Crystal Structure of Carboxylase Reaction-oriented Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from a Thermophilic Red Alga, Galdieria partita*

Hajime Sugawara, Hiroki Yamamoto, Naoki ShibataDagger , Tsuyoshi Inoue, Sachiko Okada§, Chikahiro Miyake§, Akiho Yokota§, and Yasushi Kai

From the Department of Materials Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita 565-0871, Japan and the § Graduate School of Biological Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) obtained from a thermophilic red alga Galdieria partita has the highest specificity factor of 238 among the Rubiscos hitherto reported. Crystal structure of activated Rubisco from G. partita complexed with the reaction intermediate analogue, 2-carboxyarabinitol 1,5-bisphosphate (2-CABP) has been determined at 2.4-Å resolution. Compared with other Rubiscos, different amino residues bring the structural differences in active site, which are marked around the binding sites of P-2 phosphate of 2-CABP. Especially, side chains of His-327 and Arg-295 show the significant differences from those of spinach Rubisco. Moreover, the side chains of Asn-123 and His-294 which are reported to bind the substrate, ribulose 1,5-bisphosphate, form hydrogen bonds characteristic of Galdieria Rubisco. Small subunits of Galdieria Rubisco have more than 30 extra amino acid residues on the C terminus, which make up a hairpin-loop structure to form many interactions with the neighboring small subunits. When the structures of Galdieria and spinach Rubiscos are superimposed, the hairpin region of the neighboring small subunit in Galdieria enzyme and apical portion of insertion residues 52-63 characteristic of small subunits in higher plant enzymes are almost overlapped to each other.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39)1 is the rate-limiting enzyme in the Calvin-Benson cycle of photosynthesis. It catalyzes the addition of gaseous CO2 to ribulose 1,5-bisphosphate (RuBP) and produces two molecules of 3-phosphoglycerate (3-PGA) (1, 2). However, this enzyme also catalyzes O2 addition to RuBP as the primary reaction of photorespiration. The latter reaction yields one molecule each of 3-PGA and 2-phosphoglycolate from one molecule of RuBP. The oxygenation reaction reduces the photosynthetic efficiency up to 60% (3). Thus the improvement of the carboxylation/oxygenation ratio by genetic engineering has been attempted to increase the productivity of crop plants.

For the activation of Rubisco, the reaction of CO2 with epsilon -amino group of conserved Lys-201 must occur to form carbamate, which is stabilized by Mg2+ (4). Carbamylation is necessary for both carboxylation and oxygenation reactions. Rubisco can be easily activated by including Mg2+ and bicarbonate in vitro, but in vivo activation requires a soluble chloroplast protein, Rubisco activase, and ATP (5).

Rubisco exists as a hexadecamer (L8S8) composed of eight large and eight small subunits and its molecular mass is about 550,000 Da in all eukaryotes except dinoflagellates and many bacteria (1). Another type of Rubisco is composed of only large subunits as found in some dinoflagellates and some photosynthetic bacteria, Rhodospirillum rubrum.

The primary structure of Rubisco's large subunits of non-green algae lacking chlorophyll b are highly homologous to that of the beta -purple bacterial enzyme, however, less homologous to those of the enzymes of higher plants, green algae, and cyanobacteria (6). In contrast, amino acid sequences of small subunits are less similar than those of large subunits across the Rubiscos. Small subunits of Rubisco genes (rbcS) are nuclear-encoded in green algae, Euglena and higher plants (1, 7). The small subunits of these Rubiscos have 12-18 amino acid insertions, which are not present in the plastid-encoded small subunits of non-green algae and beta -purple bacteria. On the other hand, more than 30 extra amino acid residues exist on the C terminus in these organisms (8). From phylogenetic analysis, Rubisco genes are divided into three groups, alpha -purple bacterial, beta -purple bacterial, and non-green algal, and gamma -bacterial, cyanobacterial and plant groups (9). Even though the structural difference among these groups is presumed, no crystal structure of the non-green algal group has been determined.

The gaseous substrates CO2 and O2 are the competitive inhibitors of oxygenase and carboxylase, respectively. The carboxylation/oxygenation ratio is defined as CO2/O2 relative specificity factor (Sr), VcKo/VoKc, where Vc and Vo are maximum velocities of carboxylation and oxygenation, and Ko and Kc are the Michaelis constants for O2 and CO2, respectively (10). A high Sr value means the Rubisco with an effective carbon fixation activity. Rubisco from R. rubrum with L2 composition has the low Sr value of 15. In the case of Rubiscos with L8S8 composition, Sr values are 45 for Synechococcus, 70 for Chlamydomonas, and 93 for spinach (11). Moreover, Rubiscos from marine algae have been found to exhibit high Sr values over 100 (12). Especially, Rubiscos from thermophilic red algae show exceptionally high Sr values. Rubisco from Galdieria partita has the Sr value of 238, which is the highest among the Rubiscos hitherto reported (13). The kcat(C)/KC and kcat(O)/KO values are compared between Galdieria and spinach enzymes, where kcat(C) and kcat(O) are maximum turnover numbers of carboxylation and oxygenation, respectively. kcat(C)/KC values are 0.242 and 0.182 s-1 site-1 µM-1 in Galdieria and spinach Rubiscos, respectively. There is only 1.3-fold difference in carboxylation reaction between these Rubiscos. On the other hand, kcat(O)/KO values are 0.00102 and 0.00194 s-1 site-1 µM-1 for these Rubiscos, respectively. About 2-fold difference between these Rubiscos is observed in the oxygenation reaction. The structure of Galdieria enzyme may hinder the stabilization of the transition state for the oxygenation reaction (13).

Crystal structures of the complex of activated Rubiscos from spinach, tobacco, and Synechococcus with 2-carboxyarabinitol 1,5-bisphosphate (2-CABP) have been reported (14-18). 2-CABP tightly binds to the active site of Rubisco (Kd < 10 pM) and plays an inhibitory role for the carboxylation and oxygenation of RuBP (19). If the crystal structure of Rubisco from G. partita could be solved and compared with those of other Rubisco's, it may be possible to derive the structural principle to control its high Sr value.

Here we report the crystal structure analysis of the complex of activated Rubisco from a thermophilic red alga G. partita with 2-CABP at 2.4-Å resolution. This is the first report on the crystal structure of Rubisco from beta -purple bacterial and non-green algal group.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Galdieria Rubisco was purified with the same method as described (13). Crystallization of Galdieria Rubisco has already been reported (20). However, a new crystal form of hexagonal lattice was obtained during the improvement process of crystallization conditions. The resolution of hexagonal crystal is higher than that of the reported monoclinic crystal. Crystallization was carried out using the hanging-drop vapor diffusion (21). The drops consisted of 2 µl of protein solution of 10.0 mg ml-1 comprising 20 mM MgCl2, 20 mM NaHCO3, and 2 mM 2-CABP, and 2 µl of precipitating solution suspended over a 0.5-ml reservoir containing the same precipitating solution. Two crystal forms were obtained, monoclinic and hexagonal. Crystallization conditions of monoclinic form was 100 mM Hepes (pH 7.5) containing 10% polyethylene glycol 8000 and 8% ethylene glycol for reservoir solution. By the use of 2-methyl-2,4-pentanediol instead of ethylene glycol, the hexagonal form was obtained. Finally, the hexagonal crystals were obtained at 293 K using 50 mM Hepes (pH 7.5) containing 9% polyethylene glycol 8000 and 4% 2-methyl-2,4-pentanediol for reservoir solution. Crystals grew to a typical size of 0.5 × 0.3 × 0.3 mm3 in 4-5 days.

Data collection was performed on beamline BL18B of the Photon Factory using the screenless Weissenberg camera (22). Images were indexed and evaluated by the use of DENZO (23). The space group of Rubisco was determined to be hexagonal P64 with unit cell parameters of a = b = 117.07 and c = 319.63 Å. Assuming L4S4 of Rubisco in the asymmetric unit, the Matthews constant VM (24) is calculated to be 2.55 Å3 Da-1 corresponding to a solvent content of 51.5%, which is the value for conventional protein crystals.

X-ray diffraction data were processed by using DENZO and SCALEPACK (23). A total of 277,331 measurements from 83,303 unique reflections were recorded and Rmerge was 9.7% for the data between 40 and 2.4 Å, with the completeness of 86.4%.

Crystal structure of Galdieria Rubisco was solved by the molecular replacement method. The search model was based upon both large and small subunits (L1S1) of the activated Rubisco from Synechococcus complexed with 2-CABP (PDB code 1RBL) (17). Nonconserved residues of the model were replaced by alanine. Molecular replacement calculations were performed by using AMoRe (25) from the CCP4 suite (26). A pair of L4S4 in the asymmetric unit is related by the crystallographic 2-fold axis. From the calculation of self-rotation function, non-crystallographic 2-fold axis was found with high correlation coefficient. Thus, L4S4 has two non-crystallographic 2-fold axes, which are orthogonalized to a crystallographic 2-fold axis. Then, the practical structure unit is reduced up to L2S2. From the calculation of cross-rotation and translation functions, four expected solutions have been found. The model was refined by the use of X-PLOR with 2- and 4-fold non-crystallographic symmetry restraints (27). The 4-fold axis is set in common with one of the non-crystallographic 2-fold axes. Five percent of the reflections were set aside for Rfree calculations (28). After the application of one round of simulated annealing protocol, multiple cycles of model fitting and refinements were alternated. When the weight of 2- and 4-fold non-crystallographic symmetry restraints were 300 and 100 kcal·molecule-1 Å-2, respectively, Rfree value was the lowest. Ordered water molecules were included by selecting the peaks based on Fobs - Fcalc difference Fourier maps contoured at 2.0 sigma  and 2Fobs - Fcalc density contoured at 1.0 sigma . Non-crystallographic symmetry restraints were performed throughout the refinement process. At the final stage of refinement, bulk solvent correction was applied. The quality of the final model was assessed from Ramachandran plots and analysis of model geometry with the program PROCHECK (29). The plot indicated that 90.6% of the residues lay in the favorable regions, 9.2% in the allowed regions and 0.2% in the disallowed regions (Ser-135 of small subunit). The final R and Rfree factors for all the reflections between 20.0- and 2.4-Å resolutions were 0.165 and 0.197, respectively. The root mean square deviations from ideal geometry of the bond lengths and angles were 0.010 Å and 2.55°, respectively. The estimated mean coordinate error is about 0.25 Å as deduced from the Luzzati plot (30).

    RESULTS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Overall Structure-- Compared with spinach Rubisco, the large subunits of Galdieria Rubisco have 8 and 10 prolonged amino acid residues on N and C termini, respectively. In the final model of Galdieria Rubisco, the large subunits are traced in the electron density maps for residues 7-478, while the small subunits are traced for all residues. In the crystal structures of activated spinach and Synechococcus Rubiscos complexed with 2-CABP, the electron density of the large subunits was traced for residues 9-475 (15, 17).

Galdieria Rubisco has a L8S8 structure composed of 8 L1S1 units related by approximate D4 point symmetry (Fig. 1), which is common in cyanobacterial and plant Rubiscos. Secondary structures of Galdieria Rubisco are almost the same except the extra alpha -helix in residues 77-80 and the loss of beta -strand in residues 24-26 of the large subunits (Fig. 2). Sequence identities between Galdieria and spinach or Synechococcus Rubiscos are both about 60% in the large subunits and both about 30% in the small subunits, however, the differences in their overall structures are little between these Rubiscos. The root mean square deviations in L2S2 structures between Galdieria and spinach or Synechococcus Rubiscos are 0.96 or 0.90 Å for 1138 corresponding Calpha atoms, respectively, while that between spinach and Synechococcus Rubiscos is 0.62 Å. The root mean square deviations in the large and the small subunits between Galdieria and spinach Rubiscos are 0.70 and 1.47 Å, respectively, which indicates a larger structural difference between the small subunits than that between the large ones.


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Fig. 1.   L8S8 structure of Galdieria Rubisco. Large subunits are shown in cyan and blue-gray, and small subunits are shown in blue. A, non-crystallographic 4-fold axis passes through the center of four small subunits perpendicular to the paper. B, crystallographic 2-fold axis passes through the center of the complex perpendicular to the non-crystallographic 4-fold axis on the paper. These figures were drawn by the program MidasPlus (55, 56).


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Fig. 2.   Sequence alignment of the Rubiscos and secondary structure of Galdieria Rubisco. These sequences have been aligned using the crystal structure of activated spinach and Synechococcus Rubiscos complexed with 2-CABP (14, 17). Conserved residues for substrate binding are boxed with yellow color. The residues which cause the structural changes around the active site in Galdieria Rubisco are boxed with blue. Since N-terminal residues of the large subunit in Galdieria Rubisco are disordered and quite different from the structure of spinach enzyme's structures, large subunit sequences of N-terminal residues 1-13 were not aligned. The spinach numbering has been used in Galdieria and other Rubiscos. Secondary structure in Galdieria Rubisco was determined by the program DSSP (57). Secondary structure assignment is from Knight et al. (58). Helix-O (residues 77-80) of the large subunit is only observed in Galdieria Rubisco. This figure was drawn using the program ALSCRIPT (59).

Residue 247 in large subunit is cysteine and makes disulfide bond between the large subunits composed of a L2 dimer in higher plant and Synechococcus Rubiscos (14-17). However, the corresponding residue is methionine in Galdieria Rubisco. Moreover, while tobacco Rubisco possibly forms disulfide bonds of 172-192 and 449-459 pairs (16, 31), residues 192, 449, and 459 are not cysteines in Galdieria enzyme. Thus, Galdieria Rubisco has no disulfide bond.

Structure around the Active Site-- Amino acid residues directly interacting with 2-CABP are completely conserved in Galdieria, spinach, tobacco, and Synechococcus Rubiscos (Fig. 2). In Galdieria Rubisco, the magnesium ion is coordinated by the six atoms in the same way as in other Rubiscos (carbamate oxygen of Lys-201, side chains of Asp-203 and Glu-204, C-2 and C-3 hydroxyl groups, and carboxylate oxygen of 2-CABP) (14-18). There are little differences of coordination geometry and the structure of 2-CABP between Galdieria and other Rubiscos. The binding modes of 2-CABP in Galdieria and other Rubiscos are almost the same, too.

The Flexible Loop-- Loop-6 of the large subunits consists of residues 328-339 and exists between helix-6 and strand-6. Residues 329-337 in loop-6 are completely conserved except small subunit-less Rubiscos. Crystal structure of inactivated tobacco Rubisco adopts an open conformation in loop-6 (31). The loop-6 of activated unliganded spinach Rubisco is disordered. In contrast, structures of activated Rubiscos complexed with 2-CABP adopt a closed conformation and the epsilon -amino group of Lys-334 makes ion pairs to the carboxyl group of 2-CABP and the side chain of Glu-60 (Fig. 3A) (14-17). Here, the ion pair formation is defined as the interaction between the ions within 4 Å distance. The mobility of loop-6 is necessary to bind the substrate RuBP and stabilize the reaction intermediate. Galdieria Rubisco complexed with 2-CABP also has the closed conformation. No significant difference is found in the main chain structure of loop-6 between the closed forms of Galdieria and spinach or Synechococcus Rubiscos. Temperature factors of the atoms in loop-6 are not so high in these Rubiscos suggesting that the loop is fixed in the closed structure. The side chain of Glu-336 makes a hydrogen bond to the main chain nitrogen of residue 472 in spinach, tobacco, and Synechococcus Rubiscos (14-17). Residue 472 of beta -purple bacterial and non-green algal Rubiscos is threonine, whose hydroxyl group together with the main chain nitrogen make hydrogen bonds to the side chain of Glu-336 in Galdieria Rubisco (Fig. 3A). However, the side chain orientation of Lys-334 which makes ion pair to C-2 carboxylate group of 2-CABP is little different between Galdieria and spinach Rubiscos.


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Fig. 3.   Structural comparison around the active site. Structures of Galdieria and spinach Rubiscos are superimposed (PDB code 1BUR). Galdieria and spinach Rubiscos are shown in blue-gray and cyan, respectively. Oxygen and nitrogen atoms are shown in red and blue, respectively. One of the large subunits is only labeled as B. A, structural comparison around Lys-334. B, around His-327. C, around Arg-295. D, around His-294. E, around carbamylated Lys-201. These figures were drawn using the program MidasPlus (55, 56).

C-terminal Structure of the Small Subunits-- Along the non-crystallographic 4-fold axis, there is a narrow solvent channel passing through the center of the Rubisco holoenzyme (Fig. 1A). The extra amino acid residues on the C-terminal end of the small subunit in Galdieria Rubisco make up hairpin-loop structure (Fig. 4A). So, the solvent channel of Galdieria Rubisco is narrower than spinach Rubisco. The C-terminal region of the small subunit makes many interactions with other regions, especially with the neighboring small subunits. When the structures of Galdieria and spinach Rubiscos are superimposed, the hairpin region of the neighboring small subunit in Galdieria enzyme and apical portion of extra insertion residues 52-63 in spinach enzyme are almost overlapped to each other (Fig. 4). However, the insertion residues in spinach Rubisco makes only two hydrogen bonds to the large subunit composed of L1S1 and no interaction with neighboring small subunit (14, 15). The unique C-terminal region in Galdieria Rubisco forms one hydrogen bond and one ion pair to the large subunit composed of L1S1. Moreover, this region makes two hydrogen bonds to the neighboring large subunit arranged around the 4-fold non-crystallographic symmetry.


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Fig. 4.   Superimposition of two neighboring L1S1 structures between Galdieria and spinach Rubiscos. Two neighboring L1S1 structures of Galdieria Rubisco are shown in pink and red and those of spinach enzyme are colored by cyan and blue. A, ribbon model of overlapped Galdieria and spinach Rubiscos. Opaque colors are extra C-terminal residues 124-155 of small subunits in Galdieria Rubisco and insertion residues 52-63 of small subunits in spinach enzyme. Transparent colors are other residues. This figure was drawn using the program MOLSCRIPT (60) and rendered by program RASTER3D (61). B, enlargement of overlapped region between the extra C-terminal residues of small subunits in Galdieria Rubisco and insertion residues of small subunits in spinach enzyme. Residues 258-259 of large subunits are also shown. Large and small subunits are labeled as L and S in parentheses, respectively. This figure was drawn using the program MidasPlus (55, 56).

In Galdieria Rubisco, Ser-135 of the small subunits occupies the disallowed region in the Ramachandran plot. Residues 134-137 of the small subunit forms beta -bend I and the hydroxyl group and the main chain nitrogen of Ser-135 make hydrogen bonds to the main chain oxygen atoms of Lys-258 and Asn-287 of the neighboring large subunit, respectively. These bonds may distort the two dihedral angle (phi, phi ) of Ser-135. Arg-130 of small subunit is almost conserved in beta -purple bacterial and non-green algal group (8). The side chain of Arg-130 forms two intersubunit ion pairs. Moreover, this beta -hairpin region forms 32 intersubunit ion pairs in the overall complex.

    DISCUSSION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Structural Characteristics around the Active Site and Its Relationship with Catalytic Activities-- Compared with the active site structures of Synechococcus or spinach Rubiscos (14, 17), Galdieria Rubisco has the unique structure around P-2 phosphate of 2-CABP. Especially, side chains of His-327 and Arg-295 show the significant structural differences from those of spinach Rubisco (Fig. 3, B and C). Around His-327, the terminal oxygen of Tyr-346 in Galdieria Rubisco forms a hydrogen bond to the carbonyl oxygen of Gly-329 (Fig. 3B). In higher plants and Synechococcus, residue 346 is valine instead of tyrosine. So the extra hydrogen bond in Galdieria Rubisco may affect the position of the side chain of His-327. Mutations of His-327 of R. rubrum Rubisco replaced by asparagine, glutamine, serine, and alanine increase more than 4 times the Km for RuBP compared with that of wild-type enzyme (33). Thus, His-327 appears to control the Km for RuBP.

His-298 is completely conserved except Rubiscos from non-green algae, in which residue 298 is asparagine. In Galdieria Rubisco, the side chain of Asn-298 makes a hydrogen bond to the terminal oxygen of Tyr-301 (Fig. 3C). Residue 301 is valine, isoleucine, or leucine in the Rubiscos except beta -purple bacterial and non-green algal group. Asp-302 of spinach Rubisco makes ion pair to His-298. The side chain of corresponding residue 302 in Galdieria Rubisco, serine, also makes a hydrogen bond to Asn-298. These two different types of interactions may cause the structural difference around Arg-295. Leucine and lysine mutants on Arg-295 of Rubisco obtained from cyanobacterium Anacystis nidulans are almost devoid of the carboxylation activity and are little inhibited by 2-CABP. These results suggest that Arg-295 is essential for the binding of the substrate RuBP (34). Thus, Arg-295 also seems to influence the Km for RuBP. Rubisco from the red alga Porphyridium has the value of 3.7 µM (12), which is 18% of the Km of spinach Rubisco. Porphyridium Rubisco has a high sequence identity with the Galdieria enzyme (80% identical in both large and small subunits) (35) and is regarded to have a similar Km value to that of the latter. Therefore, the unique side chain structures of Arg-295 and His-327 may reduce the Km for RuBP in Galdieria Rubisco even though the Km of Galdieria Rubisco has not been determined.

Threonine and cysteine mutants on Ser-379 of Rubisco from cyanobacterium A. nidulans are almost devoid of oxygenase activity (36). Thus, Ser-379 may control the oxygenase activity. The side chains of His-327 and Ser-379 in Galdieria Rubisco have the van der Waals contact (3.6 Å apart) and are 0.5 Å closer to each other in comparison with that in spinach Rubisco (Fig. 3B). The side chain position of His-327 in Galdieria Rubisco may be one of the structural factors to affect the specificity factor controlled by Ser-379.

His-298, a completely conserved residue except Rubiscos in beta -purple bacterial and non-green algal group, was considered to relate with the enzymatic activity. However, the corresponding alanine mutant in R. rubrum Rubisco maintains a high level of the enzymatic function with a slight decrease of activity (37). Crystal structure of activated R. rubrum Rubisco complexed with the substrate RuBP shows that the P-2 phosphate of RuBP is far from the side chains of Arg-295 and His-298 (5.1 and 5.6 Å apart, respectively) because of the approach of the side chain of Arg-295 to Ala-311 (38). This result suggests that His-298 hardly influences the enzymatic activity in R. rubrum Rubisco. Residue 311, which is phenylalanine except small subunit-less Rubiscos, restrains the side chain orientation of Arg-295 binding to P-2 phosphate of 2-CABP (14-17). The P-2 phosphate of RuBP makes ionic interactions not only to the side chain of Arg-295 but also to that of His-298 as observed in the crystal structure of spinach-activated enzyme complexed with the substrate RuBP, and Ca2+ instead of Mg2+ (39). Moreover, in the activated spinach Rubisco complexed with 3-PGA, the side chain of His-298 makes a hydrogen bond to O-1 of 3-PGA (40). These crystallographic results suggest that His-298 influences the enzymatic activity except the small subunit-less Rubiscos. When the structures of Galdieria and the activated Rubiscos binding Ca2+ are superimposed, the side chain of Asn-298 in the Galdieria enzyme seems to form no hydrogen bond with the P-2 phosphate of RuBP. The amino acids on residue 298 may be important to explain the catalytic difference between Galdieria and other Rubiscos.

Compared with other Rubiscos, the side chain of His-294 in Galdieria Rubisco also has the different structure, since the carbonyl oxygen of His-294 in Galdieria Rubisco makes a hydrogen bond to the main chain nitrogen of residue 270 because of the lack of residue 269 (Figs. 2 and 3D). This bond causes another hydrogen bond formation between the side chains of His-294 and Asn-123. Since His-294 and Asn-123 are the residues to bind 2-CABP in the active site of the enzyme, the new bond formation between these residues may modify significantly the enzymatic function of Galdieria Rubisco. The side chains of His-294 obtained from both spinach and Synechococcus Rubiscos make a hydrogen bond to the C-3 hydroxyl group of 2-CABP (Fig. 3D). In Galdieria Rubisco, the corresponding bond is also weakly formed (3.2 Å apart). In the theoretical study, transition structures for the carboxylation and oxygenation steps show little structural differences (41). The dihedral angle of O2-C2-C3-O3 is 4.40 and -7.11° for the transition structures of the carboxylation and oxygenation of RuBP, respectively, suggesting that a slight structural change around C-3 hydroxyl group may influence the catalytic activities in the carboxylation and oxygenation. The structural change of the side chain of His-294 may affect the Sr value in Galdieria Rubisco.

In Galdieria Rubisco, the amino acid residues around carbamylated Lys-201 make unique hydrogen bonds (Fig. 3E). In higher plants, residue 200 is threonine whose hydroxyl group makes a hydrogen bond to the side chain of His-238. However, in Galdieria Rubisco, residue 200 is valine, which is inactive in the hydrogen bond formation. On the other hand, residue 174 in Galdieria Rubisco is threonine, of which hydroxyl group makes a hydrogen bond to the carbonyl oxygen of carbamylated Lys-201. However, in higher plants, residue 174 is hydrophobic isoleucine. Lys-175 has the epsilon -amino group pKa of 7.9 and shows high nucleophilicity (42). Glycine mutant on Lys-175 in R. rubrum Rubisco does not catalyze the enolization reaction, suggesting that the epsilon -amino group of Lys-175 abstracts C-3 proton of RuBP (43). However, this epsilon -amino group is located too distant from C-3 proton of 2-CABP in the structures of activated Rubiscos complexed with 2-CABP (14-17). In Galdieria Rubisco, the epsilon -amino group of Lys-175 is also located too remote (6.1 Å apart) from the C-3 of 2-CABP. These crystallographic results indicate that the group of Lys-175 indirectly catalyzes the enolization reaction (2, 15). The carbamate group on Lys-201 seems to carry out the removal of C-3 proton (15, 17, 39). Moreover, Lys-175 is crucial to the protonation of the C-2 aci-acid of 3-PGA (40, 44). Thus, Lys-175, as well as carbamylated Lys-201, is an indispensable residue for the catalytic reaction, especially enolization. The hydrogen bond between Thr-174 and Lys-201 seems to cause the concerted movement of the side chains of Lys-175 and Lys-201 during the catalytic reaction. The experiment in tritiated water shows that the enolization is partially a rate-limiting step in overall carboxylase reaction (45). Moreover, the measurement of the oxygen isotope effect suggests that oxygen addition is major rate-limiting step (46). Thus, the concerted movement may affect the velocity of carboxylase reaction. On the other hand, the CO2 concentration in the chloroplast of C3 plants is 5-7 µM (47), which is 35-50% of the KC of spinach Rubisco. G. partita lives under the environment with very low CO2 concentration. That is to say, Galdieria Rubisco seems to adjust itself to the environment with low CO2 concentration. In fact, the catalytic efficiency of the carboxylase reaction in Galdieria Rubisco is 1.3-fold higher than that in spinach enzyme (13). It means that Galdieria Rubisco maintains the high velocity of the carboxylase reaction under low CO2 concentration. Therefore, the hydrogen bond between Thr-174 and Lys-201 may play a role for increasing the catalytic efficiency in vivo.

In Galdieria Rubisco, the phenyl ring position of Phe-127 which makes a van der Waals interaction with the side chain of Glu-60 is rotated about 30° from the corresponding position in spinach Rubisco (Fig. 3A). While the side chain of Phe-127 interacts with the Cgamma and Cdelta of Glu-60 in spinach Rubisco (3.4 and 3.4 Å apart, respectively), it makes the interaction with only Cgamma in Galdieria enzyme (3.7 Å). The phenyl group of Phe-127 of activated unliganded spinach Rubisco is rotated about 60° in comparison with that of activated spinach enzyme complexed with 2-CABP (14, 32). In Galdieria Rubisco, the phenyl ring of Phe-127 occupies the middle position between both forms of spinach-activated structures. The structural change of phenyl ring orientation in Galdieria Rubisco seems to be based on the hydrogen bond between the carbonyl oxygen of Phe-127 and the side chain of Arg-303, since the structures around residue 301 and 302 are different between Galdieria and spinach Rubiscos. Residue 59 next to Glu-60 is glycine in beta -purple bacterial and non-green algal Rubiscos, while the residue is conserved by alanine in the enzymes in other groups. In Galdieria Rubisco, the residues around Gly-59 can take the flexible conformation during the catalytic reactions. The glutamine mutant of Glu-60 in R. rubrum Rubisco exhibits only 3% of the wild-type Sr value (48). The side chain orientation of Glu-60 may relate with the partitioning ratio between the carboxylation and oxygenation of RuBP. The structural difference around Glu-60 may possibly affect the increase of the Sr value.

Role of C-terminal of the Small Subunit-- In Galdieria Rubisco, the extra C-terminal residues in the small subunit appear to make the intersubunit interactions more rigid. In the recently reported structure of hyperthermophilic oligomeric proteins, a large number of intersubunit ion pairs were observed (49-51). Glutamate dehydrogenase is hexamer and the numbers of intersubunit ion pairs of the enzymes from mesophile Clostridium symbiosum and hyperthermophile Pyrococcus furiosus are 18 and 62, respectively (52). G. partita is thermophilic and the optimum temperature for growth is about 45 °C. Thus Galdieria Rubisco seems to have rather high thermostability. The number of intersubunit ion pairs in Galdieria Rubisco is 136 in whole complex. The corresponding numbers in spinach Rubisco is 108 (14). Intersubunit ion pairs in Galdieria Rubisco are 1.3-fold bigger than that in spinach Rubisco. Therefore, C-terminal ion pairs appears to contribute to the thermostability of Galdieria Rubisco.

The enzymatic roles of the small subunits have not been made clear, but a single point mutation in the small subunit causes a loss of the catalytic activity despite the long distance from the active site of the large subunits (53). Hybrid Rubiscos which are different in species between the large and small subunits have been constructed to examine the role of the small subunits. The hybrid Rubisco, which consists of the large subunits from Synechococcus and the small subunits from marine diatom Cylindrotheca sp., decreases the kcat(C) value up to 5% of Synechococcus Rubisco. However, the Sr value increases up to 65 against 41 of the value in Synechococcus Rubisco (54). The small subunits of Cylindrotheca sp. Rubisco also have more than 30 extra residues on C terminus, which seems to be important for the increase of the Sr value. However, in Galdieria Rubisco, this C-terminal region is far from the substrate (more than 23 Å). Thus, it is not clear whether this extra C-terminal region contributes to an increase of the Sr value. However, 4 residues of this C-terminal region interact with large subunits in Galdieria Rubisco. Arg-130 makes an ion pair to Glu-223 of the large subunit composed of L1S1. These 2 residues are completely conserved in Rubiscos from the beta -purple bacteria and non-green algae, may form the common interactions. However, residue 223 of the large subunits in other Rubiscos interacts with the small subunits (14-17). On the other hand, the main chain nitrogen of Ser-135 of the small subunits makes a hydrogen bond with Lys-258 in Galdieria Rubisco, which also seems to be in common with Rubisco of the beta -purple bacterial and non-green algal group. The electrophoretic and crystallographic results show that spinach Rubisco has two kinds of small subunits. Residue 56 of the small subunits is leucine or histidine (14) and is included in the loop inserted in the crevice between the L2 dimers. His-56 makes a hydrogen bond to the main chain oxygen of Glu-259 of the neighboring large subunit. Glu-259 makes an ion pair to that of Arg-258 between the L2 dimers (Fig. 4B). In Galdieria Rubisco, the epsilon -amino group of Lys-139 makes a hydrogen bond to the main chain oxygen of Glu-259 of the large subunits composed of L1S1 and residues 135-139 of small subunits connect residues 258 and 259 of the large subunits instead of the direct interactions like in the spinach enzyme (Fig. 4B). Synechococcus Rubisco has neither C-terminal region of small subunits characteristic of non-green algal enzyme nor residues 52-63 of small subunits in higher plant enzymes. The loss of these residues may cause no interaction between the residues 258 and 259. It was suggested that the hydrogen bond between His-56 of small subunits and Glu-258 of large subunits relates with the regulatory communication in catalysis between the L2 dimers of the higher plant enzymes (14). The small subunits of Galdieria Rubisco also make a hydrogen bond to residue 258 of large subunit. Since Synechococcus Rubisco has the low Sr value, the hydrogen bond between residue 258 of large subunits and small subunit may possibly be related with the increase of the Sr value in Galdieria enzyme.

    ACKNOWLEDGEMENTS

We thank to Professor N. Sakabe of Tsukuba University and Drs. N. Watanabe, M. Suzuki, and N. Igarashi of the National Laboratory for High Energy Physics, for kind help in data collection at the Photon Factory. The structure refinement was performed on NEC SX-4B at the crystallographic research center, Institute for Protein Research, Osaka university. We are also grateful to Dr. G. Kurisu for the use of this supercomputer. We are also grateful to the Sakabe project of the TARA (Tsukuba Advanced Research Alliance) center at the university of Tsukuba.

    FOOTNOTES

* This work was supported in part by Grant-in-Aid 10305065 for Science Research (A) (to Y. K.) from the Ministry of Education, Science, Sports and Culture, Japan, and the Petroleum Energy Center subsidized by MITI.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (codes 1BWV and R1BWVSF) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

Dagger Present address: Dept. of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo 678-1297, Japan.

To whom correspondence should be addressed. Tel.: 81-6-6879-7408; Fax: 81-6-6879-7409; E-mail: kai{at}ap.chem.eng.osaka-u.ac.jp.

    ABBREVIATIONS

The abbreviations used are: Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase; RuBP, ribulose 1,5-bisphosphate; 3-PGA, 3-phosphoglycerate; L2, L1S1, L2S2, L4S4, L8S8, two large subunits, large and small subunits, two large and two small subunits, four large and four small subunits, holoenzyme of Rubisco; rbcS, small subunit gene of Rubisco; Sr, specificity factor; VC, maximum velocity of carboxylation; VO, maximum velocity of oxygenation; KO, Michaelis constant for O2; KC, Michaelis constant for CO2; kcat(C), maximum turnover numbers of carboxylation; kcat(O), maximum turnover numbers of oxygenation; 2-CABP, 2-carboxyarabinitol 1,5-bisphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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