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. Crystallographic 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.

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.

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.

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.

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.

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.

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.

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.

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 database based on Poisson-corrected p-distances following multiple sequence alignment using the MEGAX software package. 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.