Crystal Structure of Carboxylase Reaction-oriented Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from a Thermophilic Red Alga, Galdieria partita
1999; Elsevier BV; Volume: 274; Issue: 22 Linguagem: Inglês
10.1074/jbc.274.22.15655
ISSN1083-351X
AutoresHajime Sugawara, Hiroki Yamamoto, Naoki Shibata, Tsuyoshi Inoue, Sachiko Okada, Chikahiro Miyake, Akiho Yokota, Yasushi Kai,
Tópico(s)Microbial metabolism and enzyme function
ResumoRibulose 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 inGaldieria enzyme and apical portion of insertion residues 52–63 characteristic of small subunits in higher plant enzymes are almost overlapped to each other. 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 inGaldieria enzyme and apical portion of insertion residues 52–63 characteristic of small subunits in higher plant enzymes are almost overlapped to each other. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC4.1.1.39) 1The 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; V C, maximum velocity of carboxylation; V O, maximum velocity of oxygenation; K O, Michaelis constant for O2; K C, Michaelis constant for CO2; k cat(C), maximum turnover numbers of carboxylation; k cat(O), maximum turnover numbers of oxygenation; 2-CABP, 2-carboxyarabinitol 1,5-bisphosphate 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) (1Andrews T.J. Lorimer G.H. Hatch M.D. Boardman N.K. The Biochemistry of Plants. 10. Academic Press, New York1987: 131-218Google Scholar, 2Cleland W.W. Andrews T.J. Gutteridge S. Hartman F.C. Lorimer G.H. Chem. Rev. 1998; 98: 549-561Crossref PubMed Scopus (344) Google Scholar). 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% (3Zelitch I. Science. 1975; 188: 626-633Crossref PubMed Scopus (102) Google Scholar). 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 ε-amino group of conserved Lys-201 must occur to form carbamate, which is stabilized by Mg2+ (4Lorimer G.H. Biochemistry. 1981; 20: 1236-1240Crossref PubMed Scopus (98) Google Scholar). 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 (5Portis Jr., A.R. Biochim. Biophys. Acta. 1990; 1015: 15-28Crossref PubMed Scopus (108) Google Scholar). 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 (1Andrews T.J. Lorimer G.H. Hatch M.D. Boardman N.K. The Biochemistry of Plants. 10. Academic Press, New York1987: 131-218Google Scholar). 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 β-purple bacterial enzyme, however, less homologous to those of the enzymes of higher plants, green algae, and cyanobacteria (6Morden C.W. Golden S.S. J. Mol. Evol. 1991; 32: 379-395Crossref PubMed Scopus (57) Google Scholar). 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 (1Andrews T.J. Lorimer G.H. Hatch M.D. Boardman N.K. The Biochemistry of Plants. 10. Academic Press, New York1987: 131-218Google Scholar, 7Tabita F.R. Microbiol. Rev. 1988; 52: 155-189Crossref PubMed Google Scholar). 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 β-purple bacteria. On the other hand, more than 30 extra amino acid residues exist on the C terminus in these organisms (8Assali N.-E. Martin W.F. Sommerville C.C. Loiseaux-de Goër S. Plant Mol. Biol. 1991; 17: 853-863Crossref PubMed Scopus (41) Google Scholar). From phylogenetic analysis, Rubisco genes are divided into three groups, α-purple bacterial, β-purple bacterial, and non-green algal, and γ-bacterial, cyanobacterial and plant groups (9Assali N.-E. Mache R. Loiseaux-de Goër S. Plant Mol. Biol. 1990; 15: 307-315Crossref PubMed Scopus (57) Google Scholar). 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),V c K o/V o K c, where V c and V o are maximum velocities of carboxylation and oxygenation, and K oand K c are the Michaelis constants for O2 and CO2, respectively (10Liang W.A. Ogren W.L. Hageman R.H. Plant Physiol. 1974; 54: 678-685Crossref PubMed Google Scholar). 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 forSynechococcus, 70 for Chlamydomonas, and 93 for spinach (11Uemura K. Suzuki Y. Shinkai T. Wadano A. Jensen R.G. Chmara W. Yokota A. Plant Cell Physiol. 1996; 37: 325-331Crossref Scopus (50) Google Scholar). Moreover, Rubiscos from marine algae have been found to exhibit high Sr values over 100 (12Read B.A. Tabita F.R. Arch. Biochem. Biophys. 1994; 312: 210-218Crossref PubMed Scopus (111) Google Scholar). Especially, Rubiscos from thermophilic red algae show exceptionally high Sr values. Rubisco fromGaldieria partita has the Sr value of 238, which is the highest among the Rubiscos hitherto reported (13Uemura K. Anwaruzzaman Miyachi S. Yokota A. Biochem. Biophys. Res. Commun. 1997; 233: 568-571Crossref PubMed Scopus (137) Google Scholar). Thek cat(C)/K C andk cat(O)/K O values are compared between Galdieria and spinach enzymes, wherek cat(C) and k cat(O) are maximum turnover numbers of carboxylation and oxygenation, respectively. k cat(C)/K Cvalues are 0.242 and 0.182 s−1 site−1μm−1 in Galdieria and spinach Rubiscos, respectively. There is only 1.3-fold difference in carboxylation reaction between these Rubiscos. On the other hand,k cat(O)/K O values are 0.00102 and 0.00194 s−1 site−1μm−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 (13Uemura K. Anwaruzzaman Miyachi S. Yokota A. Biochem. Biophys. Res. Commun. 1997; 233: 568-571Crossref PubMed Scopus (137) Google Scholar). Crystal structures of the complex of activated Rubiscos from spinach, tobacco, and Synechococcus with 2-carboxyarabinitol 1,5-bisphosphate (2-CABP) have been reported (14Shibata N. Inoue T. Fukuhara K. Nagara Y. Kitagawa R. Harada S. Kasai N. Uemura K. Kato K. Yokota A. Kai Y. J. Biol. Chem. 1996; 271: 26449-26452Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 15Andersson I. J. Mol. Biol. 1996; 259: 160-174Crossref PubMed Scopus (146) Google Scholar, 16Schreuder H.A. Knight S. Curmi P.M.G. Andersson I. Cascio D. Sweet R.M. Brändén C.-I. Eisenberg D. Protein Sci. 1993; 2: 1136-1146Crossref PubMed Scopus (49) Google Scholar, 17Newman J. Gutteridge S. J. Biol. Chem. 1993; 268: 25876-25886Abstract Full Text PDF PubMed Google Scholar, 18Lundqvist T. Schneider G. J. Biol. Chem. 1989; 264: 7078-7083Abstract Full Text PDF PubMed Google Scholar). 2-CABP tightly binds to the active site of Rubisco (K d < 10 pm) and plays an inhibitory role for the carboxylation and oxygenation of RuBP (19Pierce J. Tolbert N.E. Barker R. Biochemistry. 1980; 19: 934-942Crossref PubMed Scopus (200) Google Scholar). If the crystal structure of Rubisco fromG. 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. partitawith 2-CABP at 2.4-Å resolution. This is the first report on the crystal structure of Rubisco from β-purple bacterial and non-green algal group. Galdieria Rubisco was purified with the same method as described (13Uemura K. Anwaruzzaman Miyachi S. Yokota A. Biochem. Biophys. Res. Commun. 1997; 233: 568-571Crossref PubMed Scopus (137) Google Scholar). Crystallization of Galdieria Rubisco has already been reported (20Shibata N. Yamamoto H. Inoue T. Uemura K. Yokota A. Kai Y. J. Biochem. (Tokyo). 1996; 120: 1064-1066Crossref PubMed Scopus (6) Google Scholar). 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 (21McPherson A. Preparation and Analysis of Protein Crystals. 1st Ed. John Wiley, New York1982: 96-97Google Scholar). 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 mmHepes (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 (22Sakabe N. Nucl. Instrum. Methods A. 1991; 303: 448-463Crossref Scopus (320) Google Scholar). Images were indexed and evaluated by the use of DENZO (23Otwinowski Z. Sawyer L. Issacs N. Bailey S. Proceedings of CCP4 Study Weekend 1993. Daresbury Laboratory, Warrington, England1993: 56-62Google Scholar). The space group of Rubisco was determined to be hexagonal P64 with unit cell parameters of a = b = 117.07 andc = 319.63 Å. Assuming L4S4 of Rubisco in the asymmetric unit, the Matthews constantV M (24Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7935) Google Scholar) is calculated to be 2.55 Å3Da−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 (23Otwinowski Z. Sawyer L. Issacs N. Bailey S. Proceedings of CCP4 Study Weekend 1993. Daresbury Laboratory, Warrington, England1993: 56-62Google Scholar). A total of 277,331 measurements from 83,303 unique reflections were recorded and R merge 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) (17Newman J. Gutteridge S. J. Biol. Chem. 1993; 268: 25876-25886Abstract Full Text PDF PubMed Google Scholar). Nonconserved residues of the model were replaced by alanine. Molecular replacement calculations were performed by using AMoRe (25Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5031) Google Scholar) from the CCP4 suite (26Collaborative Computational Project, Number 4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19824) Google Scholar). 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 (27Brünger A.T. Kuriyan J. Karplus M. Science. 1987; 235: 458-460Crossref PubMed Scopus (2127) Google Scholar). 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 forR free calculations (28Brünger A.T. Methods Enzymol. 1997; 277: 366-396Crossref PubMed Scopus (277) Google Scholar). 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,R free value was the lowest. Ordered water molecules were included by selecting the peaks based onF obs − F calc difference Fourier maps contoured at 2.0 ς and 2F obs −F calc density contoured at 1.0 ς. 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 (29Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). 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 Rand R free 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 (30Luzzati V. Acta Crystallogr. 1952; 5: 802-810Crossref Google Scholar). 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 ofGaldieria 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 (15Andersson I. J. Mol. Biol. 1996; 259: 160-174Crossref PubMed Scopus (146) Google Scholar, 17Newman J. Gutteridge S. J. Biol. Chem. 1993; 268: 25876-25886Abstract Full Text PDF PubMed Google Scholar). Galdieria Rubisco has a L8S8structure composed of 8 L1S1 units related by approximate D 4 point symmetry (Fig.1), which is common in cyanobacterial and plant Rubiscos. Secondary structures of Galdieria Rubisco are almost the same except the extra α-helix in residues 77–80 and the loss of β-strand in residues 24–26 of the large subunits (Fig.2). Sequence identities betweenGaldieria 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 Galdieriaand spinach or Synechococcus Rubiscos are 0.96 or 0.90 Å for 1138 corresponding Cα 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 betweenGaldieria 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.Figure 2Sequence 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 (14Shibata N. Inoue T. Fukuhara K. Nagara Y. Kitagawa R. Harada S. Kasai N. Uemura K. Kato K. Yokota A. Kai Y. J. Biol. Chem. 1996; 271: 26449-26452Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 17Newman J. Gutteridge S. J. Biol. Chem. 1993; 268: 25876-25886Abstract Full Text PDF PubMed Google Scholar). Conserved residues for substrate binding are boxedwith yellow color. The residues which cause the structural changes around the active site in Galdieria Rubisco areboxed 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 (57Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12507) Google Scholar). Secondary structure assignment is from Knight et al. (58Knight S. Andersson I. Brändén C.-I. J. Mol. Biol. 1990; 215: 113-160Crossref PubMed Scopus (282) Google Scholar). Helix-O (residues 77–80) of the large subunit is only observed in Galdieria Rubisco. This figure was drawn using the program ALSCRIPT (59Barton G.J. Protein Eng. 1993; 6: 37-40Crossref PubMed Scopus (1113) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 (14Shibata N. Inoue T. Fukuhara K. Nagara Y. Kitagawa R. Harada S. Kasai N. Uemura K. Kato K. Yokota A. Kai Y. J. Biol. Chem. 1996; 271: 26449-26452Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 15Andersson I. J. Mol. Biol. 1996; 259: 160-174Crossref PubMed Scopus (146) Google Scholar, 16Schreuder H.A. Knight S. Curmi P.M.G. Andersson I. Cascio D. Sweet R.M. Brändén C.-I. Eisenberg D. Protein Sci. 1993; 2: 1136-1146Crossref PubMed Scopus (49) Google Scholar, 17Newman J. Gutteridge S. J. Biol. Chem. 1993; 268: 25876-25886Abstract Full Text PDF PubMed Google Scholar). However, the corresponding residue is methionine in Galdieria Rubisco. Moreover, while tobacco Rubisco possibly forms disulfide bonds of 172–192 and 449–459 pairs (16Schreuder H.A. Knight S. Curmi P.M.G. Andersson I. Cascio D. Sweet R.M. Brändén C.-I. Eisenberg D. Protein Sci. 1993; 2: 1136-1146Crossref PubMed Scopus (49) Google Scholar, 31Curmi P.M.G. Cascio D. Sweet R.M. Eisenberg D. Schreuder H. J. Biol. Chem. 1992; 267: 16980-16989Abstract Full Text PDF PubMed Google Scholar), residues 192, 449, and 459 are not cysteines in Galdieria enzyme. Thus,Galdieria Rubisco has no disulfide bond. Amino acid residues directly interacting with 2-CABP are completely conserved inGaldieria, spinach, tobacco, and SynechococcusRubiscos (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) (14Shibata N. Inoue T. Fukuhara K. Nagara Y. Kitagawa R. Harada S. Kasai N. Uemura K. Kato K. Yokota A. Kai Y. J. Biol. Chem. 1996; 271: 26449-26452Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 15Andersson I. J. Mol. Biol. 1996; 259: 160-174Crossref PubMed Scopus (146) Google Scholar, 16Schreuder H.A. Knight S. Curmi P.M.G. Andersson I. Cascio D. Sweet R.M. Brändén C.-I. Eisenberg D. Protein Sci. 1993; 2: 1136-1146Crossref PubMed Scopus (49) Google Scholar, 17Newman J. Gutteridge S. J. Biol. Chem. 1993; 268: 25876-25886Abstract Full Text PDF PubMed Google Scholar, 18Lundqvist T. Schneider G. J. Biol. Chem. 1989; 264: 7078-7083Abstract Full Text PDF PubMed Google Scholar). 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. 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 (31Curmi P.M.G. Cascio D. Sweet R.M. Eisenberg D. Schreuder H. J. Biol. Chem. 1992; 267: 16980-16989Abstract Full Text PDF PubMed Google Scholar). 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 ε-amino group of Lys-334 makes ion pairs to the carboxyl group of 2-CABP and the side chain of Glu-60 (Fig.3 A) (14Shibata N. Inoue T. Fukuhara K. Nagara Y. Kitagawa R. Harada S. Kasai N. Uemura K. Kato K. Yokota A. Kai Y. J. Biol. Chem. 1996; 271: 26449-26452Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 15Andersson I. J. Mol. Biol. 1996; 259: 160-174Crossref PubMed Scopus (146) Google Scholar, 16Schreuder H.A. Knight S. Curmi P.M.G. Andersson I. Cascio D. Sweet R.M. Brändén C.-I. Eisenberg D. Protein Sci. 1993; 2: 1136-1146Crossref PubMed Scopus (49) Google Scholar, 17Newman J. Gutteridge S. J. Biol. Chem. 1993; 268: 25876-25886Abstract Full Text PDF PubMed Google Scholar). 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. GaldieriaRubisco 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 orSynechococcus 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 (14Shibata N. Inoue T. Fukuhara K. Nagara Y. Kitagawa R. Harada S. Kasai N. Uemura K. Kato K. Yokota A. Kai Y. J. Biol. Chem. 1996; 271: 26449-26452Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 15Andersson I. J. Mol. Biol. 1996; 259: 160-174Crossref PubMed Scopus (146) Google Scholar, 16Schreuder H.A. Knight S. Curmi P.M.G. Andersson I. Cascio D. Sweet R.M. Brändén C.-I. Eisenberg D. Protein Sci. 1993; 2: 1136-1146Crossref PubMed Scopus (49) Google Scholar, 17Newman J. Gutteridge S. J. Biol. Chem. 1993; 268: 25876-25886Abstract Full Text PDF PubMed Google Scholar). Residue 472 of β-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. 3 A). 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. Along the non-crystallographic 4-fold axis, there is a narrow solvent channel passing through the center of the Rubisco holoenzyme (Fig.1 A). The extra amino acid residues on the C-terminal end of the small subunit in Galdieria Rubisco make up hairpin-loop structure (Fig. 4 A). 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 (14Shibata N. Inoue T. Fukuhara K. Nagara Y. Kitagawa R. Harada S. Kasai N. Uemura K. Kato K. Yokota A. Kai Y. J. Biol. Chem. 1996; 271: 26449-26452Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 15Andersson I. J. Mol. Biol. 1996; 259: 160-174Crossref PubMed Scopus (146) Google Scholar). The unique C-terminal region in GaldieriaRubisco 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. 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 β-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 (ϕ, φ) of Ser-135. Arg-130 of small subunit is almost conserved in β-purple bacterial and non-green algal group (8Assali N.-E. Martin W.F. Sommerville C.C. Loiseaux-de Goër S. Plant Mol. Biol. 1991; 17: 853-863Crossref PubMed Scopus (41) Google Scholar). The side chain of Arg-130 forms two intersubunit ion pairs. Moreover, this β-hairpin region forms 32 intersubunit ion pairs in the overall complex. Compared with the active site structures of Synechococcus or spinach Rubiscos (14Shibata N. Inoue T. Fukuhara K. Nagara Y. Kitagawa R. Harada S. Kasai N. Uemura K. Kato K. Yokota A. Kai Y. J. Biol. Chem. 1996; 271: 26449-26452Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,17Newman J. Gutteridge S. J. Biol. Chem. 1993; 268: 25876-25886Abstract Full Text PDF PubMed Google Scholar), 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. 3 B). In higher plants and Synechococcus, residue 346 is valine instead of tyrosine. So the extra hydrogen bond in GaldieriaRubisco 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 theK m for RuBP compared with that of wild-type enzyme (33Harpel M.R. Larimer F.W. Hartman F.C. J. Biol. Chem. 1991; 266: 24734-24740Abstract Full Text PDF PubMed Google Scholar). Thus, His-327 appears to control the K m 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
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