A RuBisCO-mediated carbon metabolic pathway in methanogenic archaea

Abstract

Two enzymes are considered to be unique to the photosynthetic Calvin-Benson cycle: ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), responsible for CO₂ fixation, and phosphoribulokinase (PRK). Some archaea possess bona fide RuBisCOs, despite not being photosynthetic organisms, but are thought to lack PRK. Here we demonstrate the existence in methanogenic archaea of a carbon metabolic pathway involving RuBisCO and PRK, which we term 'reductive hexulose-phosphate' (RHP) pathway. These archaea possess both RuBisCO and a catalytically active PRK whose crystal structure resembles that of photosynthetic bacterial PRK. Capillary electrophoresis-mass spectrometric analysis of metabolites reveals that the RHP pathway, which differs from the Calvin-Benson cycle only in a few steps, is active in vivo. Our work highlights evolutionary and functional links between RuBisCO-mediated carbon metabolic pathways in methanogenic archaea and photosynthetic organisms. Whether the RHP pathway allows for autotrophy (that is, growth exclusively with CO₂ as carbon source) remains unknown.

Figure 1. Phylogenetic tree of archaeal PRK and photosynthetic PRK.

The multiple sequence alignment and phylogenetic tree were produced using CLUSTALW. Bootstrap values were inferred from 1,000 replicates and significant bootstrapping values (>75%) are shown on the nodes as black filled circles. In eukaryotic PRKs, chloroplast transit peptides were removed according to ChloroP prediction. PRKs are classified into three forms: plant-type from plants, algae and cyanobacteria (green triangle); bacterial-type from photosynthetic bacteria and chemoautotrophs (blue triangle); and archaeal-type (red triangles). Species abbreviations are as follows: P. luminescens, Photorhabdus luminescens; P. profundum, Photobacterium profundum; A. ferrooxidans, Acidithiobacillus ferrooxidans; M. oxyfera, Methylomirabilis oxyfera; T. denitrificans, Thiobacillus denitrificans; A. vinosum, Allochromatium vinosum; P. marinus, Prochlorococcus marinus; M. capsulatus, Methylococcus capsulatus; R. palustris, Rhodopseudomonas palustris BisA53; N. hamburgensis, Nitrobacter hamburgensis; A. cryptum, Acidiphilium cryptum; R. sphaeroides, Rhodobacter sphaeroides; R. rubrum, Rhodospirillum rubrum; T. crunogena, Thiomicrospira crunogena; Cyanothece sp. PCC 7424; M. aeruginosa, Microcystis aeruginosa; Synechococcus sp. PCC 7002; A. variabilis, Anabaena variabilis; N. spumigena, Nodularia spumigena; T. elongatus, Thermosynechococcus elongatus; S. elongatus PCC 7942, Synechococcus elongatus PCC 7942; P. trichocarpa, Populus trichocarpa; A. thaliana, Arabidopsis thaliana; S. oleracea, Spinacia oleracea; C. variabilis, Chlorella variabilis; C. reinhardtii, Chlamydomonas reinhardtii; G. sulphuraria, Galdieria sulphuraria; G. violaceus, Gloeobacter violaceus; G. kilaueensis, Gloeobacter kilaueensis; M. marisnigri, Methanoculleus marisnigri; M. palustris, Methanosphaerula palustris; M. boonei, Methanoregula boonei; M. liminatans, Methanofollis liminatans; M. limicola, Methanoplanus limicola; M. petrolearius, Methanoplanus petrolearius; M. hungatei, Methanospirillum hungatei; M. tarda, Methanolinea tarda; M. concilii, Methanosaeta concilii; M. thermophile, Methanosaeta thermophila; M. harundinacea, Methanosaeta harundinacea; A. profundus, Archaeoglobus profundus; A. veneficus, Archaeoglobus veneficus; F. placidus, Ferroglobus placidus and A. boonei, Aciduliprofundum boonei.

Figure 2. Structural comparison of archaeal and photosynthetic PRKs.

(a,b) Topology diagrams of the folding patterns in protomers of (a) M. hungatei PRK (MhPRK) and (b) R. sphaeroides PRK (RsPRK) are shown in yellow and blue, respectively. α-helices are denoted by cylinders, β-sheets by arrows and connecting loops by lines. Positions in the sequence that start and end each major secondary structural element are shown. (c,d) Ribbon diagrams of (c) MhPRK (Protein Data Bank (PDB) ID 5B3F) monomer and (d) RsPRK (PDB ID 1A7J) monomer. Sulphate ions are bound to the active site of MhPRK and RsPRK. Disordered regions of missing electron density are shown as dots for residues for 156–163 in MhPRK, residues 15–17 corresponding to a part of the P-loop, and residues 100–105 in RsPRK. (e,f) Active-site structures of (e) MhPRK and (f) RsPRK. Side chains of residues involved in ATP- and Ru5P-binding are shown as pink and green sticks, respectively, with oxygen atoms in red and nitrogen atoms in blue. Sulphate ions are shown in MhPRK and RsPRK, with sulphur atoms in green, and oxygen and nitrogen atoms in the same colours as those in residue side chains. Small red balls represent water molecules. Disordered regions are shown as dots, and P-loop containing residues involved in ATP binding are shown in pink. Dotted lines show interactions of active site sulphate ions.

Figure 3. Phylogenetic tree of RuBisCOs and RuBisCO-like proteins.

The phylogenetic tree was produced using CLUSTALW. Bootstrap values were inferred from 1,000 replicates and significant bootstrapping values (>75%) are shown on the nodes as black filled circles. RuBisCO clades are indicated as follows: green for form I, blue for form II, red for form III-a and purple for form III-b (methanogenic archaea). We propose the latter two novel small clades because form III is prominently divided. The form IV clade of RuBisCO-like proteins (RLPs), which function as enolases/isomerases in methionine recycling in some bacteria, is shown in yellow. Species abbreviations are as follows: C. tepidum, Chlorobium tepidum; B. subtilis, Bacillus subtilis; G. kaustophilus, Geobacillus kaustophilus; B. thuringiensis, Bacillus thuringiensis; M. burtonii, Methanococcoides burtonii; M. mahii, Methanohalophilus mahii; M. jannaschii, Methanocaldococcus jannaschii; M. acetivorans, Methanosarcina acetivorans; T. kodakarensis, Thermococcus kodakarensis; R. palustris, Rhodopseudomonas palustris and N. spumigena, Nodularia spumigena.

Figure 4. Proposed RHP pathway and related metabolic processes in Archaea.

The RHP pathway (highlighted in yellow) has metabolic links to methanogenesis, reductive acetyl-CoA pathway, pentose bisphosphate pathway, glycolysis, gluconeogenesis, and amino acid metabolism. Methanogenesis and reductive acetyl-CoA pathways share a metabolic intermediate, methylene-H₄MPT that might be synthesized by Fae with released formaldehyde from the RHP pathway (dotted black line). The successive black arrows show multiple reaction steps. The RHP pathway (red lines and arrows) is superimposed on the Calvin–Benson cycle (green lines and arrows), and reaction steps from Ru5P to F6P are common in both cycles. Missing Calvin–Benson-cycle steps in M. hungatei are indicated by grey dashed lines and arrows. Ru5P, ribulose-5-phosphate; RuBP, ribulose-1,5-bisphosphate; 3-PGA, 3-phosphoglycerate; BPG, 1,3-diphosphoglycerate; GAP, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; FBP, fructose-1,6-bisphosphate; F6P, fructose-6-phosphate; Hu6P, D-arabino-3-hexulose-6-phosphate; FA, formaldehyde; E4P, erythrose-4-phosphate; Xu5P, xylulose-5-phosphate; SBP, sedoheptulose-1,7-bisphosphate; S7P, sedoheptulose-7-phosphate; R5P, ribose-5-phosphate; NMP, nucleoside 5′-monophosphate; RiBP, ribose-1,5-bisphosphate; H₄MPT, tetrahydromethanopterin; MF, methanofuran; PGK, 3-phosphoglycerate kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FBPase, fructose-1,6-bisphosphatase; TK, transketolase; SBPase, sedoheptulose-1,7-bisphosphatase; RPE, ribulose-5-phosphate 3-epimerase; RPI, ribose-5-phosphate isomerase; AMPpase, AMP phosphorylase; RiBP isomerase, ribose-1,5-bisphosphate isomerase.

Figure 5. RuBisCO-dependent conversion of RuBP and F6P into 3-PGA in M. hungatei extracts.

Production of 3-PGA from RuBP (a) or F6P (b) by M. hungatei cell extracts, in the presence (open bars) and absence (black bars) of CABP, a RuBisCO-specific transition state inhibitor. CABP was added to the pre-activation mixture to inactivate RuBisCO. All assays were performed after reaction with the substrates for 2 h at 37 °C. Data are means±s.d. of three replicates. N.D., not detected.

Figure 6. Time-course analysis of the metabolite ¹³C fraction of M. hungatei cells.

The y axis represents the ratio of ¹³C to total carbon in each metabolite. Data are means±s.d. of two replicates. Ru5P, ribulose-5-phosphate; RuBP, ribulose-1,5-bisphosphate; 3-PGA, 3-phosphoglycerate; BPG, 1,3-diphosphoglycerate; GAP, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; FBP, fructose-1,6-bisphosphate; F6P, fructose-6-phosphate; Hu6P, D-arabino-3-hexulose-6-phosphate; FA, formaldehyde; R5P, ribose-5-phosphate; G6P, glucose-6-phosphate; G1P, glucose-1-phosphate; 2-PGA, 2-phosphoglycerate; PEP, phosphoenolpyruvate; Pyr, pyruvate; OAA, oxaloacetate.