clusters has been reported for many GRE-AEs; however, its function is still largely unknown. We anaerobically expressed and purified the 7 XSS-AEs in parallel. In accordance with previous reports, 7,26 BSS-AE Ta was found exclusively in inclusion bodies, with no observable soluble protein ( Figure 2, Lane 7). However, the BSS-AE homologs all produced some amount of soluble enzyme, although the range varied considerably, from 0.1 to 1.4 mg of protein per L of culture ( Figure 2). The amount of iron in elution fractions was analyzed to estimate how many clusters each of the homologs contained. Five XSS-AEs contained between 5 and 8.3 Fe/protein ( Figure 2), which is consistent with these AEs purifying with 1-2 [4Fe-4S] clusters. One homolog, 5, contained more than 12 Fe/protein, which would mean more than 3 [4Fe-4S] clusters could be present. Based on the Cys content of 5, this seems unlikely. It seems more plausible that experimental error in this preliminary solubility screen could be inflating this number. For these studies, instead of following up on 5, which was low yielding, we wanted to continue investigation of the highest yielding homolog, 3, using electron paramagnetic resonance (EPR) spectroscopy and LC-MS ( Figures 3A and 3B). This particular XSS-AE, known as 4-isopropylbenzylsuccinate synthase AE (IbsAE), is from a strain of Thauera that is known to degrade p-cymene. 30,31 On optimization of expression conditions for IbsAE, the protein yield was increased to 6 mg/L. Iron content was consistent with one [4Fe-4S] cluster (4.3 Fe/protein, Figure 3E). To determine whether full reconstitution of all 3 [4Fe-4S] clusters could be accomplished, we tried reconstituting the remaining 2 clusters in vitro. Similar to previous reports on similar AEs, 32 full reconstitution was not observed (7.6 Fe/protein, Figure 3E). In addition, 85-90% of the protein precipitated or aggregated as a result of reconstitution ( Figure S4).

IbsAE characterization via EPR spectroscopy and AdoMet cleavage assays
Our overarching goal was to produce high enough yields of an XSS-AE to determine conditions suitable for in vitro glycyl radical installation and hydroalkylation assays ( Figures 3C and 3D). With this goal in mind, we wanted to know if we could use IbsAE as purified instead of reconstituting, as initial attempts to optimize the reconstitution still led to dramatic losses in yield ( Figure S4). Based on previous work with other GRE-AEs, we hypothesized that the 4.3 Fe/prot we see in IbsAE corresponds to the active site [4Fe-4S] cluster. To investigate the differences between the 'IbsAE as purified' and 'IbsAE reconstituted', we turned to EPR spectroscopy. We used 5-deazariboflavin as a photoreductant, which is commonly used for GRE activation assays, to reduce the [4Fe-4S] 2+ cluster(s). We observed a signal consistent with a [4Fe-4S] 1+ cluster for the as purified IbsAE ( Figure S5). On reduction of the reconstituted IbsAE, we observe a mixture of signals, most likely corresponding to a [4Fe-4S] 1+ cluster and [3Fe-4S] 1+ cluster ( Figure S5). We reasoned that a   Figure S6).
After verifying that we did indeed have [4Fe-4S] clusters in both IbsAE as purified and IbsAE reconstituted and that we could reduce these clusters, we assessed the enzymes' ability to cleave AdoMet in the presence and absence of the corresponding XSS, 4-isopropylbenzylsuccinate synthase (IBSS) ( Figure 3B). When we incubate IbsAE with AdoMet following reduction of the [4Fe-4S] cluster, we do observe AdoMet cleavage ( Figure 4B, 1.1 mM dAdo), whereas none is observed in the control without IbsAE enzyme (Table S3) Oftentimes, low levels of AdoMet cleavage are observed without the substrate bound to IbsAE, as in this particular case; however, typically AdoMet cleavage is accelerated by addition of substrate. In iScience Article this case, the substrate for IbsAE is the protein complex IBSS. Like BSS, IBSS contains two additional [4Fe-4S]-containing subunits (IBSSb corresponds to BSSb and IBSSg corresponds to BSSg) in addition to the catalytic IBSSa subunit that harbors the glycyl radical. We obtained the genes for IBSSabg with the chaperone protein IbsE. Previous structural and proteolytic data have led to the hypothesis that BSSb could play an important role in regulating the large conformational changes that must occur to make the catalytic glycine residue physically available to BSS-AE ( Figure 1D). 6 For this reason, we wanted to be able to control the amount of the b subunit that we add to assays. We purified IBSSag as a single complex, as previous studies had demonstrated the a subunit does not solubly express without the g subunit. 23 We separately expressed and purified IBSSb and subsequently removed the N-terminal His tag with a TEV cleavage site. Repeating these assays with addition of IBSSag does lead to a large increase in AdoMet cleavage  iScience Article ( Figure 4B, 47.6 mM dAdo with as purified IbsAE and 46.7 mM dAdo with reconstituted IbsAE). As expected based on our working model ( Figure 1D), the addition of IBSSb with IBSSag yields less dAdo product in endpoint assays ( Figure 4B, 7.2 mM dAdo with as purified IbsAE and 5.4 mM dAdo with reconstituted IbsAE). The rate of AdoMet cleavage by IbsAE is also slower when IBSSb is present ( Figure 4C and Table S4).

Glycyl radical formation on IBSS
Following validation that IbsAE is able to cleave AdoMet, we next wanted to determine whether glycyl radical within IBSS could be observed by EPR spectroscopy ( Figure 3C). We tried activating IBSSag with and without IBSSb using our as purified IbsAE stock, which is missing its auxiliary [4Fe-4S] clusters. Consistent with AdoMet cleavage assays as well as our working model, we only observe significant quantities of glycyl radical without IBSSb (Figures 5A and 5B and Table S5). The observation that IbsAE is able to form a glycyl radical on IBSSag without full reconstitution of its auxiliary [4Fe-4S] clusters is consistent with work showing that 4-Hpad-AE can also activate its corresponding GRE without the auxiliary clusters. 33 However, the persistence of the glycyl radical was significantly affected in previous studies of 4-Hpad-AE, and within 16 min, most of the radical was gone. 33 Time courses of activation reactions with our as purified IbsAE demonstrate that radical persistence is not an issue with this system for at least up to 6 h ( Figure 5C and Table S6). It was also found that the as purified IbsAE installed glycyl radical within IBSSag faster than the reconstituted IbsAE ( Figure S7), which is convenient given our low yields of reconstituted IbsAE.

Cross-reactivity is observed for glycyl radical installation on BSS
To date, BSS remains the only structurally characterized XSS, and significantly more is known about the scope and mechanism for this enzyme than XSSs that function on substrates beyond toluene (e.g. IBSS). We wondered whether IbsAE could activate BSS as well. Although GRE-AEs are typically highly specific for their partner GRE, 12,23,29 we do observe an EPR signal consistent with the glycyl radical when BSSag is incubated with reduced IbsAE and AdoMet ( Figure 6A). We wanted to test the effects of BSSb on glycyl radical installation as well as the persistence of radical on BSSag. Activation time courses were performed for BSSag and BSSabg over the course of 4 h. Similar to IBSS, less radical is formed when BSSb is added to the reactions. Similar amounts of radical are formed on BSSag as IBSSag, and this radical persists for the timescale of the experiment ( Figure 6B and Table S7).

BSSb is necessary for high benzylsuccinate production
After demonstrating that BSSag can be activated, we wanted to determine whether we could observe hydroalkylation activity in vitro. We activated BSSag for 3 h and subsequently added fumarate (2 mM final conc.) and toluene (6 mM added as a solution of toluene and MeOH). BSSb was added to some reactions to test the effects of this subunit on hydroalkylation yields. We detected and quantified product formation using iScience Article high-resolution QToF-LCMS. Nine replicates of each reaction condition were conducted in parallel. In reactions with BSSag and BSSabg, a peak with the same retention time and the same exact mass as a benzylsuccinate (BS) authentic standard was observed (Figure 7). Yields increased dramatically in the presence of BSSb ( Figure 7 and Table S8, from 0.7% to 92.3% assay yield). Control reactions without BSSag produced no detectable BS in 7 of 9 samples and trace levels of BS in 2 of 9 samples (Figure 7 and Table S8).

DISCUSSION
In this work, we set out to solve the long-standing problem of in vitro glycyl radical cofactor installation in BSS. Having an in vitro system is useful for both probing the molecular mechanism of XSSs as well as for developing XSSs as biocatalysts. XSSs are crucial to anaerobic hydrocarbon degradation within microbes, and thus understanding how these enzymes work within their native cellular environments remains an important question. By developing an in vitro activation method, we were able to explore the molecular mechanism of BSS activation and catalysis in ways that were not previously possible. Prior biochemical investigations and crystal structures showed that BSS contains two accessory subunits, each with a [4Fe-4S] cluster bound. 6,23 Here, we explore hypotheses regarding the function of one of these subunits -BSSb. Based on crystallographic data, we previously proposed that BSSb is needed to control conformational dynamics of the glycyl radical domain and plug the hydrocarbon substrate channel. 6  iScience Article assess its effects on activation or catalysis. 23 Using our in vitro system, these experiments could be revisited to determine the role of BSSb 0 s [4Fe-4S] cluster. Beyond arylalkyl-succinate synthases like BSS, there are numerous alkyl-succinate synthases that functionalize saturated hydrocarbons. Even less is known about these enzymes that can directly and selectively functionalize saturated alkanes; for example, the subunit/cofactor architecture of these enzymes is still unknown and is thought to include an additional subunit. 34 Could we use a similar genome mining approach to find soluble alkyl-SS-AEs as well?
XSSs also hold the potential to be useful synthetic tools for building small molecules. They use abundant feedstocks (hydrocarbons and olefins) to form new Csp 3 -Csp 3 bonds using radical hydroalkylation. Hydroalkylation chemistry is an attractive method for forming C-C bonds as it has the potential to set multiple stereocenters at once; however, controlling the stereoselectivity remains challenging. Inside radical enzymes, substrate positioning can provide control over stereoselectivity, for example, toluene addition to fumarate to form exclusively R-benzylsuccinate in BSS. Beyond BSS, other XSSs exist that are able to perform this chemistry using a wide range of hydrocarbons, including saturated hydrocarbons. Given that the transformations they catalyze could prove so useful, why have XSSs not been widely explored as biocatalysts? One major hurdle has been activating the glycyl radical cofactor. Until now, activation has only been accomplished in whole cells. Purification of activated BSS from whole cells results in rapid loss of hydroalkylation activity. 23 The inability to generate pure, activated BSS has limited the types of studies that can be done, including characterization of variants made through mutagenesis. Recently, other groups have also developed tools to circumvent these issues. In 2021, Heider et al. developed a heterologous expression and activation system in Aromatoleum evansii, which importantly cannot degrade toluene, to assess BSS variants. 26 In addition to providing insight into the mechanism of substrate recognition, Heider et al. showed that the olefin substrate is not restricted to dicarboxylic acids. 26 Even more recently, Cirino et al. developed a heterologous system for producing alkylsuccinates in E. coli using a BSS homolog, allowing even more rapid access to substrate scope studies. 34 Although these tools will accelerate the development of XSSs as biocatalysts, a key limitation still existed -in vitro activation and subsequent hydroalkylation using purified enzymes. Whole cell activation for screening of XSS variants is attractive from a high-throughput standpoint. However, with the ability to activate in vitro, we now can conduct reactions using purified enzymes to verify findings from whole cell screening and rationalize the effects of key mutations. By combining approaches, mutagenesis studies can much more rapidly be accomplished for this enzyme class.
XSSs have fascinated and challenged scientists for decades, and numerous studies [5][6][7]21,[23][24][25][26][27][28]34 have helped to shed light on their mechanism. Methods developments reported here and described above are likely to rapidly accelerate our understanding of XSS enzyme mechanisms, XSS substrate scope, and enable XSS protein engineering efforts. We are excited to see how these future efforts will reshape the way we view XSSs and what we understand about them.

Limitations of the study
This study focuses on the development of methodology for studying XSSs but does not explore substrate scope for these enzymes. Also, it was shown that reconstitution of auxiliary clusters within the ferredoxin Figure 7. Activated BSSabg can catalyze the addition of toluene to fumarate High resolution LCMS was used to monitor formation of benzylsuccinate. A standard curve was prepared for benzylsuccinate using commercially available benzylsuccinate and L-tryptophan as an internal standard. Assay yields were determined by integrating the EIC spectrum for benzylsuccinate and calculating yield using the standard curve, with fumarate as the limiting reagent (n = 9).

OPEN ACCESS
iScience 26, 106902, June 16, 2023 9 iScience Article domain of IbsAE is not necessary for enzyme activity, but the function of this domain and its clusters was not thoroughly investigated. In addition, in Figure 2, initial yields and Fe content of XSS-AE homologs were calculated using the protein fractions shown in the gel in Figure 2, which do contain impurities that affect the accuracy of the calculated yield and Fe content. Because the results in Figure 2 were meant to serve as an initial screen to determine which homolog was the highest yielding, expression and purification of only IbsAE was optimized for improved yield and purity.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS
The authors declare no competing interests.

INCLUSION AND DIVERSITY
One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in their field of research or within their geographical location. One or more of the authors of this paper self-identifies as a gender minority in their field of research. One or more of the authors of this paper self-identifies as a member of the LGBTQIA+ community. One or more of the authors of this paper self-identifies as living with a disability. One or more of the authors of this paper received support from a program designed to increase minority representation in their field of research.

Construction of expression plasmids BSS-AE Ta homologs
The genes for the six BSS-AE Ta homologs were identified through a BLAST search using the amino acid sequence of BSS-AE Ta  iScience Article organism had been characterized as an anaerobic aromatic hydrocarbon degrader) and sequence similarity (i.e. genes with very high similarity to one another were excluded, see Table S1). The amino acid sequences for the 6 genes (see Table S1 for gene identifiers) were used for codon optimization for expression in E. coli K12. The six genes were purchased from Twist Bioscience, where they were cloned into pET28a at restriction sites BamHI and HindIII. The resulting plasmids contained both N-terminal and C-terminal hexa-His-tags. The N-terminal His-tag was removed from all AE constructs using Q5â Site-Directed Mutagenesis Kit (New England Biolabs) using the primers in Table S2. All primers were designed using NEBaseChangerä. All genes were confirmed through Sanger sequencing by Quintara Biosciences.

BSS-AE Ta
We received the gene for BSS-AE Ta from the Marsh lab. 23 The BSS-AE Ta gene was amplified and overhangs were added with complementarity to pET28a using the following primers: forward primer, 5 0 -CTTTAAGAAGGAGATATACCATGAAAATTCCATTAGTCAC-3 0 and reverse primer, 5 0 -TCGAGTGCGG CCGCAAGCTTCCTTTTCGGGTGGGTCTCTT-3 0 . The pET28a vector was amplified and overhangs were added with complementarity to BSS-AE Ta using the following primers: forward primer, 5 0 -AAGAGACCC ACCCGAAAAGGAAGCTTGCGGCCGCACTCGA-3 0 and reverse primer, 5 0 -GTGACTAATGGAATTTTCA TGGTATATCTCCTTCTTAAAG-3 0 . PCRs were conducted using the Q5â Site-Directed Mutagenesis Kit (New England Biolabs). PCR products were purified over 1% agarose gels using a Qiagen gel extraction kit. Gibson assembly reactions using NEBuilderâ HiFi DNA assembly were set up at 50 C for 1 hour with an insert:vector ratio of 3:1. A small aliquot (5 mL) of the reaction was transformed into E. coli DH5a cells (New England BioLabs). The resulting construct was verified through Sanger sequencing by Quintara Biosciences.

IBSS and BSS
The plasmids used to express BSSabg were published previously. Briefly, BSSa and BSSg were cloned into a pET-DUET vector into sites NdeI/KpnI and NcoI/HindIII, respectively. BSSa was C-terminally His 6 tagged. BSSb and TutH were cloned into a pRSF-DUET plasmid into sites NdeI/XhoI and NcoI/HindIII. For expression of BSSag with the TutH chaperone, the b-subunit was removed from the pRSF-DUET plasmid using the Q5â Site-Directed Mutagenesis Kit (New England Biolabs) and the following primers: forward primer, 5 0 -CTCGAGTCTGGTAAAGAAAC-3 0 and reverse primer, 5 0 -ATTTCGATTATGCGGCCG-3 0 . His 6 -BSSb, which included a TEV cleavage site after the N-terminal His 6 tag, was constructed using the Q5â Site-Directed Mutagenesis Kit (New England Biolabs) and the following primers: forward primer, 5 0 -AACGACC GAGAATCTTTATTTTCAGGGATCCGAGGGCAGCAACATGGAA-3 0 and reverse primer, 5 0 -GGATCG TGATGGTGATGGTGATGGCTGCTAGCCATATGTATATCTCCTTCTTATACTTAACTAATATAC-3 0 .
For the IBSS complex, the amino acid sequences (IBSSa -UniProt ID: A0A096ZNX3, IBSSb -UniProt ID: A0A096ZP03, IBSSg -UniProt ID: A0A096ZNX6) and putative chaperone protein (IbsE -UniProt ID: A0A096ZNY2) were used for codon optimization for expression in E. coli K12. The genes were purchased from Twist Bioscience as linear g-blocks with overhangs for Gibson Assembly. A pET-DUET plasmid was amplified using the Q5â Site-Directed Mutagenesis Kit (New England Biolabs) and the following primers: forward primer, 5 0 -AGCGCAGCTTAATTAACCT-3 0 and reverse primer, 5 0 -GGTATATCTCCTTCTTAAAGT TAAACAA-3 0 . PCR product was purified over 1% agarose gels using a Qiagen gel extraction kit. C-terminally His 6 tagged IBSSa and IBSSg were assembled into the linearized pET-DUET vector using NEBuilderâ HiFi DNA assembly kit. A pRSF-DUET plasmid was amplified using the Q5â Site-Directed Mutagenesis Kit (New England Biolabs) and the following primers: forward primer, 5 0 -CTCGAGTCTGGTAAAGAAAC-3 0 and reverse primer, 5 0 -CCATGGTATATCTCCTTATTAAAG-3 0 . PCR product was purified over 1% agarose gels using a Qiagen gel extraction kit. IbsE and IBSSb were assembled into the linearized pET-RSF vector using NEBuilderâ HiFi DNA assembly kit. For expression of IBSSag, the IBSSb was removed from the pRSF-DUET plasmid using the Q5â Site-Directed Mutagenesis Kit (New England Biolabs) and the following primers: forward primer, 5 0 -CTCGAGTCTGGTAAAGAAAC-3 0 and reverse primer, 5 0 -TTAGACACGCGCTTT TGC-3 0 . His 6 -IBSSb, which included a TEV cleavage site after the N-terminal His 6 tag, was constructed using the Q5â Site-Directed Mutagenesis Kit (New England Biolabs) and the following primers: forward primer, 5 0 -AACGACCGAGAATCTTTATTTTCAGGGATCCGCTAATGTGCAGACCCAG-3 0 and reverse primer, 5 0 -CAATTCATATTCTTCCTCTATATGTATACCGATCGTCGGTAGTGGTAGTGGTAGTGCTAGG-3 0 . All BSS and IBSS constructs were verified through Sanger sequencing by Quintara Biosciences.

Reconstitution of IbsAE
IbsAE purified with an intact active site cluster following the purification protocol described above. For reconstitution of the auxiliary clusters, $2 mL of purified IbsAE ($100 mM) was reconstituted at a time. iScience Article which was immediately mixed. Five molar equivalents of Na 2 S were added to the protein, which was immediately mixed. The reconstitution was allowed to incubate for 30 minutes; then 5 more equivalents of both Fe(III)Cl 3 and Na 2 S were added as described above. The reconstitution was incubated for 2 more hours, was spun at 14,000 g for 10 minutes, and was filtered (0.22 mm). After filtration, reconstituted IbsAE was purified by size exclusion chromatography on an S200 16/60 column (50 mM HEPES pH 8.0, 300 mM NaCl, 1 mM DTT). The monomer peak was collected, concentrated, and flash frozen.

BSS and IBSS large scale expression and purification
All BSS and IBSS constructs were transformed into T7 Express cells (New England BioLabs) and a single colony was used to make a glycerol stock of each. Starter cultures were inoculated from glycerol stocks and grown overnight at 37 C at 220 rpm in LB containing either 50 mg/mL kanamycin and 100 mg/mL ampicillin for IBSSag and BSSag or 50 mg/mL kanamycin for IBSSb and BSSb. Expression cultures were inoculated with 10 mL of starter culture per 1 L of LB containing the corresponding antibiotics, 150 mg iron(II) ammonium sulfate hexahydrate (CAS: 783-85-9), and 47 mg L-cysteine. Expression, TALON purification, and concentration were carried out anaerobically as described above in ''IbsAE large scale expression and purification.'' For IBSSb and BSSb, the N-terminal His-tag was cleaved with His-tagged TEV protease at a ratio of 10:1 (b subunit:TEV protease, w/w). The reaction was gently mixed and left at 4 C for $24 hours (or until >80% completion as determined by SDS-PAGE) without agitation. The reaction mixture was purified on TALON resin as detailed above. Fractions containing pure IBSSb or BSSb, with the Histag removed, were pooled and buffer exchanged into 50 mM HEPES pH 8.0, 300 mM NaCl.

Reduction of the [4Fe-4S] clusters
In many cases, flavin derivatives are used to reduce the active site cluster of GRE-AEs to initiate glycyl radical installation. Two flavin derivatives, acriflavine 36 and 5-deazariboflavin, 11 were tested for their ability to reduce the active site cluster of as purified IbsAE. In a Coy anaerobic chamber, IbsAE (60 mM) was incubated with either acriflavine and bicine (100 mM and 50 mM, respectively) or deazariboflavin (100 mM) in activation buffer (

EPR parameters
EPR spectra were collected in a Bruker EMX-Plus spectrometer at temperatures between 10-40 K with a Bruker/ColdEdge 4K waveguide cryogen-free cryostat. Xenon 1.1b.155 software was used to collect and process spectra. Spectra were recorded at 9.37 GHz with a modulation amplitude of 10 G, microwave power of 50 mW, and a 100 kHz modulation frequency. A center field of 3500 G, a sweep time of 60 s, and a sweep width of 2000 G were used. Each spectrum shown is an average of 10 scans. The double integrals of the two spectra in Figure 4A were calculated using Xenon software and compared to one another to determine the relative amount of [4Fe-4S] 1+ cluster in each. iScience Article EPR spectroscopy to quantitate glycyl radical EPR spectra of the glycyl radical was collected at 80 K. Spectra were recorded at 9.37 GHz with a modulation amplitude of 3 G, microwave power of 1.26 mW, and a 100 kHz modulation frequency. A center field of 3350 G, a sweep time of 21 s, and a sweep width of 200 G were used. Each spectrum shown is an average of 10 scans. Potassium nitrosodisulfonate (Fremy's salt, Sigma Aldrich) was used as a standard. The double integrals of each spectrum were calculated using Xenon software and compared to the double integrals obtained from Fremy's standard to obtain concentrations of glycyl radical.

AdoMet cleavage
Cluster reductions and AdoMet cleavage reactions were conducted as described above. Endpoint assays were conducted for 2 hours and time courses were conducted for 1.5-120 minutes. Reactions were quenched with one volume of methanol and 100 mM of L-tryptophan was added as an internal standard. Quenched reactions were removed from the Coy and protein was pelleted by centrifugation. The resulting supernatant was filtered through a 0.22 mm filter and used for LC/MS analysis. The LC method described above was used with the MS in positive ion mode. Extracted ion counts for 5 0 -deoxyadenosine (dAdo) and L-tryptophan were obtained, and the concentration of product was determined using a standard curve made from known amounts of dAdo and L-Trp.

Hydroalkylation
Cluster reductions and glycyl radical installation were conducted as described above. BSSb was not added to glycyl radical installation reactions. Three hours after glycyl radical installation reactions were initiated, fumarate (2 mM final conc.) and toluene (6 mM final conc. added as a stock solution in MeOH, 3% v/v) were added. Reactions were diluted such that the final conc. of BSSag was 40 mM, and BSSb (40 mM final conc.) was also added to some reactions. Control reactions contained all components except BSSag. Reactions were conducted in a Coy anaerobic chamber in a 96-well microtiter plate with a final volume of 25 mL for each reaction. Hydroalkylation reactions were quenched with two volumes of methanol and 100 mM of L-tryptophan was added as an internal standard. Quenched reactions were removed from the Coy and protein was pelleted by centrifugation. The resulting supernatant was diluted 20-fold and filtered through a 0.22 mm filter and used for LC/MS analysis. The LC method described above was used with the MS in negative ion mode. The retention times for fumarate, L-Trp, and benzylsuccinate were 1.218, 4.393, and 5.240 min, respectively. Product concentration was determined by the extracted ion count ratio of benzylsuccinate and internal standard L-Trp, multiplied by response factor 0.21, which was established via a calibration curve with known amounts of benzylsuccinate (Sigma Aldrich, CAS 884-33-3) and L-Trp. The assay yield was defined as 100x[BS]/2 mM, where 2 mM represents the initial concentration of the limiting reagent, fumarate.

QUANTIFICATION AND STATISTICAL ANALYSIS
In Figure 4B/Table S3 and Figure 4C/ Table S4, number of assays conducted for each condition was equal to 3 (n = 3), and the mean and the standard deviations were calculated in Excel. In Figure 7/ Table S8, number of assays conducted for each condition was equal to 9 (n = 9), and the mean and the standard deviations were calculated in Excel.