Preliminary crystallographic study of a ribulose-1,5-bisphosphate carboxylase-oxygenase from Chromatium vinosum

Crystals of a ribulose-1,5-bisphosphate carboxylase-oxygenase from Chromatium vinosum were obtained with the hanging-drop vapor diffusion technique, using polyethylene glycol 4000 as precipitant. The crystal belongs to the cubic system, space group I432, with unit cell dimension a = 245.9 A. An asymmetric unit includes one-quarter (L2S2, L: large subunit, S: small subunit) of a hexadecameric molecule (L8S8, 544,000 Mr), which is located on the crystallographic point symmetry 222 or 4. The crystal diffracts to at least 3.0 A resolution.     

New crystal forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Three crystal forms of the dimeric form of the enzyme ribulose-1,5-bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. Form A crystals formed from the quaternary complex comprising enzyme-activator carbamate-Mg2+-2'-carboxyarabinitol-1,5-bisphosphate are shown here to be devoid of ligands. In contrast, crystals of the quaternary complex formed with the hexadecameric L8S8 enzyme from spinach contain both the activator carbamate and 2'-carboxyarabinitol-1,5-bisphosphate. Form B crystals of the R. rubrum enzyme are monoclinic, space group P2(1) with cell dimensions a = 65.5 A, b = 70.6 A, c = 104.1 A and beta = 92.1 degrees, with two subunits per asymmetric unit. Rotation function calculations show a non-crystallographic 2-fold axis perpendicular to the monoclinic b-axis. Form C crystals are orthorhombic (space group P2(1)2(1)2(1)) with cell dimensions a = 79.4 A, b = 100.1 A and c = 131.0 A. The monoclinic crystal form diffracts to at least 2.0 A resolution on a conventional X-ray source.     

Dissociation of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach by urea

The dissociation of D-ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach, which consists of eight large subunits (L, 53 kDa) and eight small subunits (S, 14 kDa) and thus has a quarternary structure L8S8, has been investigated using a variety of physical techniques. Gel chromatography using Sephadex G-100 indicates the quantitative dissociation of the small subunit S from the complex at 3-4 M urea (50 mM Tris/Cl pH 8.0, 0.5 mM EDTA, 1 mM dithiothreitol and 5 mM 2-mercaptoethanol). The dissociated S is monomeric. Analytical ultracentrifuge studies show that the core of large subunits, L, remaining at 3-4 M urea sediments with S20, w = 15.0 S, whereas the intact enzyme (L8S8) sediments with S20, w = 17.7S. The observed value is consistent with a quarternary structure L8. The dissociation reaction in 3-4 M urea can thus be represented by L8S8----L8 + 8S. At urea concentrations c greater than 5 M the L8 core dissociates into monomeric, unfolded large subunits. A large decrease in fluorescence emission intensity accompanies the dissociation of the small subunit S. This change is completed at 4 M urea. No changes are observed upon dissociating the L8 core. The kinetics of dissociation of the small subunit, as monitored by fluorescence spectroscopy, closely follow the kinetics of loss of carboxylase activity of the enzyme. Studies of the circular dichroism of D-ribulose-1,5-bisphosphate carboxylase in the wavelength region 200-260 nm indicate two conformational transitions. The first one ([0]220 from -8000 to -3500 deg cm2 dmol-1) is completed at 4 M urea and corresponds to the dissociation of the small subunit and coupled conformational changes. The second one ([0]220 from -3500 to -1200 deg cm2 dmol-1) is completed at 6 M urea and reflects the dissociation and unfolding of large subunits from the core. The effect of activation of the enzyme by addition of MgCl2 (10 mM) and NaHCO3 (10 mM) on these conformational transitions was investigated. The first conformational transition is then shifted to higher urea concentrations: a single transition ([0]220 from -8000 to -1200 deg cm2 dmol-1) is observed for the activated enzyme. From the urea dissociation experiments we conclude that both large (L) and small (S) subunits are important for carboxylase activity of spinach D-ribulose-1,5-bisphosphate carboxylase: the L-S subunit interactions tighten upon activation and dissociation of S leads to a coupled, proportional loss of enzyme activity.     

Single and twinned crystals of ribulose-1,5-bisphosphate carboxylase-oxygenase from Alcaligenes eutrophus

Ribulose-1,5-bisphosphate carboxylase-oxygenase (L8S8) from Alcaligenes eutrophus has been crystallized by equilibrium vapor diffusion techniques with ammonium sulfate as precipitant. Crystals thus obtained either as the ternary complex with CO2 and Mg2+ or as the quaternary complex with CO2, Mg2+, and 2-carboxyarabinitol 1,5-bisphosphate, a transition state analogue, diffract at least to 2.8-A resolution. Both are essentially isomorphous to each other, having orthorhombic space group C222(1) with cell dimensions a = 159 A, b = 159 A, and c = 200 A, and there is half a molecule in the asymmetric unit. The crystals of the ternary complex are sometimes twinned about the c axis so that the space group appears to be tetragonal. In this light, our earlier report (Bowien, B., Mayer, F., Spiess, E., P?hler, A., Englisch, U., and Saenger, W. (1982) Eur. J. Biochem. 106, 405-410) on a tetragonal space group P4(2)2(1)2 with crystals obtained from the same enzyme with Mg2+ and CO2 but without 2-carboxyarabinitol 1,5-bisphosphate might be incorrect.     

Preliminary X-ray diffraction study of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum

Crystals from the dimeric enzyme ribulose-1,5-bisphosphate carboxylase of the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. The crystals are of the quarternary complex comprising enzyme: activator CO2 (as a carbamate): Mg2+: 2- carboxyarabinitol -1,5-bisphosphate (as a transition state analog). X-ray diffraction photographs show symmetry consistent with space group P4(1)2(1)2 or the corresponding enantiomorphic space group. Cell parameters are a = b = 82 A, c = 324 A with two subunits per asymmetric unit. The crystals diffract to at least 3 A resolution.     

Modulation of the tight binding of carboxyarabinitol 1,5-bisphosphate to the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase

The large subunit (L) of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) from Synechococcus PCC 6301 was expressed in Escherichia coli, purified as the octamer L8, and analyzed for its ability to tightly bind the transition state analog, 2-carboxyarabinitol 1,5-bisphosphate (CABP). [14C]CABP remained tightly bound to L8 after challenging with [12C]CABP and gel filtration, indicating that L8 alone without the small subunit (S) could tightly bind CABP. Binding of CABP to L8 induced a shift in the gel filtration profile due to apparent aggregation of L8. Aggregation did not occur with the L8S8-CABP complex nor with L8-CABP in the presence of 150 mM MgCl2. If ionic strength was increased with either KCl or MgCl2 during or after the binding of [14C]CABP to L8, [14C]CABP in the complex exchanged with [12C]CABP and was lost from the protein. Ionic strength strongly affected the rate constant (k4) for [14C]CABP dissociation from the L8-[14C]CABP complex, but had little effect on k4 for the L8S8-CABP complex. The differences in CABP binding characteristics between the L8-CABP and L8S8-CABP complexes demonstrate that S is intimately involved in maintaining the stability of the tight binding of CABP to the active site. These are the same interactions stabilizing the intermediate, 3-keto-2-carboxyarabinitol 1,5-bisphosphate, to native rubisco during CO2 fixation.     

Crystallization of the activated ternary complex of ribulose-1,5-bisphosphate carboxylase-oxygenase isolated from Rhodospirillum rubrum and from an Escherichia coli clone

Ribulose-1,5-bisphosphate carboxylase-oxygenase was purified from the photosynthetic bacterium Rhodospirillum rubrum as well as from an Escherichia coli clone overproducing the enzyme. Although the latter enzyme contains 25 additional amino acid residues at the N terminus, both preparations yielded isomorphous tetragonal, bipyramidal crystals of the ternary complex of the enzyme with CO2 and Mg2+. Crystallization is sensitive to variation in pH and to the addition of the transition state analog, 2-carboxyarabinitol-1,5-bisphosphate. The systematic absences in the X-ray diffraction photographs suggest a tetragonal space group P4(3)2(1)2 or the enantiomorph P4(1)2(1)2 with cell dimensions a = b = 83 A, c = 290 A. There is one molecule per asymmetric unit. The resolution on still photographs is 3 A. The crystals are comparable to some of those already published but differ from others.     

Crystallization and preliminary x-ray studies of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase complexed with activator and a transition state analogue

Ribulose 1,5-bisphosphate carboxylase/oxygenase has been purified from spinach and crystallized by equilibrium vapor diffusion with polyethylene glycol 6000 as a precipitant. Crystals suitable for x-ray studies were obtained from a binary complex with a transition state analogue, 2-C-carboxy-D-arabinitol 1,5-bisphosphate, and a quaternary complex with 2-C-carboxy-D-arabinitol 1,5-bisphosphate, Mg2+, and HCO-3. Two forms of crystals were obtained in the presence of 2-C-carboxy-D-arabinitol 1,5-bisphosphate. Form B crystals are plates which have orthorhombic space group P2(1)2(1)2 with unit cell dimensions a = 184 A, b = 218 A, and c = 119 A. Form C crystals are tetragonal needles with space group I422 and with cell dimensions a = b = 275 A and c = 178 A. In both forms, the asymmetric unit contains half a molecule.     

Mutations in the small subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase increase the formation of the misfire product xylulose-1,5-bisphosphate.

The small subunit (S) increases the catalytic efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) by stabilizing the active sites generated by four large subunit (L) dimers. This stabilization appears to be due to an influence of S on the reaction intermediate 2,3-enediol, which is formed after the abstraction of a proton from the substrate ribulose-1,5-bisphosphate. We tested the functional significance of residues that are conserved among most species in the carboxy-terminal part of S and analyzed their influence on the kinetic parameters of Synechococcus holoenzymes. The replacements in S (F92S, Q99G, and P108L) resulted in catalytic activities ranging from 95 to 43% of wild type. The specificity factors for the three mutant enzymes were little affected (90-96% of wild type), but Km(CO2) values increased 0.5- to 2-fold. Mutant enzymes with replacements Q99G and P108L showed increased mis-protonation, relative to carboxylation, of the 2,3-enediol intermediate, forming 2 to 3 times more xylulose-1,5-bisphosphate per ribulose-1,5-bisphosphate utilized than wild-type or F92S enzymes. The results suggest that specific alterations of the L/S interfaces and of the hydrophobic core of S are transmitted to the active site by long-range interactions. S interactions with L may restrict the flexibility of active-site residues in L.     

Purification and characterization of large and small subunits of ribulose 1,5-bisphosphate carboxylase expressed separately in Escherichia coli

Procedures were developed for 95 and 80% purification to homogeneity of the large subunit (L) and small subunit (S) of ribulose 1,5-bisphosphate carboxylase/oxygenase (L8S8) from Synechococcus PCC 6301, each expressed separately in Escherichia coli. Purified L had a low specific activity in the absence of S (0.075 mumol CO2 fixed/mg holoenzyme/min). Following elution on a Pharmacia Superose 6 or 12 gel filtration column, 50% of the purified L appeared as the octamer, L8. The rest was in equilibrium with lower polymeric species and/or was retained on the column. Large and small subunits assembled rapidly into the L8S8 holoenzyme that had high specific activities, 6.2 and 3.1 mumol CO2 fixed/mg holoenzyme/min for the homologous Synechococcus L8S8 and the hybrid Synechococcus L-pea S L8S8, respectively. The CO2 dependence for carbamylation of L8 was compared to that of L8S8 as a function of pH and CO2 concentration. The pH dependence indicated an apparent pKa for L8 of 8.28 and for L8S8 of 8.15, suggesting that S may influence the pKa of the lysine involved in carbamylation. The Kact for CO2 at pH 8.4 were similar for L8 (13.5 microM) and L8S8 (15.5 microM). L8 bound 2-[14C]carboxy-D-arabinitol 1,5-bisphosphate (CABP) tightly so that most of the bound [14C]CABP survived gel filtration. A major amount of the L8-[14C]CABP complex appeared as larger polymeric aggregates when eluted in the presence of E. coli protein.     

Isolation and characterization of two 3-phosphatases that hydrolyze both phosphatidylinositol 3-phosphate and inositol 1,3-bisphosphate.

Inositol-polyphosphate 3-phosphatase catalyzes the hydrolysis of the 3-position phosphate bond of inositol 1,3-bisphosphate (Ins(1,3)P2) to form inositol 1-monophosphate and inorganic phosphate (Bansal, V.S., Inhorn, R.C., and Majerus, P.W. (1987) J. Biol. Chem. 262, 9444-9447). Phosphatidylinositol 3-phosphatase catalyzes the analogous reaction utilizing phosphatidylinositol 3-phosphate (PtdIns(3)P) as substrate to form phosphatidylinositol and inorganic phosphate (Lips, D.L., and Majerus, P.W. (1989) J. Biol. Chem. 264, 19911-19915). We now demonstrate that these enzyme activities are identical. Two forms of the enzyme, designated Type I and II 3-phosphatases, were isolated from rat brain. The Type I 3-phosphatase consisted of a protein doublet that migrated at a relative Mr of 65,000 upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The Mr of this isoform upon size-exclusion chromatography was 110,000, suggesting that the native enzyme is a dimer. The Type II enzyme consisted of equal amounts of an Mr = 65,000 doublet and an Mr = 78,000 band upon SDS-polyacrylamide gel electrophoresis. This isoform displayed an Mr upon size-exclusion chromatography of 147,000, indicating that it is a heterodimer. The Type II 3-phosphatase catalyzed the hydrolysis of Ins(1,3)P2 with a catalytic efficiency of one-nineteenth of that measured for the Type I enzyme, whereas PtdIns(3)P was hydrolyzed by the Type II 3-phosphatase at three times the rate measured for the Type I 3-phosphatase. The Mr = 65,000 subunits of the two forms of 3-phosphatase appear to be the same based on co-migration on SDS-polyacrylamide gels and peptide maps generated with Staphylococcus aureus protease V8 and trypsin. The peptide map of the Mr = 78,000 subunit was different from that of the Mr = 65,000 subunits. Thus, we propose that the differing relative specificities of the Type I and II 3-phosphatases for Ins(1,3)P2 and PtdIns(3)P are due to the presence of the Mr = 78,000 subunit of the Type II enzyme.     

Proteins of the Bacillus stearothermophilus ribosome. Crystallization of proteins L30 and S5

Proteins L30 and S5 from the 50 S and 30 S subunits, respectively, of the Bacillus stearothermophilus ribosome have been crystallized. L30 crystals are tetragonal and the space group is P4(1)2(1)2 (or P4(3)2(1)2) with cell dimensions a = b = 46.3 A and c = 61.4 A. S5 crystals are trigonal with the space group P3(1)21 (or P3(2)21) and cell dimensions a = b = 59.3 A and c = 109.8 A. In both cases, there appears to be a single molecule in the asymmetric unit.     

A point mutation in the N-terminus of ribulose-1,5-bisphosphate carboxylase affects ribulose-1,5-bisphosphate binding

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans was used to generate novel enzymes. Two conserved residues, threonine 4 and lysine 11 in the N-terminus were changed. The substitution of threonine 4 with serine or valine had little effect on the kinetic parameters. The substitution of lysine 11 with leucine, which is non-polar, increased the K _m for ribulose-1,5-bisphosphate from 82 to 190 M but its replacement with glutamine, which has polar properties, had no appreciable effect.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - LSU large sub-unit of Rubisco - SSU small subunit of Rubisco We thank Dr. S. Gutteridge (DuPont, Wilmington, USA) for structural information and for his comments on the results described. The technical assistance of Mr. A. Cowland and Mr. I. Major was invaluable.     

Crystallization and preliminary X-ray studies of glutathione synthetase from Escherichia coli B

The glutathione synthetase from Escherichia coli B has been crystallized from 27% saturated ammonium sulfate solution (pH 5.5). The crystals are hexagonal, space group P6(2)22 or P6(4)22. The cell dimensions are a = b = 88.0 A, c = 164.2 A, and gamma = 120 degrees. The enzyme is a tetramer (Mr = 143,000) with 222 symmetry, and the asymmetric unit contains one subunit molecule (Mr = 35,600). The crystals diffract to at least 2.5 A resolution.     

Incomplete factorial search for conditions leading to high quality crystals of Escherichia coli cytidine deaminase complexed to a transition state analog inhibitor

We have used an incomplete factorial design (Carter, C. W., and Carter, C. W., Jr. (1979) J. Biol. Chem. 254, 12219-12223) to find conditions for growing high quality crystals of Escherichia coli cytidine deaminase (EC 3.5.4.5). Crystals grow at pH 6.0 in hanging or sitting drops with either 1.6 M ammonium sulfate or 2.4-2.5 M sodium phosphate as precipitant. Both conditions produce crystals with identical morphologies and unit cell constants. The space group is P3(1)21 (or its enantiomorph P3(2)21), and the unit cell constants are a = b = 120.3 A, c = 78.4 A. The asymmetric unit is most reasonably one dimer of 66,000 Mr. The crystal size is very dependent on the supersaturation ratio, S = [initial protein concentration]/[equilibrium protein concentration], exhibiting a maximum at S = 7.7. The largest crystals diffract to at least 2.5 A and have a lifetime of 4 to 5 days in the x-ray beam at room temperature. The enzyme in these crystals is complexed with the transition state analog inhibitor 1-(beta-D-ribofuranosyl)-5-fluoropyrimidin-2-one (5-fluoropyrimidin-2-one riboside). We have collected data from parent crystals and from a heavy atom derivative in which the transition state analog is replaced by the active site directed inhibitor 5-(chloromercuri)cytidine.     

Crystal structure of the unactivated form of ribulose-1,5-bisphosphate carboxylase/oxygenase from tobacco refined at 2.0-A resolution.

The structure of the unactivated form of ribulose-1,5-bisphosphate carboxylase/oxygenase was refined at a resolution of 2.0 A to an R-factor of 17.1%. The previous model (Chapman et al., 1988) was extensively rebuilt, and the small subunit was retraced. The refined model consists of residues 22-63 and 69-467 of the large subunit and the complete small subunit. A striking feature of the model is that several loops have very high B-factors, probably representing mobile regions of the molecule. An examination of the intersubunit contacts shows that the L8S8 hexadecamer is composed of four L2 dimers. The dominant contacts between these L2 dimers are formed by the small subunits. This suggests that the small subunits may be essential for maintaining the integrity of the L8S8 structure. The active site shows differences between the unactivated form and the quaternary complex. In particular, Lys334 has moved out of the active site by about 10A. This residue lies on loop 6 of the alpha beta barrel, which is a particularly mobile loop. The site of ribulose-1,5-bisphosphate carboxylase/oxygenase activation is well ordered in the absence of the carbamylation of Lys201 and Mg2+ binding. The residues are held poised by a network of hydrogen bonds. In the unactivated state, the active site is accessible to substrate binding.     

Phosphorylation in vitro of the large subunit of the ribulose-1,5-bisphosphate carboxylase and of the glyceraldehyde-3-phosphate dehydrogenase

A protein kinase activity responsible for the in vitro phosphorylation of at least six endogenous polypeptides including the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is present in the stroma (3000 X g supernatant, S30) of spinach chloroplasts. The phosphorylation of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit is strongly enhanced when sodium fluorure is used as a protein phosphatase inhibitor. Phosphorylation occurs on threonine and serine residues. The protein kinase involved is not Ca2+-dependent. There is also evidence for a protein phosphatase activity which suggests a coupled regulation by a phosphorylation-dephosphorylation process. The phosphorylating activity is drastically reduced when S30 is prepared from leaves harvested after a dark period. Phosphorylation of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit is not related to its own synthesis. The in vitro phosphorylation of the glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) is also demonstrated.     

Preliminary X-ray data for an N-terminal fragment of rabbit serum transferrin

An iron-containing fragment (Mr approximately 39,000) of rabbit serum transferrin has been crystallized from a solution of 25% (w/v) polyethylene glycol 6000, 50 mM-disodium piperazine-N,N'bis(2-ethanesulphonate) adjusted to pH 6.0 at 4 degrees C. The space group is P3(1)21 (or the enantiomorph) with a = b = 66.8(1) A, c = 137.5(3) A and Z = 6. The crystals appear as hexagonal plates, with the unique axis perpendicular to the plate. The crystals, kept at 4 degrees C, are stable in the X-ray beam for at least 130 hours and diffract to better than 1.8 A resolution.     

A binding study of the interaction of beta-D-fructose 2,6-bisphosphate with phosphofructokinase and fructose-1,6-bisphosphatase

The binding of beta-D-fructose 2,6-bisphosphate to rabbit muscle phosphofructokinase and rabbit liver fructose-1,6-bisphosphatase was studied using the column centrifugation procedure (Penefsky, H. S., (1977) J. Biol. Chem. 252, 2891-2899). Phosphofructokinase binds 1 mol of fructose 2,6-bisphosphate/mol of protomer (Mr = 80,000). The Scatchard plots of the binding of fructose 2,6-bisphosphate to phosphofructokinase are nonlinear in the presence of three different buffer systems and appear to exhibit negative cooperativity. Fructose 1,6-bisphosphate and glucose 1,6-bisphosphate inhibit the binding of fructose-2,6-P2 with Ki values of 15 and 280 microM, respectively. Sedoheptulose 1,7-bisphosphate, ATP, and high concentrations of phosphate also inhibit the binding. Other metabolites including fructose-6-P, AMP, and citrate show little effect. Fructose-1,6-bisphosphatase binds 1 mol of fructose 2,6-bisphosphate/mol of subunit (Mr = 35,000) with an affinity constant of 1.5 X 10(6) M-1. Fructose 1,6-bisphosphate, fructose-6-P, and phosphate are competitive inhibitors with Ki values of 4, 2.7, and 230 microM, respectively. Sedoheptulose 1,7-bisphosphate (1 mM) inhibits approximately 50% of the binding of fructose 1,6-bisphosphate to fructose bisphosphatase, but AMP has no effect. Mn2+, Co2+, and a high concentration of Mg2+ inhibit the binding. Thus, we may conclude that fructose 2,6-bisphosphate binds to phosphofructokinase at the same allosteric site for fructose 1,6-bisphosphate while it binds to the catalytic site of fructose-1,6-bisphosphatase.     

Artificially evolved Synechococcus PCC6301 Rubisco variants exhibit improvements in folding and catalytic efficiency

The photosynthetic CO2-fixing enzyme, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), is responsible for most of the world's biomass, but is a slow non-specific catalyst. We seek to identify and overcome the chemical and biological constraints that limit the evolutionary potential of Rubisco in Nature. Recently, the horizontal transfer of Calvin cycle genes (rbcL, rbcS and prkA) from cyanobacteria (Synechococcus PCC6301) to gamma-proteobacteria (Escherichia coli) was emulated in the laboratory. Three unique Rubisco variants containing single (M259T) and double (M259T/A8S, M259T/F342S) amino acid substitutions in the L (large) subunit were identified after three rounds of random mutagenesis and selection in E. coli. Here we show that the M259T mutation did not increase steady-state levels of rbcL mRNA or L protein. It instead improved the yield of properly folded L subunit in E. coli 4-9-fold by decreasing its natural propensity to misfold in vivo and/or by enhancing its interaction with the GroES-GroEL chaperonins. The addition of osmolites to the growth media enhanced productive folding of the M259T L subunit relative to the wild-type L subunit, while overexpression of the trigger factor and DnaK/DnaJ/GrpE chaperones impeded Rubisco assembly. The evolved enzymes showed improvement in their kinetic properties with the M259T variant showing a 12% increase in carboxylation turnover rate (k(c)cat), a 15% improvement in its K(M) for CO2 and no change in its K(M) for ribulose-1,5-bisphosphate or its CO2/O2 selectivity. The results of the present study show that the directed evolution of the Synechococcus Rubisco in E. coli can elicit improvements in folding and catalytic efficiency.