Biosynthesis of the Thiofuranose Core in Albomycin Requires a Versatile Enzyme AbmG That Catalyzes Net Dehydration via Cryptic Phosphorylation

Abstract

Albomycins are unusual sulfur-containing nucleosides from the species of Streptomyces that exhibit potent antibiotic activities against both Gram-negative and Gram-positive bacteria including clinical pathogens. Previous studies demonstrated that the twitch radical SAM enzyme AbmM catalyzes an oxidative sulfur-for-oxygen swapping reaction converting CDP to a 4′-hydroxy-4′-thiocytidine 5′-diphosphate intermediate in the initial step of albomycin biosynthesis. However, the fate of this intermediate in the biosynthetic pathway has remained elusive. Herein, the above intermediate after 5′-dephosphorylation is shown to undergo AbmG-catalyzed transformations via a cryptic double phosphorylation of its 4′-hydroxyl group followed by C–O bond cleavage to yield 5′-oxo-4′-thiocytidine. X-ray crystal structure analysis and site-directed mutagenesis of AbmG revealed Glu188 as the general base to perform C5′ deprotonation. Subsequent mechanistic studies using deuterated substrates demonstrated that the deprotonation at C5′ is pro-R specific and likely occurs concerted with elimination of pyrophosphate from C4′. This study not only highlights a unique nucleoside kinase with lyase activity to complete an overall dehydration reaction but also fills the gaps in the biosynthesis of the atypical thiofuranose core essential to the biological activities of albomycins.

Graphical Abstract:

INTRODUCTION

Albomycins (1–3) are sulfur-containing peptidyl nucleosides produced by Streptomycetes.^1–4 They contain a unique cytidyl thioheptose core which upon coupling with an L-serine residue gives rise to the active component of albomycins, SB-217452 (4) (Figure 1a).⁵ SB-217452 (4) is a potent seryl-tRNA synthetase inhibitor owing to its structural resemblance to seryl-adenylate, an intermediate during seryl-tRNA synthesis.⁵ Albomycins are also members of the sideromycin family due to the presence of the ferrochrome moiety in the structures. The conjugation of SB-217452 (4) with the iron-chelating siderophore 5 can facilitate the efficient uptake of albomycins by both Gram-negative and Gram-positive bacteria through their iron acquisition systems thereby conferring potent antibacterial activities to albomycins.^6,7

Figure 1.

(a) Proposed biosynthetic pathway of albomycins. (b) Formation of uridine-5′-aldehyde (12) in the biosynthesis of pyrimidine nucleoside natural products.

The highly unusual structure and potent biological activities of albomycins have prompted a number of investigations into their biosynthesis.^8–11 The antibiotic core SB-217452 (4) has been shown to be biosynthesized from the 5′-oxo-4′-thionucleoside intermediate 6 (Figures 1a and S1).^8,9 Elongation of the side chain of 6 is catalyzed by the PLP-dependent transaldolase AbmH via an aldol-type reaction between 6 and L-threonine.⁸ The tailoring modifications of the resulting α-amino-β-hydroxyl intermediate 7 include C6′ epimerization catalyzed by the PLP-dependent enzyme AbmD,⁸ methylation and carbamoylation of the cytosine moiety catalyzed by AbmI and AbmE, respectively,¹⁰ conjugation with a serine residue catalyzed by AbmF, and C3′ epimerization catalyzed by the radical S-adenosyl-l-methionine (SAM) enzyme AbmJ to give 4 (Figure S1).⁹ Formation of the siderophore 5 from three N-acetyl-N-hydroxylornithine units and the subsequent attachment of 5 to 4 are proposed to be catalyzed by AbmQ and AbmC, respectively; however, the details of these reactions have not been fully characterized. Importantly, the twitch radical SAM enzyme AbmM was recently demonstrated to catalyze an oxidative sulfur-for-oxygen swapping transformation of CDP (8) to generate 9,¹¹ a key step for the assembly of the thiofuranose core in albomycin. It was noted that the AbmM-catalyzed reaction not only incorporates the sulfur atom bridging C4′ and C1′, but also leaves the original ring oxygen as a hydroxyl group at C4′ as a remnant of the oxidation. This C4′ hydroxyl appendage needs to be removed to complete the construction of 6. However, how it is eliminated to transform the nascent AbmM product 9 to 5′-oxo-4′-thionucleoside (6) remains obscure and is the focus of the work reported herein.

RESULTS AND DISCUSSION

Gene Deletion of abmG and abmL.

Study of the biosynthesis of pyrimidine nucleosides has shown that the 5′-oxo form of uridine (e.g., 12), which is an analogue of 6, participates in the aldol addition that forms the C5′–C6′ bond to produce high-carbon nucleosides such as caprazamycin, liposidomycin, and muraymycin (Figure 1b).^12,13 The 5′-amination of 12 is also important for the assembly of pacidamycin and mureidomycin.¹⁴ Formation of 12 via C5′ oxidation of UMP (10) or uridine (11) is generally catalyzed by two types of enzymes, a LipL-type α-ketoglutarate (α-KG)-dependent nonheme iron oxygenase¹⁵ or a PacK-type FAD-dependent oxidase.¹⁴ However, a similar mechanism is unlikely during albomycin biosynthesis, because 9 is already in the same oxidation state as 6 and 12, and no gene encoding an α-KG-dependent nonheme iron oxygenase or an FAD-dependent oxidase can be located in or around the albomycin biosynthetic gene cluster except for abmB, a flavin-dependent oxidase that encodes an ornithine N⁵-hydroxylase.¹⁶ Furthermore, no gene candidate whose product could catalyze C4′ reduction of 9 could be found.

Among the few unassigned genes in the abm gene cluster, the protein encoded by abmG (AbmG) shares a similar sequence with enzymes of the nucleoside/nucleotide kinase (NK) superfamily (e.g., 34% identity/54% similarity to the deoxynucleoside kinase in Escherichia coli).^17,18 Thus, AbmG may be responsible for phosphorylation of the C4′-hydroxy group of 9 before or after 5′-dephosphorylation to facilitate subsequent cleavage of the C4′–O bond and rearrangement to 6. Consistent with this hypothesis, deletion of the abmG gene in S. sp. ATCC 700974 completely abolished production of albomycin δ₂ (1) (Figure S2a). Production of 1 was restored by introducing the abmG gene back into the deletion mutant (Figure S2b). Moreover, while neither 9 nor 13 was found as a metabolite produced by the wild type strain, a species that coeluted with a standard of 13, which had been prepared and fully characterized in a previously published work,¹¹ and having the same molecular mass was observed when the cell homogenate of the ΔabmG mutant was analyzed by LCMS (Figure S2c). This observation showed that in vivo conversion of 9 to 13 may be mediated by a cellular phosphatase. While abmL in the abm gene cluster is predicted to encode a phosphodiesterase suggesting that AbmL may catalyze the hydrolysis of 9 into 13, knockout of abmL in S. sp. ATCC 700974 did not abolish the production of albomycin δ₂ (1) (Figure S2d). This result indicated that AbmL need not be involved in the albomycin biosynthesis, but instead a phosphatase not encoded in the abm gene cluster may be responsible for the transformation of 9 to 13 in vivo, because bacterial phosphatases can exhibit broad substrate specificity.¹⁹ However, the possibility cannot be fully excluded that another enzyme encoded in the abm gene cluster catalyzes the hydrolysis of 9 as a secondary activity in addition to its primary biosynthetic function.

Functional Characterization of AbmG.

To test the proposed function of AbmG, N-His₆-tagged AbmG was expressed and purified from E. coli (Figure S2e). When 13 (0.39 mM) was incubated with AbmG (4.8 μM) for 20 h in the presence of adenosine 5′-triphosphate (ATP, 2.5 mM) and MgCl₂ (5.0 mM), 13 was almost completely transformed to a new species with the same molecular mass (Figures 2b and S2i). This reaction was ATP-dependent (Figure S2f). While 13 is inert to 2,4-dinitrophenylhydrazine (DNPH), the new product could be derivatized with DNPH to the corresponding hydrazone (Figure S3a,b) suggesting the presence of a carbonyl group. Furthermore, incubation of the AbmG product with AbmH led to a single product with mass equal to that of 7 (Figure S3c,d).⁸ Thus, the AbmG product was assigned as 6/18. When the AbmM-AbmG coupled reaction with 8 was conducted, 6/18 was not produced, instead 9 produced by AbmM was left in the reaction mixture (Figure S3e). This finding implied that 9 is not a substrate for AbmG and must first be dephosphorylated to 13 prior to the AbmG-catalyzed rearrangement.

Figure 2.

AbmG-catalyzed formation of 6/18. (a) Proposed mechanism of the AbmG-catalyzed conversion of 13 to 6, which is known to exist in an equilibrium with its hydrate form 18.⁸ (b) HPLC analysis of the AbmG reaction with 13. (c) HPLC analysis of the reaction of 13 with AbmG variants.

Within the first 2 h of incubating AbmG with 13, an intermediate having a mass consistent with a monophosphorylated product accumulated as the substrate (13) was consumed (Figures 2b and S2g). This intermediate was assigned as 14, because no ³J_P–H coupling was observed by ¹H NMR implying phosphorylation at 4′-O (see Supporting Information). No correlation was observed between 5′-H and 6-H of the cytosine nucleobase in the NOESY spectrum of 14 consistent with 4′S stereochemistry (Figure S14), which suggests that AbmG selectively recognizes the 4′S epimer of 13. Upon prolonged incubation, intermediate 14 was further phosphorylated to give the 4′-O-pyrophosphate 15 (Figures 2b and S2h). The presence of the diphosphate group in 15 was confirmed by a colorimetric assay using disodium molybdate(VI) (Figure S3f).²⁰ The proposed 4′S stereochemistry in 14 and 15 was verified by the crystal structure of AbmG-E188Q in complex with 15, which is discussed below.

Possible Rearrangement Mechanisms.

While deoxynucleoside kinases typically catalyze 5′-phosphorylation of nucleoside substrates in the nucleotide salvage pathway,¹⁷ and some kinases catalyze stepwise phosphorylation to yield 5′-diphosphate nucleotides,²¹ the catalytic function of AbmG is unusual, because it performs both regioselective 4′-O-phosphorylation and subsequent elimination of the 4′-O-pyrophosphate. Following the formation of 15, several possible pathways can be envisioned for the elimination step as shown in Figure 2a. Departure of pyrophosphate may result in a thiocarbenium ion (16) before a base deprotonates C5′-H to produce enol 17, which tautomerizes to form 6 (route A). The same enol intermediate (17) could also be generated via a concerted pyrophosphate elimination mechanism without the intermediary thiocarbenium ion (16) (route B). Alternatively, a hydride at C5′ in 16 could shift to the electron deficient C4′ to directly afford 6 (route C). To distinguish between these mechanisms, the AbmG reaction with 13 was conducted in buffered D₂O (>99% D). Observation of nearly complete single deuterium atom incorporation into 18 (Figure S4a) excluded the possibility of the 1,2-H shift mechanism, which would have predicted that both H atoms at C5′ of the substrate 13 be retained in the product 18 (route C).

X-ray Crystal Structure of AbmG.

The structure of AbmG was next investigated to understand how this enzyme catalyzes both 4′-O-phosphorylation and pyrophosphate elimination to effect the rearrangement of 13 to 6. While crystals of native AbmG could not be grown, AbmG lacking the seven N-terminal amino acids was found to be catalytically active and could be readily crystallized. Therefore, this construct was used to solve the structures of AbmG bound with (4′S)-13 (AbmG/(4′S)-13) and AbmG bound with cytidine and ADP (AbmG/cytidine/ADP) at 1.3 and 1.7 Å resolution, respectively. The overall structure of AbmG resembles those of deoxynucleoside kinases, which typically consists of a five-stranded parallel β sheet surrounded by nine α-helices (Figure S5).^17,22,23 The substrate binding site is located near the C-terminus of the parallel β sheet (Figures 3a and S7a). The cytosine nucleobase of (4′S)-13 is sandwiched between Phe96 and Phe127, with which it has π-π interactions, and H-bonds with Gln93, Asp124, and His131. The α-anomeric face of the thiofuranose ring of (4′S)-13 interacts with two aromatic residues Trp202 and Phe82, while the 2′-hydroxy group is anchored by Asn42 and Arg119 through a water molecule. The conserved Glu70 and Arg119 are adjacent to the β-anomeric face of the thiofuranose ring, where the phosphoryl transfer reactions take place.¹⁷

Figure 3.

Structural analysis of the AbmG-catalyzed reactions with 13. (a) The active site structure of the AbmG/(4′S)-13 binary complex. The red spheres indicate the oxygen atoms of water molecules. (b) The active site of the AbmG/15 complex.

The binding mode of cytidine in the structure of AbmG/cytidine/ADP is almost identical to that of (4′S)-13; however, the diphosphate group of ADP binds to the conserved P-loop¹⁸ (Figure S8) consisting of A44AGKTT49, which adopts a different conformation compared to that in the binary AbmG/(4′S)-13 structure (Figures S6 and S7c). Arg119 and Arg185 are positioned to H-bond with the terminal phosphate group of ATP; however, no direct interaction with ADP is observed in the AbmG/cytidine/ADP structure (Figure S6). Deprotonation of the 4′-hydroxy group in (4′S)-13 may be required for the initial phosphorylation step ((4′S)-13 → 14, Figure 2a) as is generally believed to be the case for deoxynucleoside kinases.¹⁷ While the conserved Glu70 forms salt bridges with Arg100 and Arg119, it could approach the 4′-hydroxy group of (4′S)-13 after conformational change (Figure 3a), and complete loss of AbmG activity was noted upon mutation of Glu70 to Gln (Figure 2c). Therefore, Glu70 may play an essential role in deprotonating the 4′-hydroxy group to facilitate phosphorylation of (4′S)-13.

Stereoselectivity of Pyrophosphate Elimination.

The AbmG residue Glu188 is conserved among deoxynucleotide kinases where it typically forms a H-bond with the 3′-hydroxy group of the nucleoside substrate.^17,22 In the AbmG/(4′S)-13 structure, however, Glu188 is instead at the entrance to the active site in proximity to C5′ of (4′S)-13, and the side chain carboxylate protrudes toward solvent (out-conformation) rather than interacting with the substrate (Figure 3a). Nevertheless, incubation of the AbmG-E188Q mutant with 13 led to accumulation of 15 without formation of 6/18, indicating that Glu188 is crucial for the elimination of pyrophosphate from 15 and may serve as a general base to deprotonate C5′-H of either 15 or 16 (Figures 3a and 4). To ascertain the stereochemistry of C5′-H deprotonation, the isotopologs (5′R)-[5′–²H]-13 and (5′S)-[5′–²H]-13 were chemoenzymatically synthesized and assayed with AbmG (see Supporting Information). When (5′R)-[5′–²H]-13 was used, no deuterium was found in 18, whereas the deuterium was retained in 18 after the reaction with (5′S)-[5′–²H]-13 (Figure S4b). These results indicate that the 5′-pro-R-H of 15 is selectively removed during elimination of pyrophosphate.

Figure 4.

Proposed catalytic mechanism of AbmG.

Structure Analysis of AbmG in Complex with the Diphosphate Intermediate.

To gain further structural information about pyrophosphate elimination, the crystal structure of the AbmG-E188Q mutant (with the same N-terminal truncation) complexed with intermediate 15 was solved at 1.5 Å (Figures 3b and S7b). Notably, Gln188 has the in-conformation with an O(Gln188)–C5′ (15) distance of 2.9 Å in addition to the out-conformation observed in the wild type enzyme. This is consistent with Glu188 acting as a flexible general base for C5′ deprotonation after diphosphorylation of the 4′-hydroxy group (Figure 3b). Moreover, the guanidinium side chain of Arg185 also interacts with the 5′-hydroxy group of 15, which may orient the 5′-pro-R-H atom within close proximity of Glu188. In the crystal structure, the distances between O(Gln188) and the 5′-pro-R-H versus 5′-pro-S-H are estimated to be 2.0 and 3.2 Å, respectively. Finally, the diphosphate group of 15 is coordinated by three positively charged residues (Arg119, Lys47, and Arg185) and three hydrogen bond donors (His74, Thr43, and the amide N–H of Ala44), which are expected to facilitate departure of the pyrophosphate leaving group consistent with this mechanism (Figure 4).

Deuterium KIE Analysis.

To determine whether departure of the pyrophosphate and C5′ deprotonation occur in a concerted or stepwise manner (Figure 2a, route A or B, respectively), the kinetic isotope effect (KIE) induced on V/K was measured when chemoenzymatically prepared [5′,5′–²H₂]-15 is used as the substrate. In the stepwise mechanism (15 → 16 → 17 → 6, Figure 2a), if the elimination step (15 → 16) is essentially irreversible, then it would impose a large forward commitment on the subsequent deprotonation step (16 → 17).^24–26 Consequently, any deuterium isotope effect on deprotonation of 16 measured with [5′,5′–²H₂]-15 would necessarily be masked in the observed V/K isotope effect. On the other hand, if deprotonation takes place concerted with elimination, then the corresponding deuterium isotope effect would not necessarily be masked. The V/K isotope effect was measured competitively by following changes in isotopolog enrichment of both substrate and product versus fraction of reaction when a roughly 1:1 mixture of 15 and [5′,5′–²H₂]-15 was incubated with AbmG (see Supporting Information for details). A V/K isotope effect of 2.9 ± 0.3 was thus obtained indicating a primary KIE associated with C5′ deprotonation of 15. This indicates that C5′ deprotonation and elimination of the 4′-pyrophosphate from 15 either occur in a concerted process (Figure 2a, route B, 15 → 17) or elimination of the pyrophosphate from 15 to yield the thionium 16 is readily reversible. Nevertheless, concerted elimination is also consistent with the anti-periplanar configuration between the 4′-pyrophosphate group and the 5′-pro-R-H of 15 in the AbmG active site (see Figure 4) as well as the observed stability of 15, which can be isolated without nonenzymatic elimination of pyrophosphate.

CONCLUSIONS

In summary, this study delineates the transformations of the AbmM product into the previously identified 5′-oxo-4′-thiocytidine intermediate during albomycin biosynthesis. The AbmM-catalyzed sulfur atom insertion into CDP is followed by 5′-dephosphorylation before the kinase AbmG catalyzes an unusual dehydration reaction involving 4′-O-diphosphorylation. AbmG thus serves not only as a kinase that catalyzes phosphorylation of the substrate, but also as a lyase that catalyzes subsequent C4′–O bond cleavage likely in concert with C5′-deprotonation thereby generating the aldehyde precursor to the remaining albomycin biosynthetic pathway.^8,9 Consequently, the biosynthetic pathway of the biologically active core SB-217452 in albomycins has been fully elucidated. This study also reveals a novel multifunctional enzyme in the nucleoside kinase superfamily specialized for the biosynthesis of sulfur-containing nucleoside natural products.

Supplementary Material

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.5c12827.

Additional experimental details, materials, and methods, including chemical synthesis, in vitro enzymatic assays, supplementary tables and figures, and NMR spectra of synthetic compounds (PDF)