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Published in final edited form as: ACS Chem Biol. 2024 Oct 15;19(11):2277–2283. doi: 10.1021/acschembio.4c00510

2-Deoxy-4-epi-scyllo-inosose (DEI) is the Product of EboD, A Highly Conserved Dehydroquinate Synthase-like Enzyme in Bacteria and Eustigmatophyte Algae

Samuel Tanoeyadi †,‡, Wei Zhou †,‡,⊥, Andrew R Osborn †,§, Takeshi Tsunoda †,, Arash Samadi †,#, Sachin Burade †,ƒ, Ty J Waldo †, Melanie A Higgins ζ, Taifo Mahmud †,*
PMCID: PMC11567786  NIHMSID: NIHMS2028638  PMID: 39404639

Abstract

A cryptic cluster of genes, known as the ebo cluster, has been found in a variety of genomic contexts among bacteria and algae. In Pseudomonas fluorescens NZI7, the ebo cluster (a.k.a. EDB cluster) is involved in the bacterial repellent mechanism against nematode grazing. In cyanobacteria, the cluster plays a role in the transport of the scytonemin monomer from the cytosol to the periplasm. Despite their broad distribution and interesting phenotypes, neither the pathway nor the functions of the enzymes are known. Here we show that EboD proteins from the ebo clusters in Nostoc punctiforme and Sporocytophaga myxococcoides catalyze the cyclization of mannose 6-phosphate to a novel cyclitol, 2-deoxy-4-epi-scyllo-inosose. The enzyme product is postulated to be a precursor of a signaling molecule or a transporter in the organisms. This study shed the first light onto ebo/EDB pathways and established a functionally distinct enzyme that extends the diversity of sugar phosphate cyclases.

Graphical Abstract

graphic file with name nihms-2028638-f0001.jpg

3-Dehydroquinate synthase (DHQS, AroB) is a sugar phosphate cyclase (SPC) that catalyzes the conversion of 3-deoxy-d-arabinoheptulosanate 7-phosphate (DAHP) to 3-dehydroquinate (DHQ) in the shikimate pathway.1 As a prominent metabolic pathway in plants, algae, protozoans, fungi, archaea, and bacteria, the shikimate pathway is involved in the production of many primary and secondary metabolites including aromatic amino acids, folates, ubiquinones, lignans, and flavonoids.2 In addition to DHQS, numerous variants of DHQS with different functions have been identified in bacteria, fungi, algae, and animals.3 Those include the amino-dehydroquinate synthases (aminoDHQS),4 the 2-deoxy-scyllo-inosose synthases (DOIS),5 the 2-epi-5-epi-valiolone synthases (EEVS),6 the 5-epi-valiolone synthases (EVS),7 and the 2-desmethyl-4-deoxygadusol synthases (DDGS).8, 9 AminoDHQS converts aminoDAHP to aminoDHQ, the precursor of 3-amino-5-hydroxybenzoic acid (AHBA) unit found in many bioactive natural products (e.g., mitomycin, rifamycins, ansamitocins, and geldanamycin).10 DOIS uses glucose 6-phosphate (Glc6P) as a substrate to generate 2-deoxy-scyllo-inosose (DOI), a key precursor in the biosynthesis of aminoglycoside antibiotics (e.g., gentamicin, neomycin, and kanamycin).5 EEVS, EVS, and DDGS use sedoheptulose 7-phosphate (SH7P) as a substrate, but produce 2-epi-5-epi-valiolone, 5-epi-valiolone, and 2-desmethyl-4-deoxygadusol, respectively.7 2-epi-5-epi-Valiolone or 5-epi-valiolone are precursors of pseudo-oligosaccharides (e.g., acarbose and validamycins) and related aminocyclitols,11–15 whereas 2-desmethyl-4-deoxygadusol is a precursor of the sunscreen compounds mycosporine-like amino acids.9

In addition to these SPCs, a new clade of DHQS homologues was identified more than a decade ago,8 but their function remained elusive. The genes of these DHQS-like proteins are found in many Gram-negative bacteria and eustigmatophyte algae as part of conserved cryptic gene clusters known as the ebo clusters (Figs. S1 and S2).8, 16–19

In Pseudomonas fluorescens NZI7, the ebo cluster [a.k.a. EDB cluster (from “edible”)] is involved in the bacterial repellent of Caenorhabditis elegans to avoid the nematode grazing.19 The cluster (PFL_5540–PFL_5548) appears to regulate tryptophan catabolism by the bacteria to produce indole derivatives with C. elegans repellent activity.20 In cyanobacteria, the ebo cluster (eboA–eboF) is typically located next to the scytonemin gene cluster. Inactivation of the ebo genes in Nostoc punctiforme PCC 73102 resulted in mutants that lack the ability to couple scytonemin monomers to scytonemin (Fig. S3), which takes place in the periplasm space.18 Thus, the ebo cluster was postulated to be involved in the translocation of scytonemin monomers from the cytosol to the periplasm for final oxidative dimerization (Fig. S4).18

To investigate the distribution of the new DHQS-like proteins and their frequency relative to DHQS and other SPCs, we examined 14,784 unique sequences of DHQS and its homologues reported in the NCBI database. We found that the majority of those sequences were DHQS, but there were 341 aminoDHQS, 330 DOIS, 980 EEVS, 786 DDGS, 60 EVS, and 1533 unknown DHQS-like proteins known as PFL_5540 (in P. fluorescens) or EboD (in other organisms) (Fig. 1a–c). Most of the unknown DHQS-like proteins belong to Gram-negative bacteria and some eustigmatophyte algae. Despite that the protein is more frequently found in nature than any one of the known variants of DHQS, its catalytic function is completely unknown.

Figure 1.

Figure 1.

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Distribution of DHQS and related SPCs found in the NCBI database. (a) Phylogenetic tree of DHQS and DHQS homologues; (b) The numbers of DHQS homologues separated on the basis of their putative functions; (c) Ebo and EDB gene clusters in a number of bacteria.

To investigate the catalytic function of this new enzyme, we obtained a synthetic N. punctiforme eboD gene, codon optimized for the expression in E. coli. The recombinant protein was produced in E. coli BL21(DE3), purified using Ni-NTA column (Fig. S5a), and used for EboD characterization. Although EboD has been annotated as a putative DHQS, phylogenetically it is more closely related to SH7P cyclases (e.g., DDGS and EEVS) than to DHQS. Therefore, in addition to DAHP (the substrate for DHQS) and aminoDAHP (the substrate for aminoDHQS), SH7P was also tested as potential substrates for EboD (Figs. 2a). Similar to those used in other SPC enzymes, the reactions were done in the presence of NAD+ and Co2+. However, no product was observed in the reactions using either of the C7-sugar phosphates (Fig. S6). Subsequently, we tested two other known C7-sugar phosphates, sedoheptulose 1,7-bisphosphate (SH1,7PP) and 1-deoxy-d-altro-heptulose 7-phosphate (1-deoxy-SH7P), as potential substrates (Fig. 2a). SH1,7PP was prepared from SH7P using the E. coli ATP-dependent phosphofructokinase PfkA (Fig. S7),21 whereas 1-deoxy-SH7P was synthesized from pyruvate and d-ribose 5-phosphate employing E. coli 1-deoxy-d-xylulose 5-phosphate synthase (EcDXS) (Fig. S8).22 Incubation of EboD with these C7-sugar phosphates also did not give any product.

Figure 2.

Figure 2.

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Biochemical characterization of recombinant EboD. (a–b) Chemical structures of C7- and C6-sugar phosphates investigated as potential substrates for EboD; (c) TLC analysis of EboD reactions with various C6-sugar phosphates; (d) Schematic conversion of Man6P to DEI by EboD; (e) COSY and selected HMBC and NOESY correlations of DEI; and (f) Extracted ion chromatogram of EboD reaction with Man6P.

Next, we explored if EboD can catalyze the cyclization of Glc6P (Fig. 2b), the substrate of DOIS.23 Although DOIS is relatively less similar to EboD compared to the other variants of DHQS, the possibility that a C6-sugar phosphate is a substrate for EboD cannot be ruled out. However, incubation of EboD with Glc6P did not give any products either. Subsequently, we tested other C6-sugar phosphates, i.e., fructose 6-phosphate (Fru6P), galactose 6-phosphate (Gal6P), glucosamine 6-phosphate (GlcN6P) and mannose 6-phosphate (Man6P) (Fig. 2b), as potential substrates for EboD. Although none of these C6-sugar phosphates are known to be a substrate of enzymes from the DHQS-like SPC family, their conversion to a cyclitol is mechanistically possible. Thus, Fru6P, Gal6P, GlcN6P, and Man6P were individually incubated with EboD under the same condition described above (Fig. 2b). To our delight, incubation of EboD with Man6P in the presence of NAD+ and Co2+ afforded a product, which was observed as a pink spot with p-anisaldehyde on TLC (Fig. 2c), whereas other sugar phosphates did not give any products. We confirmed that this pink spot is not simply a dephosphorylation product of Man6P (Fig. S9). In addition, another eboD gene from a Gram-negative cellulose degrading bacterium, Sporocytophaga myxococcoides DSM 11118 (Sm-eboD), was also cloned and expressed in E. coli BL21(DE3) (Fig. S5b). An enzymatic assay using this enzyme also gave the same product with an even better conversion rate, which further confirmed the catalytic activity of EboD as a Man6P processing enzyme.

To characterize the identity of the product, enzymatic reactions using Sm-EboD (50 μL × 100 tubes) were conducted, and the product was subjected to a purification process. However, earlier attempts to purify the product using various types of column chromatography were unsuccessful. Later, we discovered that the product of EboD is sensitive to light and easily oxidized or converted to its isomers. Additional precautions to avoid light and heat during enzymatic reaction, purification, and drying did result in a product with better purity (>80%, based on 1H NMR spectrum). Structure characterization using HR-MS, 1D and 2D NMR revealed the identity of the compound to be 2-deoxy-4-epi-scyllo-inosose (DEI) (Fig. 2d–e). The stereoconfiguration of DEI was determined by NOESY. Specifically, NOE correlations between H-3 and H-4 as well as H-4 and H-5 indicate that C-3 hydroxyl, which is originated from an aldehyde, adopts an R configuration (Fig. 2e). The stereoconfiguration of other hydroxyl groups are consistent with the corresponding hydroxyl groups of Man6P. LC-MS analysis of the EboD reaction with Man6P further confirmed the production of DEI (m/z 161.0455 [M-H]–) (Fig. 2f), which is missing in the reaction with boiled enzyme. Altogether, the results confirmed that EboD is a Man6P cyclase.

Similar to other DHQS-like SPC enzymes, EboD requires metal ions as co-factors. However, in contrast to other SPCs, which normally use Co2+ and/or Zn2+, EboD prefers Co2+ and Ni2+ as co-factors. It can also use other metal ions, albeit in much lower initial velocities (Fig. S10).

To determine the affinity and catalytic efficiency of EboD toward the substrate Man6P, we carried out kinetic studies. The kinetic properties were determined using the Malachite Green Phosphate Assay Kit,24, 25 and the apparent kinetic values were obtained from Michaelis-Menten and Eadie-Hofstee plots (Fig. S11). The results showed that EboD has a substrate affinity (Km 16.4 ± 2.8 μM) and catalytic efficiency (kcat/Km 0.06 ± 0.01 μM−1•min−1) at pH 7.5 comparable with other related SPCs previously characterized.7

Comparative bioinformatics analysis of EboD and related SPCs showed that they share many conserved amino acid residues across the entire proteins. However, we identified a couple of amino acid segments that appeared most useful for distinguishing EboD from the other SPCs (Fig. 3a). In the first segment, the EboD proteins contain a unique HL(M) motif, whereas the other SPCs contain a Kx or a ML motif. In the second segment, the EboD proteins contain a WxAHK motif, which is significantly different from those in the other SPCs.

Figure 3.

Figure 3.

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Proposed structure and catalytic mechanism of EboD. (a) Unique active site residues for EboD and other SPCs that may be involved in their distinct substrate recognition; (b) comparison of the sugar binding site for DOIS (green) and EboD (grey). The red star points to the C-4 hydroxyl for the dephosphorylated carbaglucose-6-phosphate inhibitor (yellow) from the DOIS structure (PDB 2GRU).26 Oxygens are shown in red, nitrogens in blue and a water molecule as a red sphere. Distances are represented in Angstroms; (c) proposed catalytic mechanism of EboD, which catalyzes the cyclization of Man6P to DEI.

Taking advantage of the available SPC crystal structures, a model structure for EboD was constructed using AlphaFold2.27, 28 The modeled structure is highly similar to the X-ray crystal structures of DOIS, EEVS, and DDGS (Fig. S12a). In addition, an AlphaFold3 model was generated for EboD in complex with NAD+ and Co2+ (Fig. S12b).29 To identify residues that could contribute to the unique substrate specificity of EboD, we analyzed the sugar binding site of EboD and compared it to DOIS, EEVS, and DDGS (Figs. 3b and S12c). We included the DOIS structure for comparison because its substrate is Glc6P, which is the most similar to the EboD substrate Man6P and there is a DOIS complex structure with the dephosphorylated analog inhibitor carbaglucose-6-phosphate.26 The DOIS structure shows H-bonding interactions between the hydroxyl groups of the cyclitol inhibitor with H246, E235, K225, and a water molecule bridged by N187 (Fig. 3b). When comparing the equivalent EboD sugar binding site, we observed that the His and Glu (H275 and E264) are conserved and could potentially have similar H-bonding interactions with the substrate. The other two substrate interacting residues are different. EboD consists of H252 and V214 instead of K225 and N187 in DOIS, respectively. H252 is from the HL(M) motif described earlier and may be able to form a H-bond with the substrate similar to DOIS K225. However, V214 will not be able to form a water bridge with the substrate. Interestingly, this water bridge is between N187 and the C-4 hydroxyl of the cyclitol inhibitor, which corresponds to the C-2 hydroxyl of the substrate Glc6P. As Man6P is a C-2 epimer of Glc6P, its C-2 hydroxyl may have an entirely new interaction with EboD that is critical for specificity and will require further structural investigations.

Given the similarity of these proteins, including their structures, active sites, and co-factors, we postulate that EboD adopts a similar catalytic mechanism as those proposed for DHQS, DOIS, EEVS, and DDGS (Fig. 3c).3, 26, 30, 31 It begins with oxidation of the alcohol at C-4 of Man6P by NAD+ to give a ketone, dephosphorylation at C-6, reduction of the C-4 ketone back to an alcohol by NADH, ring opening followed by rotation along the C-4 and C-5 bond, and aldol cyclization to give a cyclic product, DEI.

In conclusion, this study demonstrated that EboD is a new SPC that is neither a DHQS, a SH7P cyclase, nor a DOIS (Scheme 1). It appears to be a dedicated enzyme that catalyzes the cyclization of Man6P to DEI, which is part of a new biosynthetic pathway branching from the Pentose Phosphate Pathway. The latter pathway is a source of sugar phosphates that can be converted to various primary and secondary metabolites (Scheme 1). While the ebo cluster is highly conserved and widely distributed in bacteria and some algae, DEI has never been identified in nature. This may be due to its high sensitivity to light. However, in the producing organisms, this compound is expected to be modified by several other enzymes in the ebo/EDB pathways to produce active metabolites with biological functions. Therefore, the discovery and characterization of EboD, which is a gateway to important biosynthetic pathways in bacteria and algae, set the stage for full and in-depth investigations of the pathways and their products.

Scheme 1.

Scheme 1.

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Sugar phosphate cyclases involved in primary and secondary metabolism.

DHQS, 3-dehydroquinate synthase; aminoDHQS, 5-deoxy-5-amino-3-dehydroquinate synthase; DOIS, 2-deoxy-scyllo-inosose synthase; EEVS, 2-epi-5-epi-valiolone synthase; EVS, 2-epi-valiolone synthase; DDGS, 2-desmethyl-4-deoxy-gadusol synthase; and EboD, 2-deoxy-4-epi-scyllo-inosose (DEI) synthase.

METHODS

Molecular phylogenetic analysis by maximum likelihood method.

Detailed phylogenetic analysis is described in Supporting Information.

Substrates.

Substrates used in this study, including the preparation of D-sedoheptulose 1,7-bisphosphate and 1-deoxy-D-sedoheptulose 7-phosphate, are described in Supporting Information.

Construction of the expression vectors.

Detailed construction of the expression vectors is described in Supporting Information.

Recombinant EboD production and purification.

Procedures for recombinant EboD production and purification are described in Supporting Information.

Biochemical characterization of EboD.

The enzymatic reaction was done in Eppendorf tubes with a reaction volume of 50 μL each. The reaction mixture contains sugar phosphate substrate (5 mM), NAD+ (1 mM), Co2+ (20 μM), EboD (5 μM), and Tris-HCl buffer (20 mM, pH 7.5). The mixture was incubated at 37°C and the reaction was monitored by TLC [mobile phase BuOH-EtOH-H2O (5:4:4)] and LC-MS (Agilent Poroshell 120 HILIC 2.7 μM, 3.0×150 mm; solvent A, 10 mM ammonium acetate in milliQ water + 0.1% formic acid; solvent B, acetonitrile).

Determination of metal ion preference of EboD.

Determination of metal ion preference of EboD is described in Supporting Information.

Large scale enzymatic reaction and product purification.

The large-scale enzymatic reaction was done in 100 Eppendorf tubes with a reaction volume of 50 μL each. The mixtures were incubated at 37°C and the reactions were monitored by TLC [mobile phase BuOH-EtOH-H2O (5:4:4)]. Product formation was indicated by the appearance of a pink spot at Rf 0.70. After 18 h, the reactions were quenched with MeOH, centrifuged at 13000 rpm for 3 min, and the supernatants were pooled and dried. The dried sample was re-dissolved in deionized water (300–400 μL) and the solution was subjected to a Bio-Gel P-2 resin (Bio-Rad) column (length, 50 cm; diameter, 1 cm) that was completely covered with aluminum foil to minimize light exposure. The column was eluted with deionized water and the fractions were analyzed by TLC. Fractions containing the pink spot were pooled and lyophilized to give DEI (2 mg) as a white powder. 1H NMR (750 MHz, CDCl3): δ 4.35 (1H, d, J = 10 Hz, H-6), 4.14 (1H, m, H-3), 4.03 (1H, brs, H-4), 3.83 (1H, dd, J = 10, 2 Hz, H-5), 2.95 (1H, dd, J = 14, 3 Hz, H-2b), 2.37 (1H, dd, J = 14, 2 Hz, H-2a). 13C NMR (126 MHz, CDCl3): δc 208.4 (C-1), 77.1 (C-6), 74.0 (C-5), 72.3 (C-4), 69.9 (C-3), 41.9 (C-2). HRMS-QTOF (ESI+) m/z: calcd for C6H9O5 [M–H]– 161.0455, found 161.0457.

Kinetic studies of EboD.

Kinetic studies of EboD are described in Supporting Information.

Supplementary Material

Supporting Information

ACKNOWLEDGMENT

The authors thank B. Philmus and P. Reardon for technical help.

Funding Sources

This work was in part supported by Grants GM112068 and AI129957 (to T.M.) from the National Institute of General Medical Sciences (NIGMS) and the National Institute of Allergy and Infectious Diseases (NIAID), respectively, and Grant GM151137 from NIGMS (to M.A.H). A.S. was supported by Grant T32 AT010131 from National Center for Complementary and Integrative Health (NCCIH). The content is solely the responsibility of the authors and does not represent the official views of NIGMS, NIAID, NCCIH, or the National Institutes of Health (NIH).

Footnotes

Supporting Information

Experimental section, supplementary tables, and supplementary figures (ebo clusters, scytonemin biosynthetic pathway and proposed function, SDS-PAGE, enzyme assays, Michaelis-Menten and Eadie-Hofstee plots, structure comparisons, 1H, 13C, and 2D NMR spectra, and HR-ESI-MS chromatogram).

The Supporting Information is available free of charge on the ACS Publications website.

The authors declare no competing financial interest.

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Associated Data

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