Cyanobacterial antimetabolite 7-deoxy-sedoheptulose blocks the shikimate pathway to inhibit the growth of prototrophic organisms

Abstract

Antimetabolites are small molecules that inhibit enzymes by mimicking physiological substrates. We report the discovery and structural elucidation of the antimetabolite 7-deoxy-sedoheptulose (7dSh). This unusual sugar inhibits the growth of various prototrophic organisms, including species of cyanobacteria, Saccharomyces, and Arabidopsis. We isolate bioactive 7dSh from culture supernatants of the cyanobacterium Synechococcus elongatus. A chemoenzymatic synthesis of 7dSh using S. elongatus transketolase as catalyst and 5-deoxy-D-ribose as substrate allows antimicrobial and herbicidal bioprofiling. Organisms treated with 7dSh accumulate 3-deoxy-D-arabino-heptulosonate 7-phosphate, which indicates that the molecular target is 3-dehydroquinate synthase, a key enzyme of the shikimate pathway, which is absent in humans and animals. The herbicidal activity of 7dSh is in the low micromolar range. No cytotoxic effects on mammalian cells have been observed. We propose that the in vivo inhibition of the shikimate pathway makes 7dSh a natural antimicrobial and herbicidal agent.

Fig. 1

Extracts of supernatant of Synechococcus elongatus inhibits growth of Anabaena variabilis. a Agar-diffusion plate assay with the effect of organic extracts (1, methanol; 2, chloroform; 3, acetone, and 4, ethyl acetate) of lyophilized supernatant of stationary-phase S. elongatus cultures on the growth of the producer strain and A. variabilis. Neomycin (Neo, 20 µg) served as positive control. b Optical density of producer strain S. elongatus (black) and zone of A. variabilis growth inhibition (diameter) of methanol extracts of S. elongatus supernatant on agar diffusion plates (turquoise). Values represent the mean values of three biological replicates; standard deviations are indicated. Dots indicate data distribution. Source data are provided as a Source Data file

Fig. 2

Structure and chemoenzymatic synthesis of 7-deoxy-sedoheptulose (7dSh, 1). a Chemoenzymatic synthesis of 7-deoxy-sedoheptulose (7dSh). Absolute configurations of stereo-centers are indicated. b Chemical structure of 7dSh in the furanose form with given assignments of coupling constants (gray). c (1–4) ¹H NMR spectra of 7dSh (CD₃OD, 600 MHz) chromatographically purified from supernatants of stationary phase cultures of S. elongatus (1, green), of the purified 7dSh from the supernatants of Streptomyces setonensis as control (2, red), and of enzymatically synthesized 7dSh (3, black). Predicted from assigned NMR-data (4, blue) of 7dSh in the 7-deoxy-d-altro-heptulofuranose form (Bruker, TopSpin software). Additional proton NMR signals in 1–3 give evidence for the dynamic forms of 7dSh in solution (open chain tautomers, ring conformers)

Fig. 3

Effect of 7dSh (1) on the growth and photosynthetic oxygen evolution of A. variabilis cultures. a Growth of A. variabilis (OD₇₅₀) at different concentrations of 7dSh after 48 h of incubation. Cultures were inoculated to an OD₇₅₀ of 0.05, 0.2, or 0.5 (marked by dashed lines). 7dSh in aqueous solution was added at time 0. Significant differences between adjusted initial OD₇₅₀ and OD₇₅₀ after 48 h were analyzed in a one sample t-test (*p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; n.s., not significant). b Photosynthetic oxygen evolution by A. variabilis (initial OD₇₅₀ = 0.3) in the presence of 7dSh or neomycin (positive control) or without supplementation (BG11, negative control). 7dSh (ca. 50 µM) and neomycin (ca. 65 µM) were added as aqueous solution. Values in both graphs represent the mean values of three biological replicates; standard deviations are indicated. Dots indicate data distribution. Source data are provided as a Source Data file

Fig. 4

7dSh (1) prevents regeneration of resuscitating Synechocystis. a Optical density (black) and PSII quantum yield (turquoise) of chlorotic Synechocystis cultures (initial OD₇₅₀ = 0.5) regenerating in the absence or presence of 7dSh. NaNO₃ (17.3 mM) and 7dSh (ca. 260 µM) were added in aqueous solution at 0 h. Values represent the mean values of three biological replicates; standard deviations are indicated. Dots indicate data distribution. b Oxygen evolution of resuscitating Synechocystis cultures (initial OD₇₅₀ = 0.5) upon addition of nitrate (NaNO₃, 17.3 mM) in the presence or absence of 7dSh (ca. 206 µM). Significant differences between 7dSh treatment and untreated control for each timepoint were analyzed in an unpaired t-test (*p-value < 0.05; **p-value < 0.01; *** p-value < 0.001; n.s., not significant). Values represent the mean values of three biological replicates; standard deviations are indicated. Dots indicate data distribution. c Cultures of chlorotic Synechocystis (initial OD₇₅₀= 0.5) 48 h after addition of nitrate (NaNO₃, 17.3 mM) and 7dSh. Numbers indicate concentration (µg mL⁻¹) of 7dSh added to the culture. Source data are provided as a Source Data file

Fig. 5

Effects of 7dSh (1) on amino acid levels in A. variabilis cells. Levels of selected amino acids in A. variabilis (initial OD₇₅₀ = 0.5) treated with 7dSh (260 µM) for 4 h and respective untreated control cultures. Significant differences between 7dSh treatment and untreated control were analyzed in an unpaired t-test (*p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; n.s., not significant). Values represent the mean values of five biological replicates; standard deviations are indicated. Dots indicate data distribution. Source data are provided as a Source Data file

Fig. 6

7dSh (1) reduces the growth of A. thaliana seedlings. a Morphological appearance of autotrophically grown Arabidopsis thaliana seedlings 7 days after induction of germination. Seedlings were grown in constant light on agar plates without an antimetabolite (control) or in the presence of 7dSh or glyphosate. Plates were mounted vertically and illuminated from above. White arrows mark the root and shoot apical meristem. Scale bar (5 mm) applies to all images. b Measurement of the distance between root and shoot apical meristem. Significant differences between seedling sizes were analyzed in an unpaired t-test (*p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; n.s., not significant). Box-and-whisker plots represent the values of at least 16 seedlings. c Effect of 7dSh and glyphosate (each 260 µM) on the growth of A. thaliana on soil after 18 days in a day/night cycle. Statistical analysis was performed by using a one-way ANOVA. Tukey’s multiple comparison test was used as the post-hoc test. Means that were significantly different (p-value < 0.05) are marked with different capital letters in the diagram. Box-and-whisker plots represent the values of at least 58 A. thaliana seedlings. For b, c: Error bars indicate range, box bounds indicate second and third quartiles, center lines indicate median. Source data are provided as a Source Data file