A Functional Link Between RuBisCO-like Protein of Bacillus and Photosynthetic RuBisCO

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Science  10 Oct 2003:
Vol. 302, Issue 5643, pp. 286-290
DOI: 10.1126/science.1086997


The genomes of several nonphotosynthetic bacteria, such as Bacillus subtilis, and some Archaea include genes for proteins with sequence homology to the large subunit of ribulose bisphosphate carboxylase/oxygenase (RuBisCO). We found that such a RuBisCO-like protein (RLP) from B. subtilis catalyzed the 2,3-diketo-5-methylthiopentyl-1-phosphate enolase reaction in the methionine salvage pathway. A growth-defective mutant, in which the gene for this RLP had been disrupted, was rescued by the gene for RuBisCOfrom the photosynthetic bacterium Rhodospirillum rubrum. Thus, the photosynthetic RuBisCO from R. rubrum retains the ability to function in the methionine salvage pathway in B. subtilis.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant naturally occurring protein (1), functions in the Calvin cycle during photosynthesis to convert atmospheric CO2 into organic carbon. RuBisCO has several disadvantages as a biological catalyst. Besides having a low affinity for the substrate CO2 and a low turnover rate, its CO2-fixation activity is competitively inhibited by O2 (2, 3). To overcome these disadvantages, plants are obliged to invest a large amount of nitrogen to synthesize adequate levels of RuBisCO. The disadvantages may reflect the molecular evolution of RuBisCO when the Calvin cycle appeared; however, the ancestral gene remains to be identified.

RuBisCOs and proteins with sequence similarity are classified into four forms (Fig. 1A): The enzyme that is most strongly adapted to present-day atmospheric conditions represents form I, which consists of eight large and eight small subunits. It is found in photosynthetic organisms such as higher plants, algae, and autotrophic proteobacteria. Form II, with the simplest structure, is found in some photosynthetic bacteria, such as Rhodospirillum rubrum (4). Form III consists of the large subunits only, and it is found in some Archaea (5, 6). Forms I through III include the amino acid residues that are required for catalytic activity of RuBisCO, and each is capable of catalyzing both the carboxylation and the oxygenation of ribulose-1,5-bisphosphate (RuBP). Form IV has been found in Bacillus subtilis (7), Chlorobium tepidum (8, 9), and Archaeoglobus fulgidus (10). It lacks several of the amino acid residues involved in catalysis (8) (Fig. 1B). Forms III and IV are referred to as RuBisCO-like proteins (RLPs). Their functional and evolutionary relation to photosynthetic RuBisCO are unknown. We describe the functional relation between a RuBisCO-like protein of B. subtilis and photosynthetic RuBisCO.

Fig. 1.

(A) Phylogenetic tree constructed from the deduced amino acid sequences of the large subunits of RuBisCO (forms I and II) and RLPs (forms III and IV) from various organisms. The multiple sequence alignment and the tree were produced with CLUSTALW and TREE VIEW Ver. 1.5.3. Full names of species are as follows: Spinacia oleracea, Nicotiana tabacum, Synechocystis sp. PCC6803, Rhodobacter capsulatus, Methanococcus jannaschii, Pyrococcus horikoshii, Pyrococcus kodakaraensis, Chlorobium limicola, and Archaeoglobus fulgidus. L and S refer to large and small subunits, respectively. (B) Partial multiple sequence alignment of the large subunits of RuBisCO (forms I and II) and RLPs (forms III and IV). [Adapted from Hanson and Tabitha (8)] Only sequences around the residues involved in the RuBisCO reaction are aligned. Active-site residues are highlighted in black. C (yellow) and R (blue, green, and orange) above the alignment indicate residues involved in catalysis and binding of RuBP, respectively. R's in blue and green indicate residues involved in binding of the phosphate groups on C1 and C5 of RuBP, respectively. Orange residues are those that bind to RuBP but not to phosphate. Letters in red indicate active-site residues that have been replaced by other amino acids. The alignment is numbered according to the sequence of the large subunit of RuBisCO from spinach.

The ykrW gene for the RLP of B. subtilis is the first gene in the ykrWXYZ operon, which is close to the ykrTS operon (7, 1113). These operons have S-box presequences that regulate the expression of the genes involved in sulfur metabolism in B. subtilis (11, 12), and it has been predicted that the genes in these operons are involved in the methionine salvage pathway (11, 13). In this pathway, organic sulfur is salvaged from methylthioribose (MTR), which is derived from the methylthioadenosine (MTA) that is a by-product of the synthesis of spermidine. In the pathway that has been proposed in Klebsiella pneumoniae (1418) (fig. S1), MTR is phosphorylated to MTR-1-phosphate (MTR-1-P) by a kinase, and then MTR-1-P is isomerized by an aldose-ketose isomerase to methylthioribulose-1-phosphate (MTRu-1-P). MTRu-1-P then undergoes a dehydration reaction that is catalyzed by a dehydratase and yields 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P). DK-MTP-1-P is converted to 1,2-dihydroxy-3-keto-5-methylthiopentene (DHK-MTPene) via the intermediate, 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate (HK-MTPenyl-1-P), by an enolase/phosphatase. Finally, DHK-MTPene is converted to formate and 2-keto-4-methylthiobutyrate (KMTB) by a dioxygenase, and KMTB is transaminated to methionine.

In order to identify the reaction catalyzed by RLP, we analyzed the functions of the YkrS, YkrW, YkrX, YkrY, and YkrZ proteins of B. subtilis, using recombinant proteins purified from Escherichia coli cells that expressed these genes (19). MTR-1-P was generated by using recombinant YkrT, which has been reported to be an MTR kinase (20). The MTR-1-P that we obtained had the same chemical shifts (Fig. 2A) in its nuclear magnetic resonance (1H-NMR) spectrum as those reported for MTR-1-P (14), and it was used as the starting substrate for identification of the subsequent steps in the methionine salvage pathway. For this identification, we added the recombinant proteins individually to the candidate substrate and then quantified and/or analyzed the products of reactions.

Fig. 2.

1H-NMR spectra of the products generated (A to E) and UV spectral changes (G to I: YkrY, RLP, YkrX, and YkrZ) during the methionine salvage reactions (F). (A) MTR-1-P produced by YkrT from MTR and ATP. (B) Product of YkrS. (C) Product of RLP. (Inset) peaks β and γ are expanded horizontally. (D) Product of YkrX. (E) Products of YkrZ. The sequential reactions for NMR spectra (F) proceeded in D2O (99.9%; Wako, Osaka, Japan)—phosphate buffer (pD 7.5) that contained 25 mM sodium phosphate, 0.1 mM MgCl2, and 1.0 mM MTR-1-P as the starting substrate, and the total volume of each reaction mixture was 400 μl. RLP and YkrY were added to the reaction mixture simultaneously in the reaction for which results are shown in (C). The amounts of protein added for NMR were 80 μg for YkrS, 8 μg for YkrY, 65 μg for RLP, 15 μg for YkrX, and 50 μg for YkrZ. (G) The formation of DK-MTP-1-P by YkrY, from the reaction product of YkrS (MTRu-1-P), was detected by converting the reaction product to its quinoxaline in the presence of 2 mM o-phenylenediamine. (H) The RLP reaction was initiated by adding YkrY to a reaction mixture that contained MTR-1-P, YkrS and RLP. (I) YkrX was added to a mixture that had completed the RLP reaction described in (H). (J) YkrZ was added to the reaction described in (F) after completion of the YkrX reaction. Each reaction was performed at 25°C in a final volume of 100 μl, which contained 50 mM Tris-HCl (pH 7.5), 1 mM MgCl2, and 0.1 mM MTR-1-P as the starting substrate. The amounts of proteins added for measuring UV spectral changes were 15 μg for YkrS, 3 μg for YkrY, 30 μg for RLP, 3 μg for YkrX, and 2 μg for YkrZ. Spectra were recorded at 20-s intervals. The spectra are depicted in rainbow colors in the corresponding order, beginning with violet (number 1) and finishing with red (9). Because the reaction of YkrZ was not completed within 160 s, the final spectrum (number 10), obtained after a 20-min reaction, is shown in black.

YkrS formed a compound that was positive in an assay for reducing-sugars (21), when reacted with MTR-1-P. The 1H-NMR spectrum of the compound (Fig. 2B) has the same pattern of chemical shifts as that of MTRu-1-P (14), which indicates that YkrS, which has been reported to be a homolog of the α-subunit of the eukaryotic translation initiation factor 2B (22), is an MTR-1-P isomerase.

We examined the activity of MTRu-1-P dehydratase by converting the dicarbonyl group of DK-MTP-1-P to 2,3-substituted quinoxaline using o-phenylenediamine (23). The reaction of YkrY and MTRu-1-P led to an increase in the absorption of light at 320 nm of the quinoxaline (Fig. 2G), which shows that YkrY catalyzes the dehydratase reaction. DK-MTP-1-P was too unstable to allow analysis by 1H-NMR.

2,3-Diketohexyl-1-phosphate, an analog of DK-MTP-1-P, is converted to 2-hydroxy-3-ketohexenyl-1-phosphate (λmax, around 280 nm) and, subsequently, to 1,2-dihydroxy-3-ketohexene (λmax, 310 nm) by an enolase/phosphatase from Klebsiella (15). Because DK-MTP-1-P is labile, we initiated the reaction of a possible enolase/phosphatase from B. subtilis by adding YkrY to a mixture that already contained MTR-1-P, YkrS, and RLP, YkrX, or YkrZ (Fig. 2F). The absorbance of the mixture to which RLP had been added increased at 280 nm but not at 310 nm (Fig. 2H), which suggests that RLP may catalyze only the enolization of DK-MTP-1-P. The singlet peak at 2.1 ppm in the 1H-NMR spectrum of the reaction mixture is characteristic of a methylthioether (Fig. 2C). The doublet peak at 7.5 ppm can be assigned to the C1 methine proton, which is split by the phosphorous. We predicted that the C4 and C5 methylene protons would form triplets. However, the spectrum had a triplet peak at 2.88 to 2.90 ppm and a doublet peak at 2.75 to 2.77 ppm (Fig. 2C, inset). A possible explanation for this discrepancy is that one proton on C4 of DK-MTP-1-P may have been replaced by deuterium in the reaction with YkrY in the deuterated phosphate buffer. Moreover, the mass numbers of the product, HK-MTPenyl-1-P, that was formed in the deuterated buffer were 242 and 243, whereas the mass number was 241 in the absence of D2O (24). The two mass numbers obtained in the deuterated buffer may result from the partial ionization of the C2 oxygen of HK-MTPenyl-1-P. These results indicate that RLP catalyzes the DK-MTP-1-P enolase reaction and not the enolase/phosphatase reaction, and that the product of the reaction catalyzed by RLP is HK-MTPenyl-1-P. As deduced from its amino acid sequence (Fig. 1B), RLP had no RuBP-carboxylating activity.

The addition of YkrX to a reaction mixture containing the HK-MTPenyl-1-P that had been produced by RLP (Fig. 2F) resulted in a decrease in absorption at 280 nm, although a peak of absorption appeared at 310 nm with an isosbestic point at 289 nm (Fig. 2I). The final spectrum was very similar to that of 1,2-dihydroxy-3-ketohexene (15). The 1H-NMR spectrum of the reaction product was the same as that of DHK-MTPene; removal of a phosphate group from C1 of the substrate changed the doublet peak at 7.5 ppm of the substrate to a singlet peak at 8.5 ppm in the product (Fig. 2D). In addition, the amount of inorganic phosphate detected was equal to that in the starting substrate, MTR-1-P. Thus YkrX is an HK-MTPenyl-1-P phosphatase.

The deduced amino acid sequence of YkrZ is very similar to that of DHK-MT-Pene dioxygenase from Klebsiella (13). The absorbance of DHK-MTPene at 310 nm decreased in the presence of YkrZ (Fig. 2J). The 1H-NMR spectrum of the reaction product of YkrZ resembled that produced by a mixture of 3-methylthiopropionate and formate (Fig. 2E), which suggests that YkrZ is an DHK-MTPene dioxygenase.

KMTB is recycled to methionine in the methionine salvage pathway (fig. S1), but the product of the reaction catalyzed by YkrZ was 3-methylthiopropionate. It was reported that the Ni2+ form and the Fe2+ form of the dioxygenase of Klebsiella catalyze reactions that produce 3-methylthiopropionate and KMTB, respectively (17). Because we purified histidine-tagged YkrZ on a Ni2+-column, it is not surprising that YkrZ exhibited the activity of the Ni2+ form (Figs. 2E and 3, A and B). Moreover, as shown in other studies (17), replacement of Ni2+ ions with Fe2+ ions by the method in the literature (17) allowed YkrZ to produce KMTB (Fig. 3, A and C). This result demonstrates that YkrZ is a DHK-MTPene dioxygenase, as predicted for Bacillus previously (11, 13).

Fig. 3.

Analysis by high-performance liquid chromatography (HPLC) of products synthesized by the Ni2+ form and the Fe2+ form of YkrZ. (A) Authentic KMTB and 3-methylthiopropionate. (B) Products of the Ni2+ form of the enzyme. (C) Products of the Fe2+ form of the enzyme. The reaction of YkrZ (5 μg) with DHK-MTPene, produced by YkrS, YkrY, RLP, and YkrX from 1 mM MTR-1-P, was performed in 100 μl of 50 mM Tris-HCl (pH 7.5). An aliquot (20 μl) of the reaction mixture was injected into the HPLC column for analysis. A large peak at the retention time of 7.6 min in (A) was that of 3-methylthiopropionaldehyde used for the synthesis of 3-methylthiopropionate (19).

To determine whether RLP functions in the methionine salvage pathway in vivo and whether a functional link exists between photosynthetic RuBisCO and RLP, we disrupted the ykrW gene of B. subtilis by inserting a km cassette, with the transcription of the ykrX, ykrY, and ykrZ genes controlled by the spac promoter (25). Then, we examined the growth of the ykrW strain on MTA and the effects of introducing either an intact ykrW gene or the gene for RuBisCO from R. rubrum (rbcL) into the amyE gene. We designated the new strains ykrW /ykrW+ and ykrW /rbcL+, respectively. Both wild-type cells and ykrW cells grew normally on a medium that contained methionine or KMTB, but both failed to grow without sulfur in the medium (Fig. 4, A and B). MTA supported the growth of wild-type cells but not of the ykrW strain (Fig. 4B). A similar observation had been made with MTR by Sekowska and Danchin (13). Growth of the ykrW /ykrW+ strain with MTA as the sole source of sulfur (Fig. 4C) confirmed that the ykrW gene was able to rescue the ykrW mutant. The rbcL gene from R. rubrum generated active RuBisCO and also rescued ykrW mutant cells when they were grown on the MTA medium (Fig. 4C). Thus RuBisCO from R. rubrum catalyzes the same reaction as that catalyzed by RLP or DK-MTP-1-P enolase from B. subtilis. Although the ykrW /ykrW+ and wild-type cells grew at similar rates with similar lag phases, the ykrW /rbcL+ strain grew more slowly after a longer lag phase, perhaps because of slower turnover of RuBisCO in the DK-MTP-1-P enolase reaction. Actually, recombinant RuBisCO of R. rubrum catalyzed the DK-MTP-1-P enolase reaction at a much slower rate than RLP from B. subtilis (fig. S2). The RLP of B. subtilis includes both those amino acid residues of RuBisCO that are responsible for binding the phosphate on C1 of RuBP and those required for activation by CO2. However, the residues of RuBisCO that are responsible for binding the other phosphate group of RuBP and the residues of loop 6, which are essential for RuBisCO activity (2, 3), are replaced by different amino acids in RLP (Fig. 1B). The reaction catalyzed by RuBisCO consists of three sequential, partial reactions: enolization, carboxylation or oxygenation, and hydrolysis (2, 3, 26). Deletion of loop 6 from RuBisCO prevents it from catalyzing the carboxylation/oxygenation reactions (27). However, it retains the ability to catalyze the enolization reaction (27). This observation supports the hypothesis that the RLP-catalyzed enolization of DK-MTP-1-P does not require the amino acid residues that bind the phosphate group on C5 of RuBP and the loop 6. Moreover, the structure of DK-MTP-1-P is very similar to that of RuBP. In photosynthetic RuBisCO, these additional structures may hinder the DK-MTP-1-P enolase reaction, and they may also explain the slow growth of ykrW/rbcL+ cells (Fig. 4C). In this context, our results with the RLP of B. subtilis suggest that RLPs of other bacteria may also catalyze a reaction similar to one of the partial reactions of RuBisCO in a bacterial metabolic pathway.

Fig. 4.

Analysis of the function of RLP in vivo and rescue of the ykrW mutant by the gene for RuBisCO from the photosynthetic bacterium R. rubrum. The wild-type strain (A) and the ykrW mutant strain (B) were grown in the presence of 1 mM IPTG at 37°C in medium supplemented with no metabolite (rhombuses), methionine (squares), MTA (circles), and KMTB (triangles), all at 1 mM. (C) Growth in the presence of 1 mM IPTG of wild-type (filled triangles), ykrW (filled rhombuses), ykrW/ykrW + (filled squares), and ykrW /rbcL+ (filled circles) cells on minimal medium that contained 0.5 mM MTA as the sole source of sulfur. Data points show the means ± SEM of results of three independent experiments. Standard errors were not included in the figure because they fell with the respective symbols in every case.

Our analysis shows that RLP of B. subtilis functions as a DK-MTP-1-P enolase, which has no RuBP-carboxylation activity, in the methionine salvage pathway. Moreover, this function of RLP is conserved in the RuBisCO from a photosynthetic bacterium. In a standard phylogenetic tree of the large subunits of RuBisCO, the RLP from B. subtilis is not included on any branches that include RuBisCO or on branches that include other RLPs with RuBP-carboxylation activity (Fig. 1A). The codon usage and the G + C content of the gene for RLP are typical of the organism. The literature (28) suggests that genes such as the gene for RLP were probably not derived by lateral transfer of a gene for a RuBP-carboxylating enzyme from another unrelated organism, for example, in this case, an archaeon or photosynthetic bacterium. Thus, it is possible that the gene for RLP, which in B. subtilis is part of the methionine salvage pathway, and the gene for photosynthetic RuBisCO originated from a common ancestral gene (supporting online text). However, bacteria and Archaea that have RLPs first appeared on Earth (29) long before the Calvin cycle developed in photosynthetic bacteria (30), thus we suggest that RLPs may be the ancestral enzymes of photosynthetic RuBisCO.

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