Alteration of the alpha helix region of cyanobacterial ribulose 1,5-bisphosphate carboxylase/oxygenase to reflect sequences found in high substrate specificity enzymes.

The sequence at the alpha helix region of the eight-stranded beta/alpha barrel domain of the large subunit of Synechococcus sp. strain PCC 6301 ribulosebisphosphate carboxylase/oxygenase (rubisco) was altered by site-directed mutagenesis. Changes were made to match the corresponding residues in the rubisco large subunit of chromophytic and rhodophytic algae, which have considerably higher substrate specificity factors (ratio of the rate constants for the carboxylase and oxygenase reactions). A set of cumulative mutations of one to eight amino acid residues was prepared and examined and it was found that mutant enzymes which contained from one to five substitutions all exhibited substantial decreases in carboxylase activity. Mutant enzymes which contained from six to eight amino acid substitutions were inactive and failed to maintain their native quarternary structure. For enzymes which maintained their native structure, consecutive changes in the alpha helix 6 region yielded a progressive increase in the K(m) for ribulosebisphosphate, confirming the importance of this region in substrate binding. Despite these results, and previous studies which indicated the importance and potential of residues in the alpha helix 6 region to influence the ability of loop 6 to affect rubisco catalysis, simple cumulative substitution did not significantly alter the substrate specificity factor of the enzyme. The results of this study lend further credence to the idea that engineered enhancement of rubisco specificity will likely require coordination of alterations at multiple sites in the primary structure.

Mg2+ and ATP or adenosine 5'-[gamma-thio]-triphosphate (ATP gamma S) enhances intrinsic fluorescence and induces aggregation which increases the activity of spinach Rubisco activase.

ATP and Mg2+ caused a transient increase in the intrisinc fluorescence of Rubisco activase which was inhibited by the presence of ADP. Only minor changes in fluorescence were observed with ATP or Mg2+ alone. The fluorescence increase was stabilized by addition of an ATP regenerating system or by substitution of ATP with a non-hydrolyzable analog, adenosine 5'-[gamma-thio]-triphosphate (ATP gamma S). The initial rate of increase in fluorescence also depended on the concentration of protein in a manner consistent with second-order kinetics. The concentration dependence for the effect of ATP gamma S was sigmoidal, although at pH 8 the half-saturation requirements for both ATP gamma S (12 microM) and Mg2+ (1.5 mM) were not too dissimilar to the binding affinities (6 microM and 2 mM, respectively) determined indirectly with the fluorescent probe, 1-anilinonapthalene-8-sulfonate. However, the concentration dependence of ATP was about 5-fold higher than its binding affinity, also sigmoidal and quite similar to the concentration responses of ATP hydrolysis and activation of Rubisco by the protein. These characteristics of the intrinsic fluorescence indicate that it monitors a conformational change in the protein occurring after binding of the nucleotide and associated with increased aggregation. Direct evidence of increased aggregation in the presence of Mg2+ and ATP or ATP gamma S was obtained by gel-filtration chromatography. However, the apparent molecular mass was heterogeneous and also varied with temperature. The increased aggregation of the protein resulted in altered kinetic properties. The ATP hydrolysis activity of the protein increased and the half-maximal ATP concentration decreased as the protein concentration was increased in the assay. Also, a brief pretreatment of the protein with ATP and Mg2+ to increase aggregation eliminated the otherwise observed time delays in the Rubisco activation and ATP hydrolysis kinetics.

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.

Deletion of the carboxyl-terminal portion of the transit peptide affects processing but not import or assembly of the small subunit of ribulose-1,5-bisphosphate carboxylase.

Import of the small subunit of ribulose-1,5-biphosphate carboxylase/oxygenase into the chloroplast has been proposed to involve two proteolytic cleavages which convert the 20-kDa precursor (pSSU) into the mature 14-kDa subunit (SSU) via an 18-kDa intermediate. A deletion mutant (PSd48/57) of pSSU which lacks 10 amino acids in a conserved region in the carboxyl-terminal portion of the transit peptide is converted into a series of 16-18-kDa polypeptides in addition to the mature 14-kDa SSU when imported into isolated pea chloroplasts. We examined import and processing of this mutant pSSU to determine whether the 16-18-kDa SSUs undergo further maturation in the chloroplast stroma to yield 14-kDa SSU. The ratio of incorrectly processed to 14-kDa SSU is stable up to 60 min following import. This indicates that processing of PSd48/57 involves a single proteolytic cleavage which occurs during or immediately following transit across the chloroplast envelope. The carboxyl-terminal portion of the transit peptide confers either sequence specificity for the processing protease or provides a three-dimensional structure necessary for consistent cleavage at the mature amino terminus of SSU. Incorrectly processed SSUs were incorporated into the holoenzyme demonstrating that removal of the entire transit sequence is not necessary for assembly of the holoenzyme.

Identification of an assembly domain in the small subunit of ribulose-1,5-bisphosphate carboxylase.

The mature small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in higher plants contains a highly conserved sequence of 16 amino acids that is absent in the SSUs of cyanobacteria. To determine whether this region of the SSU of higher plants has a specific function, portions of the SSU genes (rbcS) of pea (Pisum sativum) and the cyanobacterium Anacystis nidulans were fused to create chimeric genes that either lacked or contained the coding sequence for the 16 conserved amino acids. Precursor proteins synthesized in vitro from the chimeric genes were incubated with isolated pea chloroplasts to assay import and assembly into the holoenzyme. Fusion proteins lacking the 16-amino acid sequence were imported and processed but failed to assemble with endogenous large subunit. Addition of the region from a pea rbcS containing the 16 amino acids to the rbcS of Anacystis enabled the imported SSU fusion protein to assemble with pea large subunit. This 16-amino acid sequence is encoded by a separate exon in certain rbcS genes of higher plants. We propose that the conserved 16-amino acid sequence constitutes a domain acquired to facilitate assembly of the eukaryotic holoenzyme.

Derivation of a physical map of chloroplast DNA from Nicotiana tabacum by two-dimensional gel and computer-aided restriction analysis.

A combined approach was used to derive a detailed physical map of Nicotiana tabacum chloroplast DNA for the restriction enzymes SalI, SmaI, KpnI, and BamHI. Complete maps for the restriction enzymes SalI, SmaI, and KpnI were derived by using two-dimensional agarose gel analysis of fragments obtained by reciprocal double digestion of chloroplast DNA. We have characterized a complete cloned library of N. tabacum chloroplast DNA which contains overlapping restriction fragments resulting from partial digestion by BamHI. With these clones and existing data, we used a novel computer-aided analysis to derive a detailed map for the enzyme BamHI. A comparison and compilation of all published N. tabacum chloroplast DNA restriction maps is presented. Differences between ours and a previously published SmaI and BamHI restriction map are discussed.