Roles of RubisCO and the RubisCO-Like Protein in 5-Methylthioadenosine Metabolism in the Nonsulfur Purple Bacterium Rhodospirillum rubrum

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the assimilation of atmospheric CO₂ into organic matter and is thus central to the existence of life on earth. The beginning of the 2000s was marked by the discovery of a new family of proteins, the RubisCO-like proteins (RLPs), which are structural homologs of RubisCO. RLPs are unable to catalyze CO₂ fixation. The RLPs from Chlorobaculum tepidum, Bacillus subtilis, Geobacillus kaustophilus, and Microcystis aeruginosa have been shown to participate in sulfur metabolism. Whereas the precise function of C. tepidum RLP is unknown, the B. subtilis, G. kaustophilus, and M. aeruginosa RLPs function as tautomerases/enolases in a methionine salvage pathway (MSP). Here, we show that the form II RubisCO enzyme from the nonsulfur purple bacterium Rhodospirillum rubrum is also able to function as an enolase in vivo as part of an MSP, but only under anaerobic conditions. However, unlike B. subtilis RLP, R. rubrum RLP does not catalyze the enolization of 2,3-diketo-5-methylthiopentyl-1-phosphate. Instead, under aerobic growth conditions, R. rubrum RLP employs another intermediate of the MSP, 5-methylthioribulose-1-phosphate, as a substrate, resulting in the formation of different products. To further determine the interrelationship between RubisCOs and RLPs (and the potential integration of cellular carbon and sulfur metabolism), the functional roles of both RubisCO and RLP have been examined in vivo via the use of specific knockout strains and complementation studies of R. rubrum. The presence of functional, yet separate, MSPs in R. rubrum under both aerobic (chemoheterotrophic) and anaerobic (photoheterotrophic) growth conditions has not been observed previously in any organism. Moreover, the aerobic and anaerobic sulfur salvage pathways appear to be differentially controlled, with novel and previously undescribed steps apparent for sulfur salvage in this organism.Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) is the key enzyme of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway. This enzyme catalyzes the primary CO₂ fixation reaction and is found in diverse organisms, including plants, most photosynthetic and chemoautotrophic microorganisms, and many archaea (25). On the basis of amino acid sequence similarities, the RubisCO family of proteins has been classified into four groups, i.e., form I, form II, form III, and form IV (Fig. ​(Fig.1).1). The enzymes classified under forms I, II, and III are all able to catalyze the RubisCO reaction, i.e., carboxylation/oxygenation of RuBP. The most recently discovered group of enzymes in the RubisCO family are the form IV or RubisCO-like proteins (RLPs). These proteins have thus far been identified in proteobacteria, cyanobacteria, archaea, and algae (2, 4, 8, 11, 12, 21, 25, 26). RLPs have been further divided into six different subgroups based on sequence similarities within the group: IV-Photo, IV-Nonphoto, IV-YkrW, IV-DeepYrkW, IV-GOS (Global Ocean Sequencing), and IV-AMC (Acid Mine Consortium) (25, 26). Despite sharing a level of sequence similarity with the bonafide RubisCOs, the RLPs are unable to carry out CO₂/O₂ fixation because their sequences contain dissimilar residues at positions analogous to RubisCO''s active-site residues (25). The structures of the Geobacillus kaustophilus and Chlorobaculum tepidum RLPs have now been solved, and there are indeed differences between the tertiary structures of these two proteins and the bonafide RubisCO enzymes (14, 17, 25). Moreover, distinct patterns of active-site residue identities among the different clades of the RLP lineage suggest that these subgroups of RLPs are likely to utilize different substrates and perform dissimilar reactions (23, 25, 26).Open in a separate windowFIG. 1.Summary of the different classes of RubisCO found in nature so far (25). Forms I, II, and III catalyze bonafide CO₂/O₂ fixation reactions by using RuBP as the substrate. Form IV RubisCO (RLP) does not catalyze RuBP-dependent CO₂/O₂ fixation and is divided into six known clades (25), with only representatives of the type IV-YkrW and IV-DeepYkrW subgroups shown to catalyze defined, yet distinct, reactions (Fig. ​(Fig.22).Previous studies performed with the Chlorobaculum tepidum RLP (of the IV-Photo group) gave the first indication that the RLPs may be involved in some aspect of sulfur metabolism (12, 13). This was later substantiated when the precise function was established for the Bacillus subtilis (2), Microcystis aeruginosa (4), and Geobacillus kaustophilus (14) RLPs of the IV-YkrW group. All three proteins catalyze a tautomerase/enolase reaction of a methionine salvage pathway (MSP) in which the substrate 2,3-diketo-5-methylthiopentanyl-1-phosphate (DK-MTP 1P) is converted to 2-hydroxy-3-keto-5-thiomethylpent-1-ene 1-phosphate (HK-MTP 1P) (Fig. ​(Fig.2).2). This reaction is very reminiscent of the enolization of RuBP catalyzed by RubisCO. Moreover, form II RubisCO from Rhodospirillum rubrum was shown to complement an RLP mutant strain of B. subtilis, with the ability to catalyze the identical tautomerase/enolase reaction (2). Interestingly, in addition to the presence of a form II RubisCO gene (cbbM), the genome of R. rubrum also encodes an RLP that clusters with the IV-DeepYkrW group (25). The function of this protein was recently determined, and it was shown to catalyze a distinct reaction that uses 5-methylthioribulose-1-phosphate as the substrate (15). Via an unprecedented 1,3-proton transfer, with two successive 1,2-proton transfers from its substrate, R. rubrum RLP catalyzes the formation of two products, i.e., 1-thiomethyl-d-xylulose-5-phosphate and 1-thiomethyl-d-ribulose-5-phosphate, at a 3:1 ratio (15) (Fig. ​(Fig.2).2). The novel reaction catalyzed by this RLP suggests that R. rubrum likely uses a different pathway to salvage sulfur compounds.Open in a separate windowFIG. 2.Distinct reactions catalyzed by type IV-YkrW (A) and type IV-DeepYkrW (B) classes of form IV RubisCO/RLP, exemplified by the proteins from B. subtilis and R. rubrum, respectively.The presence of an RLP-encoding gene triggered the search for additional genes in the R. rubrum genome that might be homologs of known enzymes that participate in a conventional MSP. Several genes were indeed identified to encode homologs of MSP enzymes. However, to this point there is no experimental evidence for the existence of a functional MSP (21) in R. rubrum. Thus, in this study, we sought to determine the role of the RLP and RubisCO protein in sulfur salvage since each protein catalyzes different reactions and RubisCO is known to be synthesized only under anaerobic conditions (6, 7). Moreover, it is well appreciated that R. rubrum possesses a versatile metabolic capacity and is able to grow under both anaerobic and aerobic conditions, using a variety of carbon sources. The involvement of RLP and RubisCO in sulfur salvage was thus determined and found to be associated with aerobic and anaerobic metabolism, respectively.