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A B12-dependent radical SAM enzyme involved in Oxetanocin-A biosynthesis

Summary Oxetanocin-A (OXT-A, 1) is a potent antitumor, antiviral, and antibacterial compound. Biosynthesis of OXT-A has been linked to a plasmid-borne, Bacillus megaterium gene cluster that contains four genes, oxsA, oxsB, oxrA, and oxrB. Here, we show that the oxsA and oxsB genes are both required for the production of OXT-A. Biochemical analysis of the encoded proteins, a cobalamin (Cbl)-dependent S-adenosylmethionine (AdoMet) radical enzyme, OxsB, and an HD-domain phosphohydrolase, OxsA, revealed that OXT-A is derived from 2′-deoxyadenosine phosphate in an OxsB-catalyzed ring contraction reaction initiated by H-atom abstraction from C2′. Hence, OxsB represents the first biochemically characterized non-methylating Cbl-dependent AdoMet radical enzyme. X-ray analysis of OxsB reveals the fold of a Cbl-dependent AdoMet radical enzyme for which there are an estimated 7000 members. Overall, this work provides a framework for understanding the interplay of AdoMet and Cbl cofactors and expands the catalytic repertoire of Cbl-dependent AdoMet radical enzymes.

Oxetanocin-A (OXT-A, 1) is a nucleoside analog produced by Bacillus megaterium NK84-0128 1 . The phosphorylated forms of OXT-A inhibit cellular and viral DNA polymerases 2 and have shown activity against hepatitis B 3 and herpes simplex viruses 1 , among others. The plasmid-borne gene cluster for OXT-A biosynthesis and resistance is located within the BglII-D fragment 4 and contains four open-reading-frames that encode two HD-domain phosphohydrolases (oxsA and oxrB), a cobalamin (Cbl)-dependent Sadenosylmethionine (AdoMet) radical enzyme (oxsB), and a pentapeptide repeat protein (oxrA). The simplicity of the cluster suggests that OXT-A may be produced through rearrangement of a purine nucleoside co-opted from a primary metabolic pathway. Since AdoMet radical enzymes catalyze some of the most challenging chemical transformations, OxsB is a likely candidate for catalyzing formation of the eponymous oxetane ring.

oxsA, B are OXT-A biosynthetic genes
A variety of nucleosides and sugars were tested for competence as substrate(s), however, no turnover was observed with any of the compounds tested (Extended Data Fig. 1). These negative results prompted us to take a genetic approach to determine if other gene products are required for OxsB activity. A non-producing, OXT-A resistant strain, B. megaterium NRS 269, and an E. coli-Bacillus shuttle vector pMM1522 were used in these experiments (Extended Data Fig. 2a). B. megaterium NRS 269 carrying the Bglα-D fragment produces OXT-A (Extended Data Fig. 2b). Deletion of oxsA or oxsB, however, abolishes OXT-A formation, whereas deletion of oxrA and oxrB has no effect on OXT-A production (Fig 1a).
Notably, both OxsA and OxrB are annotated as HD-domain phosphohydrolases, but the lack of OXT-A production in the oxsA deletion experiment indicates OxrB cannot fulfill the functional role of OxsA (Fig 1a). In a separate experiment, B. megaterium NRS 269 carrying only oxsA, oxsB, or the oxsA/oxsB pair was tested for OXT-A production. Here, OXT-A formation was observed only when both oxsA and oxsB were present, demonstrating again that oxsA and oxsB are required for OXT-A biosynthesis (Extended Data Fig. 2c).
To verify the above observation, substrate screening was retried using OxsA and reconstituted OxsB. A new product, P2, could be detected in the reaction mixture when 2′deoxyadenosine-5′-monophosphate (dAMP), or its 5′-diphosphate (dADP) or 5′triphosphate (dATP) form were employed as substrates (Extended Data Fig. 3a). P2 was characterized using MS and 1 H NMR and shown to be the aldehyde form of OXT-A 5′-monophosphate (2 in Fig. 1b, Extended Data Fig. 3b). Upon treatment with NaBD 4 , 2 is converted to OXT-A 5′-monophosphate (3 in Fig. 1b), verified based on co-elution with a chemically synthesized standard of 3 (Extended Data Fig. 3c and 4). These results confirm the requirement of OxsB and OxsA in OXT-A biosynthesis and identify dAMP, dADP and dATP as OXT-A precursors (1 in Fig. 1b). Approximately 4-10 turnovers of dATP to form 2 are observed in a 12-hr incubation using 20 μM OxsB and 20 μM OxsA (see Methods). The aldehydic group of 2 must be reduced to complete OXT-A (1) biosynthesis; indeed, when OxsB/OxsA are assayed in the presence of B. megaterium NRS 269 cell extracts or horse liver alcohol dehydrogenase, 1 is produced (Extended Data Fig. 3d,e). OxsA was recently shown to catalyze the sequential hydrolysis of mono-, di-, and tri-phosphorylated OXT-A compounds into OXT-A (3→1, Fig. 1d) 22 , supporting the ability of the OxsB/OxsA pair to turnover dAMP, dADP and dATP.
Importantly, OxsB has all of the hallmarks of an AdoMet radical enzyme; both AdoMet and reductant are required for product formation (Extended Data Fig. 3a) and 5′-dAdoH is generated (Extended Data Fig. 5a). Additionally, when OxsB is incubated with [2′-2 H 2 ]-2′-dAMP, AdoMet, OxsA and reductant, mono-deuterated 5′-dAdoH is formed (Extended Data Fig. 5b), consistent with AdoMet radical chemistry that initiates with hydrogen atom abstraction from C2′ of dAMP. Additional experiments performed using OxsB, OxsA, and [3′-2 H]-2′-dAMP show label retention in OXT-A (Extended Data Fig. 5c), further implicating C2′ as the site of hydrogen atom abstraction. All of these results are consistent with the annotation of OxsB as an AdoMet radical enzyme. Furthermore, product formation requires Cbl (Extended Data Fig. 3a), consistent with Cbl-dependent AdoMet radical chemistry. Cbl is bound to domain II, a Rossmann fold, which is similar (r.m.s.d of 3.10 Å for 565 atoms) to the Cbl-binding domain of methionine synthase (MetH) despite sharing only 14% sequence identity. As in MetH 23 , Cbl is bound to OxsB with its dimethylbenzimidazole (DMB) base displaced (i.e. "base-off") from Co (Fig. 3a, Extended Data Fig. 7). However, OxsB does not use the classic "His-on" Cbl binding sequence motif (Asp-X-His-X-X-Gly) defined by the MetH structure from which a His residue on the loop that follows β1 (II-β1 loop) replaces DMB as the lower ligand to Co 23 . In OxsB, the II-β1 loop has no His, is fourteen residues long instead of six, and is flexible, changing conformations between apo and Cbl-bound structures to cap the Cbl-binding site (Fig. 3b, Extended Fig. 8a). The closest protein residue to Co, Asn186, is too far (6.4-Å) to serve as a lower ligand, but does make through water contacts to Co (Fig. 3a). Surprisingly, Asn186 does not map to the position of the His ligand in MetH 23 , being found instead on a loop following II-β3 in the sequence Ser184-Asn186. These residues align with the highly conserved MetH residues Ser804-Leu806 23 . This Ser interacts with N3 of DMB in MetH and OxsB, whereas Asn/Leu orients the lower ligand of Cbl either by stacking (Leu/His in MetH) 23 or hydrogen bonding (Asn/H 2 O in OxsB) (Extended Data Fig. 7b-e) 23 . A water molecule (or hydroxide ion) also appears to ligate Co on the upper face of the OxsB Cbl (Extended Fig. 6c), generating a hexacoordinate form of the cofactor typically associated with a Co(III)-Cbl oxidation state 24 . This base-off, His-off mode of Cbl-binding is unlikely to be unique to OxsB given that the Cbl-dependent AdoMet radical methylase, TsrM, was reported to also bind Cbl base-off, His-off 19 .

A new mode of Cbl binding in OxsB
Overall, modifications to the Cbl-binding domain of OxsB, including the extended II-β1 loop and the absence of a protein-based ligand to Co, result in a shift of the corrin ring approximately 5-Å from the observed position in MetH and closer to the [4Fe-4S] cluster (Fig. 3b). This position of the OxsB corrin ring is approximately 90° away from the standard orientation of a corrin ring with respect to a TIM barrel; Cbl-enzymes typically position the corrin ring directly on top of the C-terminal end of a barrel in the same position that [4Fe-4S] clusters of AdoMet radical enzymes are found (Fig. 3c,d). Here we have an enzyme that accommodates both cofactors, and we find that the [4Fe-4S] cluster is in the standard position at the top of a partial TIM barrel, whereas Cbl has a novel position (Fig.  3d).

Expanded AdoMet fold accommodates Cbl
Comparison of OxsB with a classic AdoMet radical core fold exemplified by E. coli pyruvate-formate lyase activating enzyme (PFL-AE) 25 , shows an outward displacement of β-strands and helices such that the partial TIM barrel fold of OxsB is now large enough to accommodate Cbl (Fig. 4a). All previously described motifs for AdoMet and cluster binding 26,27 remain intact, albeit with a few notable variations in both their function and structure. For example, in addition to coordinating the [4Fe-4S] cluster, residues of Cys-rich loop (Pro322, His325, and Lys326 of C 313 DYSRC 318 TFC 321 PRDHK 326 ) also make contacts with Cbl ( Fig. 3a). His325 and Lys326, in particular, move 8.0-and 8.8-Å, respectively, from their positions in the apo-structure to hydrogen bond to Cbl (Extended Data Fig. 8b, c). Similarly, a residue of the "GGE" AdoMet binding motif (Ala361-Glu363) has taken on an auxiliary role; in addition to providing a backbone carbonyl to hydrogen bond with the amino group of AdoMet, the side-chain of Glu363 also interacts with an acetamide moiety of Cbl, tethering Cbl near the [4Fe-4S] cluster (Fig. 3c, Extended Data 7e). Thus, two classic AdoMet radical motifs serve to hold the [4Fe-4S] cluster, AdoMet and Cbl close to each other in OxsB.
The most substantial variation in an AdoMet radical motif is the replacement of β6 with a long loop. This loop connecting the 3 10 helix to III-α6* lies approximately where β6 ends in PFL-AE, and like β6 in PFL-AE contributes two hydrogen bonds to the AdoMet adenine ring fulfilling the AdoMet binding function (Fig. 4a, b). AdoMet radical enzyme BtrN 28 also uses a loop replacement for a missing β6, but in this case, the loop follows β5 with no intervening helices. Interestingly, OxsB has higher overall structural homology to PFL-AE than BtrN (DALI server 29 ), suggesting loss of β6 may have happened more than once.
Domain III concludes with a 25-residue loop that follows III-α6* (Fig. 4a) and serves to partially bury the [4Fe-4S] cluster. Even with this loop, however, the active-site is quite open (Extended Data Fig. 8d-f). We suspect that once substrate binds, a conformational change will close the active-site. A comparison of apo-and holo-OxsB structures reveals the helix bundle domain can swing towards the active-site as occurs in MetH 23,30,31 (Extended Data Fig. 8d).

AdoMet samples different conformations
AdoMet binds to the [4Fe-4S] cluster in two conformations, which differ by a 4.0-Å shift in the position of the AdoMet sulfur atom (Fig. 4c, Extended Data Fig. 9). In one conformation, the "radical-competent conformation", the S atom of AdoMet is 3.0-Å away from the [4Fe-4S] cluster (Fig. 4d), comparable to the average 3.4-Å S to Fe distance seen in other AdoMet radical enzymes that permits reductive cleavage of AdoMet 27 . In the second orientation, AdoMet does not engage with the GGE motif and does not appear competent to perform radical chemistry as the S atom is 6.5-Å away from the cluster (Fig. 4d). Instead, this binding mode places AdoMet approximately where one would expect it to bind to methylate Cbl; the methyl is 5.8-Å from Co (Fig. 4e), a similar distance to that found in the reactivation complex of MetH 32 . As OxsB does not catalyze a methylation reaction, this second AdoMet conformation is unlikely to be catalytically relevant. However, it could represent an orientation sampled to minimize uncoupled AdoMet cleavage. Regardless, these structural data on OxsB show that the active site of a Cbl-dependent AdoMet radical enzyme is designed such that two AdoMet binding modes can be populated, one in which AdoMet is positioned for radical chemistry and one in which the methyl group of AdoMet is close to Co. Indeed, recent studies 19,20 performed on TsrM describe a new mode of AdoMet binding; AdoMet binds close to the [4Fe-4S] cluster, but not with traditional coordination through either the amino or carboxylate moieties.
Although we do not have OxsB structures with substrate bound, structures of other AdoMet radical enzymes show that substrate sits in the lateral opening of the barrel such that the site of hydrogen atom abstraction is 3.7-4.0 Å from the 5′ position of AdoMet 27 . Using the substrate-bound structure of PFL-AE 25 as a guide, we can map the approximate site of hydrogen-atom abstraction to a position in OxsB that is equidistant from the 5′-carbon of AdoMet (the radical-competent orientation) and Co of Cbl (Fig. 4f, Extended Data Fig. 8g). This close juxtaposition of the putative substrate-binding site to both cofactors (3.7-Å in both cases) suggests that a hydrogen-atom abstraction step catalyzed by 5′-dAdo• could be followed by a Cbl-dependent reaction without any movement of the substrate or protein. Thus, OxsB may use two common strategies of radical enzymes: a conformational change seals an open active site prior to radical generation, and an arrangement of cofactors that limits the need for conformational changes once highly reactive radical intermediates are formed.

Proposed mechanisms for OXT-A synthesis
The above structural and biochemical data support the involvement of Cbl-dependent AdoMet radical chemistry in formation of the four-membered ring of OXT-A from phosphorylated forms of deoxyadenosine, thereby taking advantage of prevalent compounds in the cell (Fig. 1d, 5). Based on the deuterium labeling experiments, catalysis is likely initiated via hydrogen atom abstraction from C2′ of dAMP, dADP or dATP by 5′-dAdo•. Rearrangement of the resulting substrate radical (4) to the product radical (6) may proceed via an intermediate enol radical (4→5→6) (Fig. 5), analogous to the Cbl-dependent mutases 33 . Steps 4→6 are expected to be thermodynamically unfavorable due to contraction of a five-membered furanosyl ring to an oxetanyl ring. Coupling of this unfavorable reaction to the favorable OxsA phosphate hydrolysis reaction could shift the equilibrium toward product. Additionally, Cbl could play a role in lowering the activation energy barrier for these steps, either through a direct or indirect coordination of a substrate-bound radical. Steps 6 → 2 require an electron acceptor, and Cbl is one possible acceptor; formation of a Cbl-product radical complex could provide a conduit by which the unpaired electron in 6 is transferred to Cob(II) to form 2 and Cob(I). Subsequent electron transfer from Cob(I) to [4Fe-4S] 2+ would reset the redox state of the catalytic center. Alternatively, an external electron acceptor could be involved 34 , or [4Fe-4S] 2+ could accept the electron from 6 as has been proposed for DesII 35 . Finally, the aldehyde product 2 is converted to 3 by a cellular component, which along with dephosphorylation by OxsA completes OXT-A production.

Discussion
This work confirms that oxsA and oxsB are the essential OXT-A biosynthetic genes; we show here that OxsB uses AdoMet radical chemistry to catalyze ring contraction of a deoxyadenosine phosphate and recently showed that OxsA catalyzes hydrolysis of the resulting phosphorylated OXT-A compounds 22 . Although we do not know the roles of the remaining two genes located within the OXT-A gene cluster, our genetic experiments confirm that they are not required for OXT-A production. We suspect that oxrA and oxrB are resistance genes in agreement with their annotation. However, it is interesting to consider why resistance genes are necessary in light of our observation that OxsB requires OxsA for activity. The coupling of the two enzyme activities in itself protects the producing organism from the toxic phosphorylated forms of OXT-A; OxsB does not produce phosphorylated OXT-A unless OxsA can dephosphorylate and render the compound inert. However, OXT-A must be quickly exported before endogenous kinases can re-phosphorylate the compound. We hypothesize that the gene products of oxrA and oxrB represent the organism's safety net if export is hindered.
We further demonstrate that OxsB catalysis requires Cbl, although the exact role of Cbl in this nonmethylase is not yet clear. The fact that the apo-OxsB structure is almost identical (0.98 Å r.m.s.d) to holo-OxsB argues against a structural role for Cbl, and the location of Cbl in the OxsB active site is certainly consistent with a catalytic one. The OxsB structure reveals adaptions of both the classic AdoMet radical and Cbl motifs that result in the adjacent positioning of cofactors in the active site. These structural features may have evolved to allow for AdoMet radical-mediated Cbl-methylation, and may have been appropriated to assist in OxsB's complex radical-rearrangement, with Cbl retained to facilitate rearrangement or to accept an electron. The latter possibility would seem to be a mundane use for a cofactor that requires 30 enzymes in its biosynthesis.
We still have much to learn about the approximately 7000 enzymes that comprise the Cbldependent AdoMet radical enzyme superfamily. Already, with only a handful of these enzymes characterized, an impressive list of functions has emerged, ranging from production of antiviral compounds to bacteriochlorophylls. Outside of this enzyme family, there is no precedent for radical-based methylation, or for any of the functions suggested here for Cbl of OxsB. Thus, regardless of the mechanistic details that emerge, new chemical territory will be charted as we continue to explore this impressive enzyme family.

Extended Data
Extended Data Figure 1

. Putative substrates tested in the OxsB and OxsA/OxsB reactions
Reductants used to test these substrates were sodium dithionite, hexa-ammineruthenium(II) chloride, NADPH/methylviologen, titanium(III) citrate/methyl viologen, and a flavodoxin/ flavodoxin-NADPH reductase system 35,47,48 .  Residues highlighted in blue are conserved interactions between Cbl and MetH or OxsB, which are not from the standard Cbl-binding motifs. Residues shown in black form interactions with Cbl, but are not conserved between the proteins. (e) Stereoview of the contacts that Cbl makes with the Cbl-binding (pink) and AdoMet radical (cyan) domains of OxsB. All of the residues that form interactions with Cbl are highlighted as sticks, but only residues from the AdoMe radical domain are labeled for clarity.  Cbl. (b,c) In the absence of a [4Fe-4S] cluster, the Cys residues of the cluster-binding loop in the apo-structure are oriented similarly to those in the reconstituted structure. Cys318 and Cys321 in the apostructure, however, exhibit a partial occupancy disulfide linkage. At the end of the cluster binding loop, there are more substantial differences between the structures; His325 and Lys326 of OxsB Cbl/[4Fe-4S]/AdoMet move 8.0-and 8.8-Å, respectively, from their positions in the apo-structure to interact with the nucleotide tail of Cbl. Presumably, positively charged residues are needed to accommodate the negatively charged phosphate moieties of substrate and an aromatic residue may stack with substrate adenine. (g) An overlay of the β-strands from the AdoMet radical domains of OxsB (cyan) with those from a peptide-bound (green) stucture of PFL-AE 25 (gray) was used to map the approximate substrate binding site in OxsB. The yellow sphere, which corresponds to the Cα of the substrate peptide Gly residue, is 3.7-Å away from the 5′-carbon of AdoMet and 3.7-Å away from Co of Cbl. Extended Data Figure 9. AdoMet interactions (a) A stereoview of the AdoMet radical domain of OxsB. Each of the AdoMet radical motifs 26,27 is highlighted including the GGE (E363), ribose (E436), GXIXGXXE (I474), and the β6 (adenine-binding) motif (E545). The GGE motif provides a carbonyl to hydrogen bond with the amino moiety of AdoMet. In OxsB, E363 also contacts a Cbl acetamide. The ribose motif is found at the C-terminal loop following III-β4 where Glu436 forms two hydrogen bonds with the AdoMet ribose hydroxyl moieties. In the same loop, two residues upstream, the backbone amide of Gly434 contributes a hydrogen bond to the carboxyl group of AdoMet. Following a short III-α4a helix that connects III-β4 and III-α4, Lys448 interacts with the AdoMet carboxylate similar to what was previously observed in QueE 60 , HemN 61 , HydE 62 , PylB 63 , anSMEcpe 64 , and BtrN 28 . In terms of the GXIXGXXE motif, Ile474 from a loop following III-β5 provides hydrophobic contacts to the adenine ring of AdoMet as observed previously. However, instead of the backbone of Ile474 being stabilized by a polar residue on III-α5, as found in other AdoMet radical enzymes, the backbone of Ile474 is stabilized through interactions with the side chains of Gln442 and Tyr446 from III-α4a and the backbone of ribose motif residue Glu436. The final motif, the so-called β6 motif, is present although β6 is not. A loop substitutes for β6, with backbone atoms of E545 making hydrogen bonds to the adenine ring of AdoMet. Additional residues F320, M544, I546, and L547 that provide hydrophobic interactions to the adenine ring of AdoMet and a hydrogen bond to N6 are also shown. (f) A simulated annealing composite omit electron density map calculated after the "radical-competent" orientation of AdoMet was omitted from the refined structure of OxsB Cbl/[4Fe-4S]/AdoMet . This map is contoured at ±3.0 σ around the "radical-competent" orientation of AdoMet. (g) A similar simulated annealing composite omit electron density map was calculated after the "non-radical competent" orientation of AdoMet was omitted from the refined structure of OxsB Cbl/[4Fe-4S]/AdoMet . This map is also contoured at ±3.0 σ around the observed AdoMet conformation. Table 1 Data collection and refinement statistics of OxsB

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.     Mechanistic proposal for generation of OXT-A aldehyde 2.