Dioxane Bridge Formation during the Biosynthesis of Spectinomycin Involves a Twitch Radical S-Adenosyl Methionine Dehydrogenase That May Have Evolved from an Epimerase

Abstract

Spectinomycin is a dioxane-bridged, tricyclic aminoglycoside produced by Streptomyces spectabilis ATCC 27741. While the spe biosynthetic gene cluster for spectinomycin has been reported, the chemistry underlying construction of the dioxane ring is unknown. The twitch radical SAM enzyme SpeY from the spe cluster is shown here to catalyze dehydrogenation of the C2′ alcohol of (2′R,3′S)-tetrahydrospectinomycin to yield (3′S)-dihydrospectinomycin as a likely biosynthetic intermediate. This reaction is radical-mediated and initiated via H atom abstraction from C2′ of the substrate by the 5′-deoxyadenosyl radical equivalent generated upon reductive cleavage of SAM. Crystallo-graphic analysis of the ternary Michaelis complex places serine-183 adjacent to C2′ of the bound substrate opposite C5′ of SAM. Mutation of this residue to cysteine converts SpeY to the corresponding C2′ epimerase mirroring the opposite phenomenon observed in the homologous twitch radical SAM epimerase HygY from the hygromycin B biosynthetic pathway. Phylogenetic analysis suggests a relatively recent evolutionary branching of putative twitch radical SAM epimerases bearing homologous cysteine residues to generate the SpeY clade of enzymes.

Graphical Abstract

INTRODUCTION

The majority of reactions catalyzed by radical S-adenosyl-l-methionine (SAM) enzymes involve coupling the reduction of SAM to a radical-mediated transformation of the substrate.^1–3 These reactions involve a catalytic [Fe₄S₄] cluster that participates in the reduction of SAM and initiation of the transformation.^4,5 In some cases, the substrate is oxidized concomitant with the reduction of SAM; however, several enzymes have instead been reported to catalyze redox neutral transformations.^2,6 In the latter case, SAM may be either regenerated after each catalytic cycle or undergo net reduction by an additional source of reducing equivalents.^7–10 However, in vitro observations to date have suggested that the regeneration of SAM during redox neutral transformations of the substrate is actually quite rare.¹⁰ An interrelated subgroup of radical SAM enzymes coordinate one or two auxiliary [Fe₄S₄] clusters in addition to the aforementioned [Fe₄S₄] cluster that is characteristic of all radical SAM enzymes.^11–14 These SPASM/twitch radical SAM enzymes catalyze both redox neutral and oxidative transformations. Moreover, the SPASM/twitch enzymes have drawn particular attention, because the auxiliary clusters may participate in the redox handling of radical intermediates during the catalytic cycles of these enzymes.^15–19

HygY is a twitch radical SAM enzyme that catalyzes the epimerization of galacamine to talamine (1 → 2) during the biosynthesis of hygromycin B (see Figure 1A).^20,21 A recent report, however, has demonstrated that HygY can be converted from an epimerase to a dehydrogenase (see 1 → 3, Figure 1A) via the mutation of a single catalytic cysteine residue (cysteine-183).²⁰ This change in the fundamental redox chemistry of HygY emphasized the importance of a local reductant (e.g., an H atom donor) for redox neutral transformations. Moreover, this observation also suggested a potential mechanism by which an oxidative twitch radical SAM enzyme could evolve from a predecessor catalyzing a redox neutral reaction or vice versa. HygY is also closely related to the putative twitch radical SAM enzyme SpeY, which is encoded in the gene cluster responsible for biosynthesis of the clinically useful aminoglycoside antibiotic spectinomycin (6, see Figure 1B).^22–26 Despite nearly 50% sequence identity between HygY and SpeY, alignment reveals that the catalytic cysteine critical for the epimerization catalyzed by HygY is replaced by a serine residue in SpeY (serine-183, see Figure S16 for overlay). This suggests that SpeY may be a naturally selected homologue of HygY that catalyzes oxidative as opposed to redox neutral (e.g., epimerase) chemistry.

Figure 1.

(A) Epimerization (1 → 2) and dehydrogenation (1 → 3) reactions catalyzed by HygY and its C183A mutant, respectively. (B) Spectinomycin (6) is composed of an actinamine ring (7, green) linked to an actinospectose ring (8, blue) via a dioxane bridge (red). (C) Examples of natural products in addition to spectinomycin that also possess dioxane ring systems.

The biosynthesis of spectinomycin (6) is of particular interest in its own right due to its tricyclic structure, which is composed of a central dioxane core that bridges actinamine (7) and actinospectose (8) moieties via hemiacetal and glycosidic linkages. While the initial steps of actinamine biosynthesis have been studied in some detail,^{22,23,25–27} little is known regarding the assembly of actinospectose (8) and the mechanism by which the central dioxane ring is constructed. Moreover, there are few reports describing the biosynthesis of dioxane heterocycles despite their appearance in several natural products such as citrinal A (9),²⁸ botrylactone (10),²⁹ sinenside A (11),³⁰ and affinoside A (12)³¹ (Figure 1C). Inspection of the central dioxane of spectinomycin, however, suggests that elimination of the C6 alcohol from the C2′ hemiacetal would yield a pseudodisaccharide (5) that bears a striking resemblance to the immediate dehydrogenation product (3) generated in the reaction catalyzed by the HygY-C183A mutant (see Figures 1A,B). Consequently, SpeY and HygY may be the result of evolutionary divergence of an ancestral twitch radical SAM enzyme leading to two different yet related enzyme activities in two separate aminoglycoside biosynthetic pathways.

RESULTS AND DISCUSSION

In an effort to understand the origin of the dioxane ring in spectinomycin, SpeY was isolated and characterized in vitro. Recombinant SpeY from Streptomyces spectabilis ATCC 27741 was heterologously expressed in E. coli and purified aerobically as an N-His₆-tagged construct. The purified protein was reconstituted under anaerobic conditions with Na₂S and Fe(NH₄)₂(SO₄)₂ for 3 h followed by gel-filtration to yield a protein solution with a dark olive-green color (see Supporting Information, section S2.3). The reconstituted SpeY exhibited an absorption shoulder at 420 nm, which is characteristic of proteins containing bound [Fe₄S₄] clusters (Supporting Information, Figure S1).^32,33 Iron titration³⁴ of the reconstituted protein indicated 8.7 ± 0.1 mol Fe per SpeY monomer. Residues C24, C28, and C31 make up the conserved radical SAM CX₃CX₂C motif in the SpeY primary sequence,^2,35 and mutation of these residues to alanine produced the Δrad-SpeY triple mutant that contained only 3.7 ± 0.2 mol Fe per monomer after reconstitution. These results are consistent with the presence of two [Fe₄S₄] clusters bound per SpeY monomer, which was confirmed by X-ray crystallography as described below.

Potential spectinomycin biosynthetic pathways can be grouped according to whether the bridging glycoside linkage is introduced before or after the oxidation of C2′ to yield the carbonyl in 5. The latter pathways were considered, initially leading to two biosynthetic models where TDP-2′-keto-actinospectose (14) serves as a biosynthetic intermediate (see Figure 2). Route a mirrors that of desosamine biosynthesis and the aminolyase reaction catalyzed by the nontwitch radical SAM enzyme DesII (i.e., 17 → 20).^36,37 Subsequent dehydrogenation (20 → 14) could then be catalyzed by one of the two putative NADP-dependent oxidoreductases SpeH or SpeI, which are also encoded by the gene cluster. Alternatively, 17 may first undergo dehydrogenation followed by deamination possibly facilated by deprotonation of the acidic C3′ carbon in 18 (Figure 2, route b). These pathways would also explain the deoxygenation of C4′ that is observed in spectinomycin (6).

Figure 2.

Oxidation of C2′ may take place prior to formation of the glycosidic linkage. Compositional analysis of spectinomycin (6) into two possible biosynthetic precursors actinamine (7) and TDP-2′-keto-actinospectose (14), which may be coulped in a reaction catalyzed by the putative glycosyltransferase SpcG. Two possible pathways were considered that lead to the formation of 14 from TDP-glucose (15) are also shown. TDP-4,6-dideoxy-4-amino-d-glucose (17) may be produced from TDP-d-glucose (15) in reactions catalyzed by SpcE and SpcS1, which are respectively annotated as a TDP-glucose 4,6-dehydratase and a transaminase.

To test these hypotheses, TDP-4-amino-4,6-dideoxy-d-glucose (17) was prepared as previously described.³⁷ SpeH and SpeI were likewise cloned from Streptomyces spectabilis ATCC 27741, heterologously expressed in E. coli, and purified as N-His₆-tagged proteins. However, no substrate consumption or product formation was observed by LC-MS analysis after 5 μM reconstituted SpeY was incubated for 3 h with 0.5 mM 17, 1 mM SAM, 2 mM sodium dithionite, and 2 mM dithiothreitol (DTT) in 50 mM Tris·HCl buffer (pH 8.0, Supporting Information, Figure S5). SpeH and SpeI were similarly unreactive toward 17 in the presence of 0.5 mM NAD(P) (Figures S3 and S4). Finally, no reaction was noted when compound 20, which could be generated from 17 using DesII,^36,37 was incubated with SpeY, SpeI, or SpeH under analogous conditions.

An alternative biosynthetic pathway involves formation of the glycosidic linkage before introduction of the C2′ carbonyl in 5 (Figure 3A). This pathway is inspired by the structural resemblence between galacamine (1) and 2′S-dihydrospectinomycin ((2′S)-21) as well as the sequence similarity between HygY and SpeY. Thus, in direct analogy to the HygY-C183A mutant, SpeY may catalyze C2′-dehydrogenation of 21 to yield 5, which upon intramolecular hemiacetal formation would produce spectinomycin (see Figure 3A). Compound (2′S)-21 was thus synthesized according to the scheme shown in Figure 3B,C, which involves the use of silver triflate and N-iodosuccinamide to couple N-benzyloxycarbonyl (Cbz)-protected actinamine (22) with 27 to yield 28 (see Supporting Information, section S4). The final step in this synthesis involves Cbz-deprotection via hydrogenation catalyzed by palladium over carbon to provide (2′S)-21 (calcd for C₁₄H₂₆N₂O₇ [M + H]⁺ 335.1813, obsd 335.1811); however, overreduction of the C3′ keto group in (2′S)-21 also took place, leading to low levels of (2′R,3′S)-30 and (2′R,3′R)-30 as minor side products (calcd for C₁₄H₂₈N₂O₇ [M + H]⁺ 337.1969, obsd 337.1963) that were left unseparated.

Figure 3.

(A) Dehydrogenation of the C2′ alcohol may occur after formation of the pseudodisaccharide (2′S)-21 analogous to the oxidation of galacamine catalyzed by HygY-C183A (1 → 3, Figure 1A). (B) Preparation of Cbz-protected actinamine (22) from spectinomycin (6). (C) Preparation of 21 from Cbz-protected actinamine (22) and 1-methylglucoside (23). The final Cbz-deprotection step (29 → (2′S)-21) results in partial reduction of the C3′ carbonyl yielding (2′R,3′S)-30 and (2′R,3′S)-30 as minor contaminants.

No apparent consumption of (2′S)-21 or spectinomycin (6) formation was observed by LC-MS analysis following a 3 h incubation of 0.25 mM (2′S)-21/30 with 5 μM reconstituted SpeY, 0.5 mM SAM, 0.5 mM sodium dithionite, and 1 mM DTT in 200 μL 50 mM HEPES (pH 8.0). However, comparison of assays containing SpeY versus boiled enzyme demonstrated a significant decrease in the MS signal intensity arising from the reduced minor species 30 (m/z 337.2) relative to that of (2′S)-21 (m/z 335.2) (Figure 4A,B, Supporting Information, Figure S8). This observation indicated that an isomer of the contaminating species (i.e., (2′R,3′R)-30 or (2′R,3′S)-30) as opposed to (2′S)-21 had instead been consumed during the incubation with SpeY.

Figure 4.

LC-MS analysis following incubation of (2′S)-21 (along with (2′R,3′S)-30 and (2′R,3′S)-30 as minor components) using (A) supernatant of boiled SpeY and (B) SpeY under the assay conditions described in the text. (C) Preparation and isolation of four diastereomers of 30. Conversion of 6 to 31 was accomplished using a set of three reactions: 1) NaBH₄, MeOH; 2) Cbz-Cl, NaHCO₃, acetone/H₂O (3:2); 3) H₂, Pd/C, MeOH/H₂O/HOAc (3:3:1). Cbz protection in the second step facilitated chromatographic separation prior to deprotection in the third step (see Supporting Information). The same protocols were also applied to convert each isolated diastereomer of 31 to the individual diastereomers of 30. (D) Extracted positive ion chromatograms at m/z 603.3 (corresponding to protonated 32) for (a) (3′S)-32 standard, (b) SpeY incubation with (2′R,3′S)-30 followed by benzyl chloroformate derivatization, (c) coinjection of a and b. (E) Time-dependent consumption of SAM (33) and (2′R,3′S)-30 with concomitant production of 5′dAdoH (35) during the SpeY-catalyzed dehydrogenation of (2′R,3′S)-30. Assays were performed by incubating 5 μM SpeY with 0.33 mM (2′R,3′S)-30, 0.5 mM SAM, and 0.5 mM sodium dithionite. The reaction was terminated by adding an equal volume of ethanol at different time points prior to analysis by HPLC and LC-MS. Error bars represent one standard deviation about the mean of two duplicate assays.

To establish the substrate specificity of SpeY, four diastereomers of 30 were prepared differing with respect to their stereochemistry at C2′ and C3′ (see Figure 4C and Supporting Information, section S4). Of the four diastereomers prepared, only (2′R,3′S)-30 (250 μM) showed significant consumption during a 3 h incubation with SpeY, SAM, DTT, and dithionite under the aforementioned conditions. The resulting product had a mass consistent with loss of H₂ from (2′R,3′S)-30 (calcd [M + H]⁺ m/z 335.1813; obsd 335.1814), suggesting dehydrogenation of either the C2′ or C3′ hydroxyl group. Derivatization with benzyl chloroformate yielded a species identified as (3′S)-32 based on coelution with a synthetic standard during LC-MS analysis (see Figure 4D). ¹H NMR spectroscopy following isolation and deprotection was indicative of (3′S)-31 (see Supporting Information, Figures S30–S33). Consequently, SpeY appears to be specific for the (2′R,3′S) diastereomer of 30 and catalyzes regiospecific dehydrogenation of the C2′ hydroxyl group to yield (3′S)-31.

Dehydrogenation of the C2′ alcohol in (2′R,3′S)-30 immediately yields the bicyclic species 37 prior to intramolecular cyclization to generate the 1,4-dioxane core in (3′S)-31 (see Figure 5A). The cyclization involves hemiacetal formation and could in principle proceed with addition of the C6-OH of actinamine at either the re or si face of the newly introduced carbonyl at C2′ to yield two different diastereomers. However, only (3′S)-31 was observed as the cyclized product, which is consistent with the observed stereochemistry of isolated spectinomycin and the synthetic preparations of (3′S)-31. Gas-phase calculation (RB3LYP/6–31G**) of the optimized dioxane configurations of norspectinomycin and nordihydrospectinomycin implied that inversion at C2′ leads to 2.5 and 7.2 kcal/mol changes in free energy favoring the observed tricyclic diastereomers, respectively (see Supporting Information). Assuming that the two conformers have equivalent free energies of solvation, this implies that essentially all (i.e., >98%) of the equilibrated hemiacetals will reside in the experimentally observed conformation (i.e., (3′S)-31) at 300 K. Consequently, dioxane ring formation likely proceeds nonenzymatically following dehydrogenation of C2′.

Figure 5.

(A) Two possible mechanisms for the SpeY-catalyzed dehydrogenation of (2′R,3′S)-30. (B) ESI-MS of (3′S)-31 (m/z [M + H]⁺ 335.2) generated in the reaction of SpeY with (1) natural abundance (2′R,3′S)-30, and (2) [2′-²H]-(2′R,3′S)-30. (C) ESI-MS of 5′dAdoH (35, m/z [M + H]⁺ 252.1) generated in the reaction of SpeY with (1) natural abundance (2′R,3′S)-30 and (2) [2′-²H]-(2′R,3′S)-30.

SpeY-catalyzed dehydrogenation of (2′R,3′S)-30 is dependent on the presence of SAM, which is consumed roughly one-to-one with (2′R,3′S)-30 to generate 5′-deoxyadenosine (5′dAdoH, 35, see Figure 4E). At later time points, additional SAM consumption can be observed in excess of (2′R,3′S)-30, suggesting a low level of SAM reduction uncoupled from dehydrogenation of the C2′ hydroxyl upon the accumulation of product. These observations are consistent with net reduction of SAM via an intermediary 5′-deoxyadenosyl radical equivalent (5′dAdo•, 34) and H atom transfer to the C5′ methyl of 35 (Figure 5A). However, the dehydrogenation could proceed via hydrogen atom abstraction from either C2′ (route a) or C3′ (route b) to generate radical intermediate 36 or 38, respectively (Figure 5A). Subsequent deprotonation and one-electron oxidation possibly via electron transfer to the oxidized auxiliary cluster would then introduce the corresponding ketone (36 → 37 → (3′S)-31 or 38 → (2′S)-21). The putative C3′ ketone 21 from route b could then undergo tautomerization to give 37 prior to cyclization.

To identify the site of H atom abstraction from (2′R,3′S)-30, the corresponding C2′-deuterated isotopologue [2′-²H]-(2′R,3′S)-30 was prepared (99% atom D, see Supporting Information, section S4). Incubation of the isotopologue with 5 μM SpeY, 1 mM SAM, 2 mM sodium dithionite, and 2 mM DTT in 50 mM HEPES buffer (pH 8.0) for 1 h resulted in >95% monodeuterated 5′-deoxyadenosine (35) with no observable retention of the label in the product (3′S)-31 (Figure 5B,C). Moreover, the product was unlabeled when the reaction was repeated with natural abundance (2′R,3′S)-30 in buffered D₂O (Figure S11). Finally, SpeY was unable to catalyze the tautomerization of (2′S)-21 to 37 and (3′S)-31 as alluded to above. These findings indicated that SpeY-catalyzed dehydrogenation of (2′R,3′S)-30 proceeds via H atom abstraction from C2′ (route a) by the putative 5′dAdo radical or its equivalent (34) without the intermediacy of an enediol.

The C2′ α-hydroxyalky radical (36) must be oxidized and the ${[{\text{Fe}}_{\text{4}}{\text{S}}_4]}_{\text{rad}}^{2+}$ cluster reduced back to the ${[{\text{Fe}}_{\text{4}}{\text{S}}_4]}_{\text{rad}}^{1+}$ state to complete the catalytic cycle. Previous work on other SPASM/twitch radical SAM dehydrogenases has suggested that oxidation of the α-hydroxyalkyl radical may take place via electron transfer to the oxidized auxiliary cluster.^15–19 Moreover, an external intermediary such as flavodoxin may also be required to recycle the reducing equivalent from the auxiliary cluster back to the catalytic cluster.¹⁶ In contrast, evidence has been presented suggesting that DesII, which is not a member of the SPASM/twitch subgroup and lacks an auxiliary [Fe₄S₄] cluster, can catalyze dehydrogenation of TDP-quinovose with oxidation of the α-hydroxyalkyl radical by ${[{\text{Fe}}_{\text{4}}{\text{S}}_4]}_{\text{rad}}^{2+}$.³⁷ Furthermore, there are examples of oxidative radical SAM enzymes such as HemN/CgdH³⁸ that do not bind auxiliary [Fe₄S₄] clusters. Consequently, an auxiliary cluster and an external redox carrier may not necessarily be essential features for the catalysis of dehydrogenation reactions by twitch radical SAM enzymes.

To assess the importance of the auxiliary cluster to the SpeY-catalyzed dehydrogenation, the four cysteine residues that coordinate the auxiliary cluster (i.e., C175, C194, C241, and C244, see below) were mutated to alanine. The resulting quadruple mutant and the C175A single mutant were both inactive, being unable to catalyze either dehydrogenation of (2′R,3′S)-30 or reduction of SAM. Moreover, incubation of the mutants with SAM and [2′-²H]-(2′R,3′S)-30 did not show significant deuterium transfer to SAM or depletion of deuterium from the residual substrate. Consequently, the intact auxiliary cluster may play a more general role in organizing the active site for catalysis, given the apparent inability of this mutant to facilitate even the reductive cleavage of SAM.

To determine whether SpeY catalysis can take place in the absence of an external source of reducing equivalents, the enzyme was first preincubated with 0.4 mM sodium dithionite for 15 h, thereby allowing the excess reductant to decompose.^37,39 Subsequent addition of 0.25 mM substrate, 0.5 mM SAM, and 1 mM DTT led to only a single turnover of (2′R,3′S)-30 to (3′S)-31 per protein monomer (see Figure S7). In contrast, unreduced SpeY was inactive both in the absence of dithionite and when combined with the deproteinized filtrate from the overnight incubation of SpeY with dithionite. However, the prereduced SpeY could be induced to catalyze multiple turnovers by including 0.5 mM fresh dithionite in the assay. Consequently, the putative α-hydroxyalkyl radical does not appear to reduce the catalytic cluster either directly or via the auxiliary cluster. This is consistent with the hypothesis that an external redox carrier participates in SPASM/twitch radical SAM-catalyzed dehydrogenation reactions.¹⁶

Additional insight into the catalytic mechanism of SpeY was provided by crystal structures of the SpeY·SAM binary complex (2.02 Å, PDB: 7X0B) as well as the ternary complexes bound with substrate (SpeY·SAM·[(2′R,3′S)-30], 1.90 Å, PDB: 7WZV) and product (SpeY·SAM·37, 1.98 Å, PDB: 7WZX) as shown in Figures 6A and S15 (statistics provided in Table S3). These crystal strutures were determined using the SpeY-E269A/E270A double mutant, which was identified using surface entropy reduction analysis⁴⁰ and found to crystallize better than the wildtype (see Supporting Information, section S5.1 and Figure S14). Both E269 and E270 are on the protein surface, and no significant difference in activity was observed between the wildtype and E269A/E270A mutant. The structure of SpeY·SAM is similar to that of SpeY·SAM·[(2′R,3′S)-30] (rmsd 0.343 Å, Figure S15B), which suggests that a significant conformational change is not required for substrate binding at least in crystallo.

Figure 6.

Crystal structure of the SpeY·SAM·[(2′R,3′S)-30] ternary complex. (A) Homodimeric quaternary structure. (B) Secondary and tertiary structure of the SpeY monomer. (C) Putative binding interactions between SAM and SpeY within the active site including the CX₃CX₂C motif (C24, C28, and C31 binding with the [Fe₄S₄]_rad cluster; S30 and A32 are within hydrogen bonding distance of the adenine moiety of SAM), GGE motif (G68, G69, and E70), ribose motif (E117, S119, Y121, and Y150 originating from β4 and β5), GXIXGXXE and the β6 motif (F153 and R154) (bound (2′R,3′S)-30 has been hidden to improve visualization). (D) Putative binding interactions between (2′R,3′S)-30 (tan) and SpeY within the active site. (E) The 2Fo–Fc density maps for the bound substrate ((2′R,3′S)-30) and product demonstrating that the product binds in the bicyclic form (37). (F) Relative orientation of SAM (cyan) and (2′R,3′S)-30 (tan) with respect to the radical SAM ([Fe₄S₄]_rad) and auxiliary ([Fe₄S₄]_aux) clusters in the SpeY active site. (G) Orientation of Ser183, substrate C2′, and SAM C5′ in the ternary complex.

The radical SAM domain (residues 1–154) ligates the [Fe₄S₄]_rad cluster via residues C24, C28, and C31, which make up the conserved CX₃CX₂C motif. SpeY lacks the complete β₆/α₆ radical SAM core fold⁴¹ and instead exhibits a β₆/α₄ structure similar to that of BtrN (Figures 6B and S17).¹¹ SAM is thus bound within the SpeY active site via contacts with the GGE, β4 ribose, GXIXGXXE, and β6 motifs as observed in other radical SAM enzymes (Figure 6C).⁴² The SpeY twitch domain (residues 165–244) consists of a β-hairpin (β1′ and β2′) and four α-helixes (Figure 6B, see Supporting Information, sections S5.4 and S15). All four iron centers of the auxiliary [Fe₄S₄] cluster are ligated by C175, C194, C241, and C244.^11,13,14 The shortest distance between the catalytic and auxiliary clusters is 14.2 Å, placing them roughly equidistant from C2′ of the bound substrate at 8.9 and 9.8 Å, respectively (Figure 6F). The C-terminal domain (residues 251–302) is composed of two β-sheets (β3′ and β4′) and a helix (α5′), which lies adjacent to the two [Fe₄S₄] clusters. These two β-sheets make up β-hairpins with β6 and β5, respectively.

The substrate (2′S,3′S)-30 binds in the SpeY active site in a chair conformation. The actinospectose moiety of (2′S,3′S)-30 is stabilized by H-bond interactions with E17, H33, and K65, while the actinamine component is stabilized via interactions with N148, Y150, and W181 (Figure 6D). Only E17 and K65 are within hydrogen bonding distance of the C2′ hydroxyl group (2.6 and 2.8 Å, respectively) and thus may serve as proton acceptors during oxidation of the C2′-hydroxyl group (Figure S18). The product binds in a similar, bicyclic conformation (i.e., as 37), consistent with cyclization taking place nonenzymatically after release from the active site as discussed above (Figures 6E and S19); however, it should be noted that SpeY·SAM·37 differs from the actual product complex, which is expected to have l-methionine and 5′-deoxyadenosine bound instead of SAM. SAM binds deeper in the binding pocket where it appears to be locked in place by the substrate, which suggests that (2′S,3′S)-30 binds after SAM. The C2′ carbon of the bound substrate is roughly colinear with the C5′ methylene of SAM (4.0 Å) and the hydroxyl group of serine-183 (5.0 Å, see Figure 6G). This renders C2′ of (2′S,3′S)-30 the most accessible site for H atom abstraction by the 5′-deoxyadenosyl radical equivalent following reductive cleavage of SAM, which is consistent with the deuterium transfer experiments involving [2′-²H]-(2′R,3′S)-30.

Given the high degree of sequence identity (48%) between SpeY and HygY, the S183C mutant of SpeY was prepared to determine whether it would catalyze an analogous epimerization reaction. Thus, 0.5 mM (2′S,3′S)-30, 1 mM SAM, 2 mM sodium dithionite, and 2 mM dithiothreitol (DTT) were incubated overnight (16 h) with 5 μM SpeY-S183C in 50 mM HEPES buffer (pH 8.0) prior to derivatization with benzyl chloroformate and LC-MS analysis. As shown in Figure 7, complete consumption of (2′S,3′S)-30 was observed along with the formation of a new product that coeluted with the Cbz-derivatized C2′ epimer (2′S,3′S)-30 (i.e., (2′S,3′S)-39, calcd [M + H]⁺ m/z 605.2705; obsd 605.2725). The C2′ dehydrogenation product could also be observed but only as a minor product (Figure S13). When the experiment was repeated in buffered D₂O, the epimerized product ((2′S,3′S)-30) acquired a single deuterium label with no significant deuterium incorporation into the residual substrate (Figure S12). This result was corroborated when overnight incubation of [2′-²H]-(2′R,3′S)-30 with the SpeY-S183C mutant in buffered H₂O led to loss of the deuterium label in the epimerized product but not the residual substrate (Figure S12). These results imply that the putative α-hydroxyalkyl radical is stereoselectively reduced within the mutant active site and not prematurely released.

Figure 7.

Epimerization activity of the SpeY-S183C mutant. (a) Cbz-derivatized (2′S,3′S)-30 standard (i.e., (2′S,3′S)-39). (b) Cbz-derivatized (2′R,3′S)-30 standard (i.e., (2′R,3′S)-39). (c) EIC of Cbz-derivatized (2′S,3′S)-30 following overnight incubation of 30 with the SpeY-S183C mutant in buffered D₂O. (d) EIC of Cbz-derivatized (2′S,3′S)-30 and Cbz-derivatized [2′-²H]-(2′R,3′S)-30 following overnight incubation of [2′-²H]-(2′R,3′S)-30 with the SpeY-S183C mutant.

The stereoselectivity of SpeY-S183C-catalyzed epimerization is consistent with the location of S183/C183 in the crystal structure of wildtype SpeY (Figures 6G and S6). Moreover, this reaction is analogous to the HygY-catalyzed epimerization of galacamine (1) to talamine (2) during the biosynthesis of hygromycin B (see Figure 1A).²⁰ To obtain some insight regarding the most recent common ancestor of HygY and SpeY, a phylogenetic analysis of SpeY and HygY was performed using sequences of similar length from the radical SAM Megacluster-1-1 (SPASM/twitch) database⁴³ (see section S6 of the Supporting Information for details). A minimum evolution tree constructed from the 50 sequences nearest SpeY (Poisson corrected p-distances)⁴⁵ identified adjacent clades in the genus Streptomyces representative of SpeY and HygY isoforms bearing homologous Ser and Cys residues, respectively (Figure 8). In comparison to the HygY isoforms, homologues more distant from the SpeY isoforms grouped into clades largely in the phylum Proteobacteria with homologous cysteine residues. A similar result was obtained when the analysis was centered on HygY (see Supporting Information, section S6). This patterning suggests a relatively recent Cys → Ser mutation of an enzyme encoded within the genome of an ancestral form of Streptomyces following putative horizontal gene transfer from the Proteobacteria lineage.

Figure 8.

Minimum evolution tree constructed on the 50 unique sequences of similar length nearest to SpeY in the Megacluster-1-1 (twitch/SPASM) sequence database⁴³ based on Poisson-corrected p-distances following multiple sequence alignment using the MEGAX software package.⁴⁴ Extant taxa have been grouped according to the genus or phylum (Ph) of the producing strains and labeled with the residue that aligns with serine-183 of SpeY in the multiple sequence alignment. Ancestral taxa have been labeled with the proposed homologous residue. The outgroup included BtrN and NeoN as described in Supporting Information. The analogous tree centered on HygY is provided in Figure S2.

CONCLUSIONS

SpeY is a twitch radical SAM dehydrogenase that catalyzes C2′ oxidation of a pseudodisaccharide precursor during the biosynthesis of spectinomycin. While this reaction can facilitate intramolecular hemiacetal formation resulting in the characteristic dioxane bridge, additional work will be required to established its precise biosynthetic role and in particular how the actinospectose moiety (8) is constructed. Nevertheless, both (3′S)-31 and (3′R)-31 are known impurities in commercial preparations of spectinomycin consistent with a potential role in biosynthesis.⁴⁶ Mutation of serine-183 to cysteine converts SpeY from a C2′ dehydrogenase to a C2′ epimerase. This significant change in the redox chemistry can be rationalized from the crystal structure of the ternary Michaelis complex where the S_γ/O_γ center of residue-183 forms a roughly colinear array with C2′ of the substrate and C5′ of SAM. This configuration may therefore facilitate formation of the putative C2′ α-hydroxyalkyl radical intermediate (36) as well as its subsequent reduction via H atom transfer from the cysteine residue in the S183C mutant (Figure S6). In the wildtype S183 variant, however, the intermediary radical is instead oxidized in a process that may involve the auxilary [Fe₄S₄] cluster. The mechanistic details of SpeY catalysis are currently under investigation.

The catalytic interchangeability of SpeY via the S183C mutation directly mirrors that of the homologous twitch radical SAM epimerase HygY.²⁰ Consequently, SpeY represents a naturally selected variant of the artificially constructed HygY-C183A mutant, which likewise acts as a dehydrogenase. Phylogenetic analysis suggests that SpeY and HygY are the descendents of a twitch radical SAM enzyme bearing a catalytic cysteine residue, which is suggestive of redox-neutral (e.g., epimerization) as opposed to oxidative chemistry (e.g., dehydrogenation) in the ancestral twitch radical SAM enzyme. On the basis of these observations, SpeY and its nearest homologues are proposed to represent an enzymological clade that diverged as oxidases from a lineage of twitch radical SAM enzymes catalyzing epimerizations or other redox neutral transformations. How other mutations contributed to the diversification of these radical SAM enzyme activities as well as the mechanisms by which the gene clusters were inherited remain questions under investigation.