Previous and current view on glycolytic routes, glycolytic shunts and the CBB cycle in Synechocystis

(A) The central carbohydrate metabolism in Synechocystis as described by Chen et al (1). The shown GDH/GK bypass and ED pathway were assumed to be present based on the misinterpretation of experimental data (1). It was unclear at that time how glycolytic routes and the CBB cycle are organized under photomixotrophic conditions on glucose in continuous light. (B) Updated view where glycolytic routes and the CBB cycle are intertwined under photomixotrophic conditions so that glucose is fed via the glycolytic PGI and OPP shunt into the CBB cycle based on flux analyses (3). ED pathway and GDH/GK bypass are omitted based on results of this study. CBB, Calvin–Benson–Bassham; ED, Entner-Doudoroff; EDA, Entner-Doudoroff aldolase; EDD, Entner-Doudoroff dehydratase, EMP, Embden–Meyerhoff–Parnass; GDH, glucose dehydrogenase; GK, gluconate kinase; GND, 6-phosphogluconate dehydrogenase; HK, hexokinase; KDPG, 2-keto-3-deoxy-6-phosphogluconate; OPP, oxidative pentose phosphate; PFK, phosphofructokinase; PGI, phosphoglucose isomerase; ZWF, glucose-6-phosphate dehydrogenase.

Evaluation of the proposed glucose dehydrogenase/gluconate kinase (GDH/GK) bypass in Synechocystis and representative cyanobacteria.

(A) GDH activity measurements in crude cell extracts of Synechocystis WT, Δzwf and a strain overexpressing putative GDH1 (sll1709:oe) under photoautotrophic or photomixotrophic conditions. As a positive control, 0.05 U glucose dehydrogenase from Pseudomonas sp. was added to WT cell extract. (B) GDH activity measurements in crude cell extracts of photomixotrophically grown Δzwf cultures and with purified putative GDH1 (Sll1709). NADP+, NAD+, and the two quinones DMB (2,6-Dimethoxy-1,4-benzochinon) and DQ (2,3-Dimethoxy-1,4-naphthochinon) were tested as electron acceptors. (C) Gluconate kinase activity measurements in cell extracts of Synechocystis WT and Δzwf under photoauto-or photomixotrophic conditions. As a positive control, 0.05 U gluconate kinase from E. coli was added to WT cell extract. (D) Blast analyses of putative glucose dehydrogenase (GDH) in cyanobacteria. Protein databases of 261 cyanobacteria that also carry a putative gluconate kinase (GK) were analyzed for GDH homologues.

Specific activities of the two cyanobacterial GDHs from Spirulina and Lyngbya and the gluconate kinase from Lyngbya.

Enzyme assays were performed after recombinant expression in E. coli and following purification.

Utilization of gluconate and glucose as carbon source for growth in WT, Δhk (Δsll0539), and Δhk::hk.

(A) Growth of WT in the absence and presence of gluconate. (B) Hexokinase activity in WT and Δhk with 10 mM glucose. (C) Photomixotrophic growth of WT, Δhk, and Δhk::hk. (D) Glucose concentration (%) in the growth medium after inoculation (day 0; 10 mM glucose) and on day 4 in WT, Δhk, and Δhk::hk.

The accumulation of 6-phosphogluconate (6PG) in ΔedaΔgnd in contrast to Δeda and Δgnd is not a result of a missing ED pathway flux but a secondary mutation in the zwf gene in Δgnd.

(A) 6PG accumulation in WT, ΔedaΔgnd, and ΔedaΔgndΔzwf under photoautothrophic conditions. (B) Zwf activity measurements in cell crude extracts of WT, Δzwf, Δgnd, Δeda, and ΔedaΔgnd. reveals no Zwf activity in Δgnd. The values are normalized to protein content. (C) Sequencing of the zwf genes of WT, Δgnd, Δeda, and ΔedaΔgnd revealed the insertion of an adenine in position 399 in Δgnd, which results in a premature stop codon. (D) Immunoblots of soluble cell extracts of WT, Δzwf, Δgnd, Δeda, and ΔedaΔgnd with antibodies specific against ZWF (αZwf). An antibody against 30S ribosomal protein (αRps) was utilized as a loading control. The WT was loaded in three different concentrations (10%, 50%, 100%) to facilitate relative quantification of the αZwf signals.

Structural comparison and sequence alignments of EDA enzymes including the genome-annotated version and an N-terminally extended version with an alternative start codon of the Synechocystis enzyme.

(A) Structures of E. coli and C. crescentus EDA enzymes are compared with AlphaFold 3 models of the N-terminally extended (long) and genome-annotated (short) Synechocystis EDA variants. The conserved N-terminal α-helix is highlighted in red. Amino acid sequences were retrieved from KEGG, AlphaFold 3 models were generated using AlphaFold Server v3.0 (22) and structures were visualized and edited using UCSF ChimeraX. (B) Sequence alignment highlighting the N-terminal residues of the conserved α-helix in red. The catalytic lysine responsible for Schiff-base formation and the glutamate acting as the acid/base catalyst are indicated in cyan. The alignment was performed using Clustal Omega (24) and manually visualized in BioEdit.

Substrate specificity and characterization of EDA (Sll0107) from Synechocystis.

(A) Substrate specificity was assessed with 5 mM of each KDPG, oxaloacetate, FBP, and KHG using the continuous coupled assay (see Methods). EDA exhibited high catalytic activity toward KDPG and reduced activity toward oxaloacetate and KHG, while no detectable cleavage of fructose 1,6-bisphosphate (FBP) was observed. This finding contrasts with the reported FBP cleavage activity of EDA from Glycine max (2). (B-D) Enzymatic characterization of EDA using KDPG, OAA and KHG as substrates. The activity of EDA was measured across a range of (B) KDPG, (C) OAA and (D) KHG concentrations (0–1 mM), (0-10 mM) and (0-20 mM) respectively. Colored bands indicate 95% confidence and data are shown as mean ± standard deviations from three technical replicates. The kinetic parameters of KDPG (Vmax = 8.28 U mg⁻¹; Km = 0.13 mM), OAA (Vmax = 4.87 U mg⁻¹; Km = 3.19 mM) and KHG (Vmax = 1.05 U mg⁻¹; Km = 2.18 mM) were determined by fitting the classical Michaelis–Menten equation using Wolfram Mathematica v14.2. (E) Effect of NADP+ on EDA activity. Different NADP+ concentrations (0, 1, 2 and 3 mM) were tested for their effect on EDA activity. Assays were performed with sub-saturating KDPG concentration (0.2 mM). The relative activity (%) in comparison to the control without effector (100%; specific activity of 4.3 U/mg) is shown. (F) Effect of K147G mutation on EDA activity. Enzymatic activity of wild-type EDA and the catalytic mutant EDAK147G was measured with 5 mM KDPG, oxaloacetate, FBP, and KHG using continuous coupled assays (see Methods). Data are presented as mean ± SD from three technical replicates (n = 3).

Kinetic parameters of EDA (Sll0107) from Synechocystis

Proposed metabolic map of the central carbon metabolism in Synechocystis with all suggested in vivo roles of the promiscuous aldolase Sll0107 (EDA) highlighted in green, based on substrate promiscuity and activities observed in vitro.

ACO, Acontinase; AGP, ADP-glucose pyrophosphorylase; CBB, Calvin–Benson–Bassham; DHAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; EMP, Embden–Meyerhof–Parnas; EDA, Enter-Doudoroff aldolase; ENO, enolase; F6P, fructose 6-phosphate; FBA, fructose-bisphosphate-aldolase; FBP, fructose 1,6-bisphosphate; FBPase, fructose-1,6-bisphosphatase; F/SBPase, fructose-1,6-biphosphatase/sedoheptulose-1,7-biphosphatase; FUM, fumarase; GAP, glyceraldehyde 3-phosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GBP, glucose 1,6-bisphosphate; GND, 6-phosphogluconate dehydrogenase; GP, glycogen phosphorylase; GS, glycogen synthase; HK, hexokinase; IDH, isocitrate dehydrogenase; ME, malic enzyme, MDH, malate dehydrogenase; OPP, oxidative pentose phosphate; PDH, pyruvate dehydrogenase complex, PEPC, phosphoenolpyruvate carboxylase, PEPS, phosphoenolpyruvate synthetase, PFK, phosphofructokinase; PGAM, phosphoglycerate mutase; PGI, phosphoglucose isomerase; PGK, phosphoglycerate kinase; PGL, 6-phosphogluconolactonase; PGM, phosphoglucomutase; PRK, phosphoribulokinase; PUTA, proline dehydrogenase; PYK, pyruvate kinase; R-5P, ribose 5-phosphate; RPE, ribulose-5-phosphate epimerase; RPI, ribose-5-phosphate isomerase; SDH, succinate dehydrogenase; TalB, transaldolase; TKT, transketolase; TPI, triosephosphate isomerase; X-5P, xylulose 5-phosphate; ZWF, glucose-6-phosphate dehydrogenase.