Archaeal Type III RuBisCOs Function in a Pathway for AMP Metabolism

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Science  16 Feb 2007:
Vol. 315, Issue 5814, pp. 1003-1006
DOI: 10.1126/science.1135999


The type III ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) present in the archaeon Thermococcus kodakaraensis was found to participate in adenosine 5′-monophosphate (AMP) metabolism, a role that is distinct from that of classical RuBisCOs of the Calvin-Benson-Bassham cycle. Genes annotated as thymidine phosphorylase (deoA) and eucaryal translation initiation factor 2B (e2b2) were found to encode AMP phosphorylase and ribose-1,5-bisphosphate isomerase, respectively. These enzymes supplied the RuBisCO substrate, ribulose-1,5-bisphosphate, from AMP and phosphate. Archaea with type III RuBisCOs all harbor both DeoA and the corresponding E2b2 homologs. In this pathway, adenine was released from AMP and the phosphoribose moiety entered central-carbon metabolism.

Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) is presumed to be the most abundant enzyme on our planet (1) and is widely known as the key CO2-fixing enzyme of the Calvin-Benson-Bassham (CBB) cycle, which is present in all plants, algae, cyanobacteria, and many other autotrophic bacteria (2, 3). Besides the classical type I and II RuBisCOs of the CBB cycle, two groups of proteins that are structurally related to the type I and II enzymes have been identified and designated as type III (4, 5) and type IV RuBisCOs (6, 7). Type IV RuBisCOs, also called the RuBisCO-like proteins, lack several residues necessary for conventional RuBisCO catalysis. Thus, type IV RuBisCO from Bacillus subtilis instead exhibits 2,3-diketo-5-methylthiopentyl-1-phosphate enolase activity and operates in the methionine (Met) salvage pathway in B. subtilis (7).

The type III RuBisCOs are found only in the Archaea, and they harbor all of the residues necessary for ribulose-1,5-bisphosphate (RuBP) carboxylase-oxygenase activity. Proteins from Thermococcus kodakaraensis (4, 810), Methanocaldococcus jannaschii, Archaeoglobus fulgidus, and other archaea (5, 11) exhibit RuBisCO activity. However, a functional CBB pathway has not been identified in Archaea, and archaeal genome sequences do not support the presence of a CBB pathway. In particular, homologs of phosphoribulokinase, another key enzyme of the CBB pathway that supplies RuBP for RuBisCO, cannot be found on the archaeal genomes. Hence, we first investigated whether archaeal RuBisCOs were involved in Met salvage, similar to type IV RuBisCOs.

Many organisms use Met as a precursor for polyamine elongation with concomitant generation of methylthioadenosine (MTA). The Met salvage pathway is present in some bacteria and eukaryotes and recycles MTA to Met (fig. S1) (1214). Homologs of enzymes involved in the conversion of Met to MTA are present on the T. kodakaraensis genome (15). Although homologs of several steps in the Met salvage pathway, including three YkrS homologs, are present, YkrX and YkrZ homologs cannot be identified on the T. kodakaraensis genome.

The absence of a complete Met salvage pathway in T. kodakaraensis was confirmed by examining a mutant that lacks the ability to synthesize Met de novo. Two adjacent genes presumed to encode the components of Met synthase, metE1 (TK1446) and metE2 (TK1447), were disrupted (16). The resulting strain, ΔmetE21-1, displayed the expected genotype (ΔpyrF, ΔmetE21::pyrF) and could grow only in the presence of exogenous Met. The ΔmetE21-1 strain could not grow when Met was substituted with various concentrations of MTA. We then disrupted the T. kodakaraensis (Tk)–RuBisCO gene (Tk-rbc) itself and subsequently isolated the gene-disruption mutant (Δrbc-1AA), which indicated that Tk-rbc is not essential for growth. We did, however, consistently observe a slight decrease in the cell yield of Δrbc-1AA as compared to the host strain when cells were grown in nutrient-rich medium.

We then shifted our attention to the three YkrS homologs present on the T. kodakaraensis genome and considered various possibilities for the roles of these homologs. The three homologs, Tk-E2b1 (45% identical with YkrS from B. subtilis), Tk-E2B2 (32% identical), and Tk-Gcd2 (27% identical), are members of the eukaryotic translation initiation factor 2B (eIF2B) α/β/δ family (1719). However, the functions of the eIF2B homologs in the Archaea are not known. The B. subtilis YkrS is amemberofthis family, and it catalyzes the isomerization of methylthioribose 1-phosphate (MTR–1-P) to methylthioribulose 1-phosphate (MTRu–1-P) (7). We noticed structural similarities between MTR–1-P and ribose 1,5-bisphosphate (R15P) and between MTRu–1-P and RuBP, indicating that these archaeal IF2B (aIF2B) proteins might be involved in the generation of RuBP. The isomerization of R15P to RuBP also corresponds to the reaction proposed to be responsible for RuBP synthesis in methanogenic Archaea (20).

A phylogenetic tree was constructed with 49 aIF2B homologs from 27 archaeal genomes. The homologs composed several subgroups, and we found that one branch (shaded in Fig. 1) included homologs only from organisms harboring type III RuBisCOs, with the only exception being AAG20059 from Halobacterium sp. NRC-1. The biased distribution did not correlate at all with the phylogenetic relationships of the organisms themselves. The correlation prompted us to examine experimentally whether there was a functional relationship between this subgroup of aIF2B homologs, which includes Tk-E2b2 and type III RuBisCO.

Fig. 1.

Phylogenetic tree of archaeal proteins annotated as members of the aIF2B family. aIF2B family proteins present in the 27 complete archaeal genome sequences were aligned with the ClustalW program available at the DNA Data Bank of Japan, and a phylogenetic tree was constructed with the maximum-likelihood method by means of PHYML (26, 27). The shaded branch harbors the aIF2B homologs from archaeal strains that possess a type III RuBisCO. A. pernix, Aeropyrum pernix K1; A. fulgidus, Archaeoglobus fulgidus VC-16; H. marismortui, Haloarcula marismortui ATCC 43049; M. jannaschii, Methanocaldococcus jannaschii DSM2661; M. burtonii, Methanococcoides burtonii DSM6242; M. maripaludis, Methanococcus maripaludis S2; M. kandleri, Methanopyrus kandleri AV19; M. acetivorans, Methanosarcina acetivorans C2A; M. barkeri, Methanosarcina barkeri str. Fusaro; M. mazei, Methanosarcina mazei Go1 OCM88; M. hungatei, Methanospirillum hungatei JF-1; M. stadtmanae, Methanosphaera stadtmanae DSMZ3091; M. thermautotrophicus, Methanothermobacter thermautotrophicus ΔH; N. pharaonis, Natronomonas pharaonis DSM2160; P. torridus, Picrophilus torridus DSM9790; P. aerophilum, Pyrobaculum aerophilum IM2; P. abyssi, Pyrococcus abyssi GE5; P. furiosus, Pyrococcus furiosus DSM3638; P. horikoshii, Pyrococcus horikoshii OT3; S. solfataricus, Sulfolobus solfataricus P2; S. tokodaii, Sulfolobus tokodaii 7; S. acidocaldarius, Sulfolobus acidocaldarius DSM639; T. kodakaraensis, Thermococcus kodakaraensis KOD1; T. acidophilum, Thermoplasma acidophilum DSM1728; T. volcanium, Thermoplasma volcanium GSS1.

The Tk-e2b2 and Tk-e2b1 genes were expressed in Escherichia coli and heat-treated. An initial examination of RuBP synthesis activity from R15P was performed using an R15P solution enzymatically prepared with glucose 1,6-bisphosphate (G16P), ribose 5-phosphate (R5P), and phosphoglucomutase. Extracts containing Tk-E2b1 displayed only negligible levels of activity that were equivalent to those found in extracts from E. coli harboring a mock plasmid, pET21a(+) (0.001 to 0.002 μmol min–1 mg–1). In contrast, although expression levels were substantially lower than those of Tk-E2b1, extracts containing Tk-E2b2 displayed levels of RuBP-synthesizing activity that were over 10 times higher than those of Tk-E2b1. Purified Tk-E2b2 generated RuBP from the R15P solution with activity levels of 0.4 μmol min–1 mg–1. High-performance liquid chromatography analyses confirmed that only R15P, and not G16P or R5P, was the substrate for RuBP synthesis (fig. S2).

In order to identify a protein that supplies R15P, we searched for genes that were specifically present in archaeal strains harboring type III RuBisCO. This led to the identification of a putative thymidine phosphorylase gene (deoA). The only exception was Methanococcus maripaludis, which harbors a deoA gene but not a RuBisCO (Table 1). DeoA is known to release the thymine base from thymidine while phosphorylating the 1′-carbon of the ribose moiety (21). If DeoA could phosphorylate nucleoside monophosphates, this would generate R15P. We expressed the Tk-deoA gene (TK0352) in E. coli, purified the protein, and found that it produced high levels of R15P in the presence of phosphate and adenosine 5′-monophosphate (AMP) (Table 2). Tk-DeoA displayed a specific activity of 22.3 μmol min–1 mg–1 for the reaction AMP + Pi → R15P + adenine. The stoichiometric ratio of the concentration of AMP consumed ([AMP consumed]) and [adenine produced] was confirmed as 1:1 (fig. S3). The equilibrium constant K ([R15P][adenine]/[AMP][Pi]) was 6.02 × 10–3 ± 0.46 × 10–3 at 85°C. Using Tk-DeoA, we were able to synthesize large amounts of R15P and to test the R15P isomerase activity of Tk-E2b2 in the presence of abundant substrate. We observed that 1.80 ± 0.07 mol of 3-phosphoglycerate (3-PGA) were produced for every mole of R15P consumed when Tk-E2b2 and Tk-RuBisCO were coupled at 85°C, strongly supporting a stoichiometric ratio of [R15P consumed]:[3-PGA produced] = 1:2. Tk-E2b2 exhibited a specific activity of 32.3 μmol min–1 mg–1 for the reaction R15P → RuBP (Table 2).

Table 1.

Distribution of RuBisCO, aIF2B, DeoA, and ADP-dependent sugar kinase homologs on archaeal genomes. Shaded boxes indicate the presence of a homolog on the genome.

OrganismType III RuBisCOaIF2B homologView inlineDeoA homologADP-dependent sugar kinase
Aeropyrum pernix
Archaeoglobus fulgidus
Haloarcula marismortui
Halobacterium sp. NRC-1
Methanocaldococcus jannaschii
Methanococcoides burtonii
Methanococcus maripaludis
Methanosarcina acetivorans
Methanosarcina barkeri
Methanosarcina mazei
Methanosphaera stadtmanae
Methanospirillum hungatei
Methanothermobacter thermautotrophicus
Methanopyrus kandleri
Nanoarchaeum equitans
Natronomonas pharaonis
Picrophilus torridus
Pyrobaculum aerophilum
Pyrococcus abyssi
Pyrococccus furiosus
Pyrococcus horikoshii
Sulfolobus acidocaldarius
Sulfolobus solfataricus
Sulfolobus tokodaii
Thermococcus kodakaraensis
Thermoplasma acidophilum
Thermoplasma volcanium
  • View inline* Homologs included in the shaded branch of Fig. 1.

  • Table 2.

    Specific activities of Tk-DeoA and Tk-E2b2. Synthesis of 3-PGA was not observed in the absence of protein or substrate.

    ProteinSubstrateSpecific activity (μmol min-1 mg of protein)
    Tk-DeoA AMP 22.3 ± 0.2
    GMP 0.01
    CMP 0.14 ± 0.01
    UMP 0.06 ± 0.01
    TMP 0.01
    Tk-E2b2 R15PView inline 32.3 ± 0.4
  • View inline* Produced with Tk-DeoA.

  • The protein proposed to catalyze the isomerization between R15P and RuBP in M. jannaschii is the product of MJ0601, but the recombinant protein does not seem to be active in vitro (20). Likewise, the MJ0601 homolog in T. kodakaraensis (Tk-thi4, TK0434) was expressed in E. coli and, again, activity was not detected. This indicates that at least in T. kodakaraensis, Tk-E2b2 is the enzyme primarily responsible for RuBP synthesis from R15P.

    Our results showed that in T. kodakaraensis, a putative thymidine phosphorylase is actually an AMP phosphorylase, whereas an aIF2B homolog is actually a R15P isomerase. Because the Archaea possessing a type III RuBisCO are all anaerobic, it is most likely that only the carboxylase activity of RuBisCO, and not the oxygenase activity, is biologically relevant in these strains. Thus, Tk-DeoA, Tk-E2b2, and the RuBisCO convert AMP, phosphate, CO2, and H2O, respectively, to adenine and two molecules of 3-PGA (Fig. 2). This pathway hence salvages the adenine base of AMP and diverts the ribose moiety into central-carbon metabolism.

    Fig. 2.

    A metabolic pathway composed of homologs of DeoA, aIF2B, and type III RuBisCOs.

    Many of the Archaea that possess this pathway are organisms that use adenosine 5′-diphosphate (ADP)–dependent (AMP-forming) sugar kinases for glycolysis (22, 23). These enzymes would produce a larger intracellular pool of AMP as compared with organisms possessing classical ATP-dependent kinases. Because the product of the pathway, 3-PGA, can be used for ATP generation, the pathway may enable anaerobic Archaea to use AMP when energy levels are low and/or intracellular AMP is in excess. In A. fulgidus, which does not harbor ADP-dependent sugar kinases, the pathway may be involved in the conversion of the AMP generated by adenosylphosphosulfate reductase (24) during sulfate reduction. Another possibility is that AMP is produced from 5-phosphoribosyl 1-pyrophosphate (PRPP) by adenine phosphoribosyltransferase, whose homolog is present in T. kodakaraensis. This reaction would recycle the adenine molecule released by Tk-DeoA and also complete a cyclic pathway composed of (i) pentose synthesis, (ii) conversion of PRPP to 3-PGA, and (iii) conversion of 3-PGA to fructose 6-phosphate (gluconeogenesis). If the non-oxidative branch of the pentose phosphate pathway were to be responsible for pentose synthesis, the cyclic pathway would constitute a CO2 fixation pathway (fig. S4). Although their genome sequences are not available, there are several autotrophic strains in the Crenarchaeota that exhibit RuBisCO activity despite lacking phosphoribulokinase activity (25).

    Type III RuBisCOs seem to have a function distinct from that of the classical RuBisCOs in the CBB pathway of Bacteria and Eucarya. When considering that the organisms harboring type III RuBisCOs represent relatively deep and short lineages in evolution, it may well be that the carboxylase activity of RuBisCO originally evolved to function in this pathway.

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    Materials and Methods

    Figs. S1 to S4


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