Yohtaro Saito

A new rubisco-like protein coexists with a photosynthetic rubisco in the planktonic cyanobacteria microcystis

Two genes encoding proteins related to large subunits of Rubisco were identified in the genome of the planktonic cyanobacterium Microcystis aeruginosa PCC 7806 that forms water blooms worldwide. The rbcLI gene belongs to the form I subfamily typically encountered in cyanobacteria, green algae, and land plants. The second and newly discovered gene is of the form IV subfamily and widespread in the Microcystis genus. In M. aeruginosa PCC 7806 cells, the expression of both rbcLI and rbcLIV is sulfur-dependent. The purified recombinant RbcLIV overexpressed in Escherichia coli cells did not display CO2 fixation activity but catalyzed enolization of 2,3-diketo-5-methylthiopentyl-1-phosphate, and the rbcLIV gene rescued a Bacillus subtilis MtnW-deficient mutant. Therefore, the Microcystis RbcLIV protein functions both in vitro and in vivo and might be involved in a methionine salvage pathway. Despite variations in the amino acid sequences, RbcLIV shares structural similarities with all members of the Rubisco superfamily. Invariant amino acids within the catalytic site may thus represent the minimal set for enolization, whereas variations, especially located in loop 6, may account for the limitation of the catalytic reaction to enolization. Even at low protein concentrations in vitro, the recombinant RbcLIV assembles spontaneously into dimers, the minimal unit required for Rubisco forms I–III activity. The discovery of the coexistence of RbcLI and RbcLIV in cyanobacteria, the ancestors of chloroplasts, enlightens episodes of the chaotic evolutionary history of the Rubiscos, a protein family of major importance for life on Earth.

Crystal structure of 5‐methylthioribose 1‐phosphate isomerase product complex from Bacillus subtilis: Implications for catalytic mechanism

The methionine salvage pathway (MSP) plays a crucial role in recycling a sulphahydryl derivative of the nucleoside. Recently, the genes and reactions in MSP from Bacillus subtilis have been identified, where 5‐methylthioribose 1‐phosphate isomerase (M1Pi) catalyzes a conversion of 5‐methylthioribose 1‐phosphate (MTR‐1‐P) to 5‐methylthioribulose 1‐phosphate (MTRu‐1‐P). Herein, we report the crystal structures of B. subtilis M1Pi (Bs‐M1Pi) in complex with its product MTRu‐1‐P, and a sulfate at 2.4 and 2.7 Å resolution, respectively. The electron density clearly shows the presence of each compound in the active site. The structural comparison with other homologous proteins explains how the substrate uptake of Bs‐M1Pi may be induced by an open/closed transition of the active site. The highly conserved residues at the active site, namely, Cys160 and Asp240 are most likely to be involved in catalysis. The structural analysis sheds light on its catalytic mechanism of M1Pi.

Enzymatic Characterization of 5-Methylthioribose 1-Phosphate Isomerase from Bacillus subtilis

The product of the mtnA gene of Bacillus subtilis catalyzes the isomerization of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P). The catalysis of MtnA is a novel isomerization of an aldose phosphate harboring a phosphate group on the hemiacetal group. This enzyme is distributed widely among bacteria through higher eukaryotes. The isomerase reaction analyzed using the recombinant B. subtilis enzyme showed a Michaelis constant for MTR-1-P of 138 μM, and showed that the maximum velocity of the reaction was 20.4 μmol min−1 (mg protein)−1. The optimum reaction temperature and reaction pH were 35 °C and 8.1. The activation energy of the reaction was calculated to be 68.7 kJ mol−1. The enzyme, with a molecular mass of 76 kDa, was composed of two subunits. The equilibrium constant in the reversible isomerase reaction [MTRu-1-P]/[MTR-1-P] was 6. We discuss the possible reaction mechanism.

Structural and Functional Similarities between a Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO)-like Protein from Bacillus subtilis and Photosynthetic RuBisCO

The sequences classified as genes for various ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO)-like proteins (RLPs) are widely distributed among bacteria, archaea, and eukaryota. In the phylogenic tree constructed with these sequences, RuBisCOs and RLPs are grouped into four separate clades, forms I-IV. In RuBisCO enzymes encoded by form I, II, and III sequences, 19 conserved amino acid residues are essential for CO2 fixation; however, 1-11 of these 19 residues are substituted with other amino acids in form IV RLPs. Among form IV RLPs, the only enzymatic activity detected to date is a 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) enolase reaction catalyzed by Bacillus subtilis, Microcystis aeruginosa, and Geobacillus kaustophilus form IV RLPs. RLPs from Rhodospirillum rubrum, Rhodopseudomonas palustris, Chlorobium tepidum, and Bordetella bronchiseptica were inactive in the enolase reaction. DK-MTP-1-P enolase activity of B. subtilis RLP required Mg2+ for catalysis and, like RuBisCO, was stimulated by CO2. Four residues that are essential for the enolization reaction of RuBisCO, Lys175, Lys201, Asp203, and Glu204, were conserved in RLPs and were essential for DK-MTP-1-P enolase catalysis. Lys123, the residue conserved in DK-MTP-1-P enolases, was also essential for B. subtilis RLP enolase activity. Similarities between the active site structures of RuBisCO and B. subtilis RLP were examined by analyzing the effects of structural analogs of RuBP on DK-MTP-1-P enolase activity. A transition state analog for the RuBP carboxylation of RuBisCO was a competitive inhibitor in the DK-MTP-1-P enolase reaction with a Ki value of 103 μm. RuBP and d-phosphoglyceric acid, the substrate and product, respectively, of RuBisCO, were weaker competitive inhibitors. These results suggest that the amino acid residues utilized in the B. subtilis RLP enolase reaction are the same as those utilized in the RuBisCO RuBP enolization reaction.

Enzymatic Characterization of 5-Methylthioribulose-1-phosphate Dehydratase of the Methionine Salvage Pathway in Bacillus subtilis

5-Methylthioribulose-1-phosphate (MTRu-1-P) dehydratase catalyzes the reaction from MTRu-1-P to 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) in the methionine salvage pathway in Bacillus subtilis. The properties of this enzyme remain to be determined. We characterized these properties using a recombinant protein. The enzyme, with a molecular mass of 90 kDa, was composed of four subunits. The K m and V max of the enzyme were 8.9 μM and 42.7 μmole min−1 mg protein−1 at 25 °C respectively. Maximum activity was observed at pH 7.5 to 8.5 and 40 °C. The activation energy of the reaction from MTRu-1-P to DK-MTP-1-P was 63.5 kJ mol−1. The reaction product DK-MTP-1-P was labile, and decomposed at a rate constant of 0.048 s−1 to an unknown compound that was not utilized by DK-MTP-1-P enolase, the enzyme catalyzing the next step. The function of this enzyme in the pathway is discussed.

Plausible Novel Ribose Metabolism Catalyzed by Enzymes of the Methionine Salvage Pathway in Bacillus subtilis

The methionine salvage pathway (MSP) recycles reduced sulfur from 5-methylthioribose. Here we propose a novel ribose metabolic pathway performed by MSP enzymes of Bacilli. MtnK, an initial catalyst of MSP, had significant ribose kinase activity, with Vmax and Km values of 2.9 µmol min(-1) mg of protein(-1) and 4.8 mM. Downstream enzymes catalyzed the isomerization of ribose-1-phosphate and subsequent dehydration, enolization, dephosphorylation, and dioxygenation.

Crystallization and preliminary X-ray analysis of methylthioribose-1-phosphate isomerase from Bacillus subtilis

Methylthioribose-1-phosphate isomerase (MtnA) from Bacillus subtilis, the first enzyme in the downstream section of the methionine-salvage pathway, was crystallized using the sitting-drop vapour-diffusion method. Crystals grew using ammonium sulfate as the precipitant at 293 K. They diffracted to 2.5 A at 100 K using synchrotron radiation and were found to belong to the tetragonal space group P4(1), with unit-cell parameters a = b = 69.2, c = 154.7 A. The asymmetric unit contains two molecules of MtnA, with a VM value of 2.4 A3 Da(-1) and a solvent content of 48%.

His267 is involved in carbamylation and catalysis of RuBisCO-like protein from Bacillus subtilis

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and RuBisCO-like protein (RLP) from Bacillus subtilis catalyze mechanistically similar enolase reactions. Both enzymes require carbamylation of the ε-amino group of the active site lysine during activation to generate the binding site of the essential Mg(2+) ion. His267 forms a possible hydrogen bond with the carbamate of the active site Lys176 in B. subtilis RLP. This active site histidine is completely conserved in RLPs and RuBisCO. H267Q, H267N and H267A mutant enzymes required higher CO(2) concentrations for maximal activity than wild-type enzyme, suggesting that the histidine is involved in high affinity for activator CO(2) in Bacillus RLP. These mutations showed weak effects on the catalysis of RLP, whereas this residue is reportedly essential for catalysis in RuBisCO but is not involved in the carbamylation. The different functions of the active site histidine in RLP and RuBisCO are discussed.

Crystallization and preliminary X-ray analysis of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase from Bacillus subtilis

2,3-Diketo-5-methylthiopentyl-1-phosphate enolase (DK-MTP-1P enolase) from Bacillus subtilis was crystallized using the hanging-drop vapour-diffusion method. Crystals grew using PEG 3350 as the precipitant at 293 K. The crystals diffracted to 2.3 A resolution at 100 K using synchrotron radiation and were found to belong to the monoclinic space group P2(1), with unit-cell parameters a = 79.3, b = 91.5, c = 107.0 A, beta = 90.8 degrees. The asymmetric unit contained four molecules of DK-MTP-1P enolase, with a V(M) value of 2.2 A(3) Da(-1) and a solvent content of 43%.

Evolutionary Potential of Rubisco-Like Protein in Bacillus subtilis: Interaction with Transition-State Analog of Rubisco

Rubisco-like protein (RLP) from Bacillus subtilis catalyzes the 2,3-diketo-5- methylthiopentyl- 1-phosphate (DK-MTP-1-P) enolase reaction in the methionine salvage pathway. This reaction resembles that of the first step in the Rubisco reaction; enolization of ribulose-1,5-bisphosphate (RuBP). The chemical structure of DK-MTP-1-P is very similar to that of RuBP. However, the proton Abstracted in the enolization reaction is different between the two enzymes. We are interested in the inter-relationship of the reactions catalyzed by Rubisco and RLP, and here analyzed the general enzymatic properties of the RLP using the recombinant B. subtilis enzyme. B. subtilis RLP (BsRLP) was a homodimer and had the Michaelis constant for DK-MTP-1-P of 19.3 μM and the maximum reaction velocity of 112.5 μmol min−1 (mg protein)−1. The optimum temperature and pH for the reaction were 35°C and 8.2, respectively. BsRLP required a divalent cation for catalysis and was specific for Mg2+. Interestingly,the reaction intermediate analog 2-carboxyarabinitol- 1,5-bisphosphate and the reaction product phosphoglycerate of Rubisco exhibited competitive inhibition with respect to DK-MTP-1-P, but 2-carboxyribitol- 1,5-bisphosphate had no effect at all. BsRLP conserves essential residues, K175, K201, D203 and E204, for enolization reaction of Rubisco. Mutational analysis showed that these residues also played key roles in RLP. The present study revealed that bona fide Rubisco and BsRLP resembled each other not only in their primary sequences of the proteins and catalytic reactions but also in the active site structures and catalytic mechanisms. Thus, the gene for BsRLP may be closely related to that forphotosynthetic Rubisco.