Substitutions at the Asp-473 Latch Residue of Chlamydomonas Ribulosebisphosphate Carboxylase/Oxygenase Cause Decreases in Carboxylation Efficiency and CO2/O2 Specificity

Abstract

The loop between α-helix 6 and β-strand 6 in the α/β-barrel active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) plays a key role in discriminating between gaseous substrates CO2 and O2. Based on numerous x-ray crystal structures, loop 6 is either closed or open depending on the presence or absence, respectively, of substrate ligands. The carboxyl terminus folds over loop 6 in the closed conformation, prompting speculation that it may trigger or latch loop 6 closure. Because an x-ray crystal structure of tobacco Rubisco revealed that phosphate is located at a site in the open form that is occupied by the carboxyl group of Asp-473 in the closed form, it was proposed that Asp-473 may serve as the latch that holds the carboxyl terminus over loop 6. To assess the essentiality of Asp-473 in catalysis, we used directed mutagenesis and chloroplast transformation of the green alga Chlamydomonas reinhardtii to create D473A and D473E mutant enzymes. The D473A and D473E mutant strains can grow photoautotrophically, indicating that Asp-473 is not essential for catalysis. However, both substitutions caused 87% decreases in carboxylation catalytic efficiency (Vmax/Km) and ∼16% decreases in CO2/O2 specificity. If the carboxyl terminus is required for stabilizing loop 6 in the closed conformation, there must be additional residues at the carboxyl terminus/loop 6 interface that contribute to this mechanism. Considering that substitutions at residue 473 can influence CO2/O2 specificity, further study of interactions between loop 6 and the carboxyl terminus may provide clues for engineering an improved Rubisco. The loop between α-helix 6 and β-strand 6 in the α/β-barrel active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) plays a key role in discriminating between gaseous substrates CO2 and O2. Based on numerous x-ray crystal structures, loop 6 is either closed or open depending on the presence or absence, respectively, of substrate ligands. The carboxyl terminus folds over loop 6 in the closed conformation, prompting speculation that it may trigger or latch loop 6 closure. Because an x-ray crystal structure of tobacco Rubisco revealed that phosphate is located at a site in the open form that is occupied by the carboxyl group of Asp-473 in the closed form, it was proposed that Asp-473 may serve as the latch that holds the carboxyl terminus over loop 6. To assess the essentiality of Asp-473 in catalysis, we used directed mutagenesis and chloroplast transformation of the green alga Chlamydomonas reinhardtii to create D473A and D473E mutant enzymes. The D473A and D473E mutant strains can grow photoautotrophically, indicating that Asp-473 is not essential for catalysis. However, both substitutions caused 87% decreases in carboxylation catalytic efficiency (Vmax/Km) and ∼16% decreases in CO2/O2 specificity. If the carboxyl terminus is required for stabilizing loop 6 in the closed conformation, there must be additional residues at the carboxyl terminus/loop 6 interface that contribute to this mechanism. Considering that substitutions at residue 473 can influence CO2/O2 specificity, further study of interactions between loop 6 and the carboxyl terminus may provide clues for engineering an improved Rubisco. Like many α/β-barrel-domain enzymes (1Wierenga R.K. FEBS Lett. 2001; 492: 193-198Google Scholar), the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, 1The abbreviations used are: Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; Bicine, N,N-bis(2-hydroxyethyl)glycine; CABP, 2-carboxy-d-arabinitol 1,5-bisphosphate; RuBP, ribulose 1,5-bisphosphate; Ω, CO2/O2 specificity factor. 1The abbreviations used are: Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; Bicine, N,N-bis(2-hydroxyethyl)glycine; CABP, 2-carboxy-d-arabinitol 1,5-bisphosphate; RuBP, ribulose 1,5-bisphosphate; Ω, CO2/O2 specificity factor. EC 4.1.1.30) has a loop (i.e. between β-strand 6 and α-helix 6) that folds over substrate during catalysis (reviewed in Refs. 2Spreitzer R.J. Salvucci M.E. Annu. Rev. Plant Biol. 2002; 53: 449-475Google Scholar, 3Parry M.A.J. Andralojc P.J. Mitchell R.A. Madgwick P.J. Keys A.J. J. Exp. Bot. 2003; 54: 1321-1333Google Scholar, 4Andersson I. Taylor T.C. Arch. Biochem. Biophys. 2003; 414: 130-140Google Scholar). Numerous studies have indicated that loop 6 plays a major role in discriminating between CO2 and O2 in the competing RuBP carboxylation and oxygenation reactions of Rubisco (reviewed in Refs. 2Spreitzer R.J. Salvucci M.E. Annu. Rev. Plant Biol. 2002; 53: 449-475Google Scholar and 3Parry M.A.J. Andralojc P.J. Mitchell R.A. Madgwick P.J. Keys A.J. J. Exp. Bot. 2003; 54: 1321-1333Google Scholar), and Lys-334 at the apex of loop 6 interacts with the C-2 carboxyl group of the transition state analog CABP in various Rubisco x-ray crystal structures (5Schreuder H.A. Knight S. Curmi P.M.G. Andersson I. Cascio D. Sweet R.M. Branden C.I. Eisenberg D. Protein Sci. 1993; 2: 1136-1146Google Scholar, 6Newman J. Gutteridge S. J. Biol. Chem. 1993; 268: 25876-25886Google Scholar, 7Andersson I. J. Mol. Biol. 1996; 259: 160-174Google Scholar, 8Sugawara H. Yamamoto H. Shibata N. Inoue T. Okada S. Miyake C. Yokota A. Kai Y. J. Biol. Chem. 1999; 274: 15655-15661Google Scholar, 9Taylor T.C. Backlund A. Bjorhall K. Spreitzer R.J. Andersson I. J. Biol. Chem. 2001; 276: 48159-48164Google Scholar). However, Rubisco may be unique among α/β-barrel enzymes in that the carboxyl terminus folds over loop 6 and appears to stabilize its closed conformation. This arrangement of loop 6 and the carboxyl terminus is also observed in the crystal structure of unactivated Rubisco that contains RuBP in the active site (10Taylor T.C. Andersson I. J. Mol. Biol. 1997; 265: 432-444Google Scholar). In vivo, Rubisco activase is responsible for facilitating the opening of the closed structure to release this RuBP (reviewed in Refs. 2Spreitzer R.J. Salvucci M.E. Annu. Rev. Plant Biol. 2002; 53: 449-475Google Scholar and 11Portis A.R. Photosynth. Res. 2003; 75: 11-27Google Scholar), thereby allowing spontaneous carbamylation of an active site Lys-201 and introduction of Mg2+ to produce the active form of the enzyme (reviewed in Ref. 12Andrews T.J. Nat. Struct. Biol. 1996; 3: 3-7Google Scholar). The change from closed to open conformation in the carboxyl-terminal, α/β-barrel domain of the large subunit is accompanied by movement of the amino-terminal domain of a neighboring large subunit (reviewed in Ref. 4Andersson I. Taylor T.C. Arch. Biochem. Biophys. 2003; 414: 130-140Google Scholar), which also contributes residues to the active site and may contain the primary site of interaction with Rubisco activase (13Larson E.M. O'Brien C.M. Zhu G. Spreitzer R.J. Portis A.R. J. Biol. Chem. 1997; 272: 17033-17037Google Scholar, 14Ott C.M. Smith B.D. Portis A.R. Spreitzer R.J. J. Biol. Chem. 2000; 275: 26241-26244Google Scholar). In the absence of RuBP or CABP, the carboxyl terminus is disordered, and loop 6 is usually disordered or misfolded into an open conformation depending on the source or treatment of the Rubisco used for crystallization (15Curmi P.M. Cascio D. Sweet R.M. Eisenberg D. Schreuder H. J. Biol. Chem. 1992; 267: 16980-16989Google Scholar, 16Taylor T.C. Andersson I. Nat. Struct. Biol. 1996; 3: 95-101Google Scholar, 17Hansen S. Vollan V.B. Hough E. Andersen K. J. Mol. Biol. 1999; 288: 609-621Google Scholar, 18Duff A.P. Andrews T.J. Curmi P.M.G. J. Mol. Biol. 2000; 298: 903-916Google Scholar, 19Okano Y. Mizohata E. Xie Y. Matsumura H. Sugawara H. Inoue T. Yokota A. Kai Y. FEBS Lett. 2002; 527: 33-36Google Scholar). An open crystal structure of tobacco Rubisco was solved in which phosphate was observed to reside at a site normally occupied by the carboxyl group of Asp-473 in the closed structure (18Duff A.P. Andrews T.J. Curmi P.M.G. J. Mol. Biol. 2000; 298: 903-916Google Scholar). This prompted speculation that Asp-473 may serve as a latch that holds the carboxyl terminus over loop 6 in the closed conformation (18Duff A.P. Andrews T.J. Curmi P.M.G. J. Mol. Biol. 2000; 298: 903-916Google Scholar). In plant-like Rubisco enzymes, comprised of eight ∼55-kDa large and eight ∼16-kDa small subunits, Asp-473 is 100% conserved, but the large subunit carboxyl terminus is quite variable in length and sequence identity (Fig. 1). Some bacterial Rubisco enzymes exist as only large subunit dimers (reviewed in Ref. 20Tabita F.R. Photosynth. Res. 1999; 60: 1-28Google Scholar), and without electron density for the carboxyl terminus (21Andersson I. Knight S. Schneider G. Lindqvist Y. Lundqvist T. Branden C.I. Lorimer G.H. Nature. 1989; 337: 229-234Google Scholar), it is more difficult to assign a residue comparable with Asp-473 in their divergent sequences (i.e. Rhodospirillum rubrum, Fig. 1). In previous studies, the role of the large subunit carboxyl terminus in Rubisco function was investigated by using biochemical or genetic methods that deleted or added residues (22Spreitzer R.J. Goldschmidt-Clermont M. Rahire M. Rochaix J.D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5460-5464Google Scholar, 23Portis A.R. Arch. Biochem. Biophys. 1990; 283: 397-400Google Scholar, 24Ranty B. Lundqvist T. Schneider G. Madden M. Howard R. Lorimer G. EMBO J. 1990; 9: 1365-1373Google Scholar, 25Gutteridge S. Rhoades D.F. Herrmann C. J. Biol. Chem. 1993; 268: 7818-7824Google Scholar, 26Zhu G. Jensen R.G. Bohnert H.J. Wildner G.F. Schlitter J. Photosynth. Res. 1998; 57: 71-79Google Scholar) or by using directed mutagenesis to replace cyanobacterial carboxyl-terminal residues with those characteristic of land plants (25Gutteridge S. Rhoades D.F. Herrmann C. J. Biol. Chem. 1993; 268: 7818-7824Google Scholar). Although some alterations were observed in carboxylation catalytic efficiency or holoenzyme stability (22Spreitzer R.J. Goldschmidt-Clermont M. Rahire M. Rochaix J.D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5460-5464Google Scholar, 23Portis A.R. Arch. Biochem. Biophys. 1990; 283: 397-400Google Scholar, 24Ranty B. Lundqvist T. Schneider G. Madden M. Howard R. Lorimer G. EMBO J. 1990; 9: 1365-1373Google Scholar, 25Gutteridge S. Rhoades D.F. Herrmann C. J. Biol. Chem. 1993; 268: 7818-7824Google Scholar, 26Zhu G. Jensen R.G. Bohnert H.J. Wildner G.F. Schlitter J. Photosynth. Res. 1998; 57: 71-79Google Scholar), only the deletion of as many as 10 residues from the carboxyl terminus of the Synechococcus enzyme caused a substantial reduction in CO2/O2 specificity (Fig. 1, residues 466-475) (25Gutteridge S. Rhoades D.F. Herrmann C. J. Biol. Chem. 1993; 268: 7818-7824Google Scholar). If the carboxyl terminus plays a role in stabilizing loop 6 during catalysis, and loop 6 determines the differential stabilization of the carboxylation and oxygenation transition states (27Chen Z. Spreitzer R.J. Planta. 1991; 183: 597-603Google Scholar, 28Chen Z. Spreitzer R.J. Photosynth. Res. 1992; 31: 157-164Google Scholar), one might expect that specific carboxyl-terminal residues may influence CO2/O2 specificity. However, the function of individual residues was not assessed by directed-mutagenesis substitutions in previous studies. The green alga Chlamydomonas reinhardtii serves as a useful model organism for the study of Rubisco because photosynthesis-deficient mutants can be maintained with acetate as an alternative source of carbon and energy. Mutant strains that lack functional genes for the large (chloroplast rbcL) or small (nucleus rbcS) subunit can be complemented by transformation (reviewed in Ref. 29Spreitzer R.J. Rochaix J.D. Goldschmidt-Clermont M. Merchant S. The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Dordrecht, Netherlands1998: 515-527Google Scholar), and the wild-type rbcL gene can be replaced with a mutant rbcL gene regardless of whether the mutant large subunit can produce a functional holoenzyme (e.g. Ref. 30Zhu G. Spreitzer R.J. J. Biol. Chem. 1994; 269: 3952-3956Google Scholar). Furthermore, because mutant genes exist in vivo, genetic selection can be used to recover second-site suppressor mutations as a means for identifying complementing structural interactions (29Spreitzer R.J. Rochaix J.D. Goldschmidt-Clermont M. Merchant S. The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Dordrecht, Netherlands1998: 515-527Google Scholar, 31Du Y.C. Hong S. Spreitzer R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14206-14211Google Scholar). With this in mind, and considering the evolutionary conservation and potential critical function of Asp-473, we decided to examine the essentiality of Asp-473 by directed mutagenesis and Chlamydomonas chloroplast transformation. D473A and D473E substitutions did not prevent the photosynthetic growth of the Chlamydomonas transformants, indicating that Asp-473 is not essential for Rubisco function. However, both substitutions caused substantial decreases in catalytic efficiency and CO2/O2 specificity. Strains and Culture Conditions—C. reinhardtii 2137 mt+ is the wild-type strain (32Spreitzer R.J. Mets L. Plant Physiol. 1981; 67: 565-569Google Scholar). Mutant MX3312, which lacks the rbcL gene that encodes the Rubisco large subunit, was obtained from Dr. Genhai Zhu (Maxygen, Inc., Redwood City, CA) and used as the host for chloroplast transformation. This mutant was created via chloroplast transformation of wild-type 2137 mt+ with the 786-bp coding region of the bacterial aadA gene (33Hollingshead S. Vapnek D. Plasmid. 1985; 13: 17-30Google Scholar) flanked precisely by the Chlamydomonas rbcL 5′- and 3′-untranslated regions (34Dron M. Rahire M. Rochaix J.D. J. Mol. Biol. 1982; 162: 775-793Google Scholar). Because chloroplast transformation occurs by homologous recombination in Chlamydomonas, mutant MX3312 has a photosynthesis-deficient, acetate-requiring phenotype due to the complete loss of the rbcL coding sequence, but it is resistant to aminoglycosides such as spectinomycin and streptomycin due to the expression of aadA (35Goldschmidt-Clermont M. Nucleic Acids Res. 1991; 19: 4083-4089Google Scholar). All strains are maintained at 25 °C in darkness with 10 mm acetate medium containing 1.5% Bacto-agar (32Spreitzer R.J. Mets L. Plant Physiol. 1981; 67: 565-569Google Scholar). For biochemical analysis, cells were with of acetate medium at 25 °C on a in and a containing the Chlamydomonas rbcL gene Y.C. Spreitzer R.J. J. Biol. Chem. 2000; 275: Scholar), directed mutagenesis was using and a mutagenesis from C. J. 1996; 9: Scholar). To create the D473A the sequence was to which also a site useful for The D473E was created by to In this the mutant also a to at the for that an site useful for The mutant and were into the chloroplast of rbcL mutant MX3312 by using a Plant 1992; Scholar), and were on medium in the by methods Y.C. Spreitzer R.J. J. Biol. Chem. 2000; 275: Scholar, G. Spreitzer R.J. J. Biol. Chem. 1996; Scholar). of and enzyme were to the of the D473A and D473E mutant genes R.J. Scholar). The mutant rbcL genes were and to that only the mutations were G. Spreitzer R.J. J. Biol. Chem. 1994; 269: 3952-3956Google Scholar, Y.C. Spreitzer R.J. J. Biol. Chem. 2000; 275: Scholar, G. Spreitzer R.J. J. Biol. Chem. 1996; Scholar). The mutant Chlamydomonas strains were D473A and and was from cells as R.J. Scholar) and Biochem. Scholar). were to with a in the Scholar). were either with or to with Rubisco and by G. Zhu G. Spreitzer R.J. 1994; Scholar). holoenzyme was from by in mm 10 mm 10 mm and mm R.J. Scholar). The stability of Rubisco was as Y.C. Spreitzer R.J. J. Biol. Chem. 2000; 275: Scholar, Z. Hong S. Spreitzer R.J. Plant Physiol. 1993; Scholar). RuBP and were via the of from Z. R. Spreitzer R.J. Proc. Natl. Acad. Sci. U. S. A. Scholar). The of carboxylation to oxygenation at of CO2 and O2 is by the CO2/O2 specificity and are the for carboxylation and and and are the for CO2 and O2 Plant Physiol. 54: Scholar). of and Rubisco was by and in and mm in reactions at 25 °C Plant Physiol. 1981; 67: Scholar, R.J. FEBS Lett. 1982; Scholar). and were by methods Plant Physiol. 1981; 67: Scholar, T.C. Biochem. J. Scholar). was by of Rubisco to containing mm mm 10 mm and or RuBP at 25 °C (27Chen Z. Spreitzer R.J. Planta. 1991; 183: 597-603Google Scholar). To the for the transition state analog CABP (27Chen Z. Spreitzer R.J. Planta. 1991; 183: 597-603Google Scholar, 30Zhu G. Spreitzer R.J. J. Biol. Chem. 1994; 269: 3952-3956Google Scholar, G. Spreitzer R.J. J. Biol. Chem. 1996; Scholar), Rubisco was at 25 °C for in mm 10 mm 10 mm mm and The was on a that at 25 °C with mm 10 mm 10 mm and mm The was with the and were and for and G. Spreitzer R.J. J. Biol. Chem. 1996; Scholar). The was with a of at 25 °C for the was CABP and were by methods J. R. 19: Scholar, G. Jensen R.G. Plant Physiol. 1990; Scholar). and Mutant the functional of Asp-473, directed mutagenesis was used to its group by with or to its without by with rbcL mutant and were into the chloroplast of the rbcL deletion mutant MX3312, were on medium in the at to that of transformation with the wild-type rbcL gene Y.C. Spreitzer R.J. J. Biol. Chem. 2000; 275: Scholar, G. Spreitzer R.J. J. Biol. Chem. 1996; Scholar). Furthermore, of a of via (32Spreitzer R.J. Mets L. Plant Physiol. 1981; 67: 565-569Google Scholar) at 25 or °C on medium or acetate medium the or in the revealed in growth to that of has useful for identifying rbcL mutants with various in Rubisco catalysis, or stability (reviewed in Ref. 29Spreitzer R.J. Rochaix J.D. Goldschmidt-Clermont M. Merchant S. The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Dordrecht, Netherlands1998: 515-527Google Scholar). the critical role proposed for Asp-473 in Rubisco catalysis, with or caused major alterations in holoenzyme function or stability in and in of mutants D473A and D473E were on in the of Rubisco holoenzyme were observed to that of not and also that the mutants have wild-type of Rubisco (Fig. To further that the D473A and D473E mutants not the structural stability of Rubisco, were Y.C. Spreitzer R.J. J. Biol. Chem. 2000; 275: Scholar, Z. Hong S. Spreitzer R.J. Plant Physiol. 1993; Scholar). in Fig. the D473A and D473E mutant enzymes are not from wild-type Rubisco with to stability in the Asp-473 group or its has on Rubisco holoenzyme stability in or in of Rubisco from and large subunit mutants D473A and D473E Rubisco in of mm 10 mm 10 mm and mm was at for The were on and for RuBP at 25 °C Y.C. Spreitzer R.J. J. Biol. Chem. 2000; 275: Scholar, Z. Hong S. Spreitzer R.J. Plant Physiol. 1993; Scholar). for enzyme were the of the °C mutant mutant D473A and D473E mutant enzymes were to have and decreases in Ω, in the decreases in and 87% decreases in carboxylation catalytic efficiency are responsible for the The D473A and D473E mutant enzymes also have decreases in the for CABP which is from the carboxylation and the of which is of a change in the of the carboxylation and oxygenation transition states (27Chen Z. Spreitzer R.J. Planta. 1991; 183: 597-603Google Scholar, 28Chen Z. Spreitzer R.J. Photosynth. Res. 1992; 31: 157-164Google Scholar). However, is for the mutant enzymes This that the of RuBP to the a to both carboxylation and is also by the mutant of Rubisco from and mutants D473A and are the means of enzyme are the means of enzyme are the means of enzyme are the means of enzyme are the means of enzyme are the means of enzyme in a of CABP to Rubisco from and mutants D473A and in a Rubisco the of but it has a carboxylation of only a and is by O2. of RuBP which is the in the that to the loss of Rubisco an for genetic engineering at (reviewed in Refs. 2Spreitzer R.J. Salvucci M.E. Annu. Rev. Plant Biol. 2002; 53: 449-475Google Scholar and 3Parry M.A.J. Andralojc P.J. Mitchell R.A. Madgwick P.J. Keys A.J. J. Exp. Bot. 2003; 54: 1321-1333Google Scholar). However, the in the and of Rubisco enzymes from divergent Nature. 1981; Scholar), Rubisco catalysis on a of active site residues (reviewed in Refs. 2Spreitzer R.J. Salvucci M.E. Annu. Rev. Plant Biol. 2002; 53: 449-475Google Scholar, 4Andersson I. Taylor T.C. Arch. Biochem. Biophys. 2003; 414: 130-140Google Scholar, 12Andrews T.J. Nat. Struct. Biol. 1996; 3: 3-7Google Scholar). to a Rubisco on of of the of the enzyme some from the active With subunits, and in x-ray crystal structures (reviewed in Ref. 4Andersson I. Taylor T.C. Arch. Biochem. Biophys. 2003; 414: 130-140Google Scholar), it is a to regions of structure from the active site that may influence catalysis. by genetic and selection in (29Spreitzer R.J. Rochaix J.D. Goldschmidt-Clermont M. Merchant S. The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Dordrecht, Netherlands1998: 515-527Google Scholar), or mutagenesis Protein 1998; Scholar, F.R. J. Mol. Biol. 2003; Scholar, R.J. Y.C. 2001; Scholar), of sequence Y.C. Spreitzer R.J. J. Biol. Chem. 2003; Scholar), of divergent x-ray crystal structures (18Duff A.P. Andrews T.J. Curmi P.M.G. J. Mol. Biol. 2000; 298: 903-916Google Scholar) may it be to regions of Based on an of Rubisco crystal structures, (18Duff A.P. Andrews T.J. Curmi P.M.G. J. Mol. Biol. 2000; 298: 903-916Google Scholar) proposed that Asp-473 in the carboxyl-terminal region of the large subunit may serve as a critical latch residue that for the of the carboxyl terminus over loop 6 during catalysis. Because the large subunit carboxyl terminus is one of the variable regions of Rubisco structure with to both length and sequence identity I. Taylor T.C. Arch. Biochem. Biophys. 2003; 414: 130-140Google Scholar, 26Zhu G. Jensen R.G. Bohnert H.J. Wildner G.F. Schlitter J. Photosynth. Res. 1998; 57: 71-79Google Scholar) (Fig. Asp-473 might serve as a critical by which the in the carboxyl terminus influence loop 6 catalysis. Although we to chloroplast transformation and genetic selection in Chlamydomonas to critical residues that for the loss of Asp-473, it was to that D473A and D473E substitutions did not the of Chlamydomonas to grow This indicated that Asp-473 is not essential for catalysis, and further that Rubisco stability in (Fig. or in (Fig. However, both mutant enzymes 87% decreases in carboxylation catalytic efficiency and ∼16% decreases in CO2/O2 specificity and In a previous study that on the expression of Synechococcus Rubisco in (25Gutteridge S. Rhoades D.F. Herrmann C. J. Biol. Chem. 1993; 268: 7818-7824Google Scholar), deletion of the carboxyl-terminal residues Asp-473, Fig. caused a in but did not change This that the decreases in from the D473A and D473E substitutions in the Chlamydomonas enzyme may be due to alterations in structure the loss of the by either or a may prevent a of the carboxyl terminus with loop thereby interactions between residues (Fig. Because the biochemical of the D473A and D473E enzymes are quite one might also that structural The that interactions between the carboxyl terminus and loop 6 may a role in Rubisco function is by the previous study with the Synechococcus enzyme (25Gutteridge S. Rhoades D.F. Herrmann C. J. Biol. Chem. 1993; 268: 7818-7824Google Scholar). of additional and (Fig. carboxylation and caused a in (25Gutteridge S. Rhoades D.F. Herrmann C. J. Biol. Chem. 1993; 268: 7818-7824Google Scholar). the which Lys-334 from (Fig. also plays an role in the interactions between the carboxyl terminus and loop 6. In the x-ray crystal structure of Chlamydomonas Rubisco, numerous interactions can be observed between the carboxyl terminus and loop 6 in the closed form of the enzyme (Fig. Because Asp-473 is in with and and its a with the group of alterations in the interactions with loop 6 residues may be to for the observed decreases in that from the D473A and D473E However, Chlamydomonas to the residue to land Rubisco or on catalysis G. Spreitzer R.J. J. Biol. Chem. 1996; Scholar). Asp-473 is also in with and and an with (Fig. All of residues are conserved, and but are in with loop 6 in Asp-473 might also be to loop 6 via alterations in such In a previous study with the Synechococcus enzyme G. P.J. Madgwick P.J. M.A.J. Biochem. J. 1998; Scholar), substitutions at also caused decreases in The group of with the of both carboxyl-terminal and loop 6 (Fig. and a in Chlamydomonas Rubisco was to a in Z. Spreitzer R.J. J. Biol. Chem. 1989; Scholar). Based on it that a of carboxyl-terminal residues might influence Rubisco CO2/O2 specificity via interactions with residues that also with loop 6. are in the of because it is a of the differential stabilization of the carboxylation and oxygenation transition states at the of both reactions (27Chen Z. Spreitzer R.J. Planta. 1991; 183: 597-603Google Scholar, 28Chen Z. Spreitzer R.J. Photosynth. Res. 1992; 31: 157-164Google Scholar). substitutions that influence may be potential for genetic engineering at Rubisco. may be from the the D473E and D473A mutant enzymes not the carboxylation transition state analog CABP as as the wild-type enzyme However, the mutant enzymes also have in and a was observed for the Synechococcus carboxyl-terminal deletion mutant that caused a in (25Gutteridge S. Rhoades D.F. Herrmann C. J. Biol. Chem. 1993; 268: 7818-7824Google Scholar). Although loop 6 is not essential for the of RuBP in the to both carboxylation and oxygenation E.M. Scholar), with the structural of substrate (18Duff A.P. Andrews T.J. Curmi P.M.G. J. Mol. Biol. 2000; 298: 903-916Google Scholar), indicating that loop 6 and the carboxyl terminus are in a closed state to the of CO2 or O2 in the of carboxylation or This may a as to the by which CO2 and O2 the active study of the interactions between the carboxyl terminus and loop 6 may the role of this structural arrangement in catalysis and may also provide useful to the of an improved Rubisco Dr. Genhai Zhu for the mutant strain and Dr. R. Portis of for a of