Ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO 2 fixation and, thus, limits agricultural productivity. However, Rubisco enzymes from different species have different catalytic constants. If the structural basis for such differences were known, a rationale could be developed for genetically engineering an improved enzyme. Residues at the bottom of the large-subunit ␣͞␤-barrel active site of Rubisco from the green alga Chlamydomonas reinhardtii (methyl-Cys-256, Lys-258, and Ile-265) were previously changed through directed mutagenesis and chloroplast transformation to residues characteristic of land-plant Rubisco (Phe-256, Arg-258, and Val-265). The resultant enzyme has decreases in carboxylation efficiency and CO 2͞O2 specificity, despite the fact that land-plant Rubisco has greater specificity than the Chlamydomonas enzyme. Because the residues are close to a variable loop between ␤-strands A and B of the small subunit that can also affect catalysis, additional substitutions were created at this interface. When largesubunit Val-221 and Val-235 were changed to land-plant Cys-221 and Ile-235, they complemented the original substitutions and returned CO 2͞O2 specificity to the normal level. Further substitution with the shorter ␤A-␤B loop of the spinach small subunit caused a 12-17% increase in specificity. The enhanced CO 2͞O2 specificity of the mutant enzyme is lower than that of the spinach enzyme, but the carboxylation and oxygenation kinetic constants are nearly indistinguishable from those of spinach and substantially different from those of Chlamydomonas Rubisco. Thus, this interface between large and small subunits, far from the active site, contributes significantly to the differences in catalytic properties between algal and land-plant Rubisco enzymes.catalysis ͉ Chlamydomonas ͉ chloroplast ͉ photosynthesis ͉ ribulosebisphosphate carboxylase͞oxygenase C O 2 and O 2 compete at the active site of ribulose-1,5-bisphosphate (RuBP) carboxylase͞oxygenase [Ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco), Enzyme Commission 4.1.1.39] for either the carboxylation or oxygenation of RuBP (reviewed in refs. 1-3). Whereas carboxylation is responsible for the accumulation of carbon in the biosphere, oxygenation is a nonessential reaction that leads to the loss of fixed carbon via the photorespiratory pathway. The ratio of the catalytic efficiencies (V max ͞K m ) of carboxylation (V c ͞K c ) and oxygenation (V o ͞K o ) defines the CO 2 ͞O 2 -specificity kinetic constant ⍀ (4), which is determined by the differential stabilization of the carboxylation and oxygenation transition states for the rate-limiting partial reactions (5). However, net CO 2 fixation is determined by the difference between the velocities of carboxylation and oxygenation at the CO 2 and O 2 concentrations that occur in vivo (4, 6). Because of its pivotal role in catalyzing the rate-limiting step of photosynthesis, genetic engineering of Rubisco aimed at increasing net CO 2 fixa...

Apurinic/apyrimidinic endonuclease (APE-1) is essential for base excision repair (BER) of damaged DNA. Here molecular dynamics (MD) simulations of APE1 complexed with cleaved and uncleaved damaged DNA were used to determine the role and position of the metal ion(s) in the active site before and after DNA cleavage. The simulations started from an energy minimized wild-type structure of the metal-free APE1/damaged-DNA complex (1DE8). A grid search with one Mg2+ ion located two low energy clusters of Mg2+ consistent with the experimentally determined metal ion positions. At the start of the longer MD simulations, Mg2+ ions were placed at different positions as seen in the crystal structures and the movement of the ion was followed over the course of the trajectory. Our analysis suggests a "moving metal mechanism" in which one Mg2+ ion moves from the B- (more buried) to the A-site during substrate cleavage. The anticipated inversion of the phosphate oxygens occurs during the in-line cleavage reaction. Experimental results, which show competition between Ca2+ and Mg2+ for catalyzing the reaction, and high concentrations of Mg2+ are inhibitory, indicate that both sites cannot be simultaneously occupied for maximal activity.

Comparison of subunit sequences and X-ray crystal structures of ribulose-1,5-bisphosphate carboxylase/oxygenase indicates that the loop between beta-strands A and B of the small subunit is one of the most variable regions of the holoenzyme. In prokaryotes and nongreen algae, the loop contains 10 residues. In land plants and green algae, the loop is comprised of approximately 22 and 28 residues, respectively. Previous studies indicated that the longer betaA-betaB loop was required for the assembly of cyanobacterial small subunits with plant large subunits in isolated chloroplasts. In the present study, chimeric small subunits were constructed by replacing the loop of the green alga Chlamydomonas reinhardtii with the sequences of Synechococcus or spinach. When these engineered genes were transformed into a Chlamydomonas mutant that lacks small-subunit genes, photosynthesis-competent colonies were recovered, indicating that loop size is not essential for holoenzyme assembly. Whereas the Synechococcus loop causes decreases in carboxylation V(max), K(m)(O(2)), and CO(2)/O(2) specificity, the spinach loop causes complementary decreases in carboxylation V(max), K(m)(O(2)), and K(m)(CO(2)) without a change in specificity. X-ray crystal structures of the engineered proteins reveal remarkable similarity between the introduced betaA-betaB loops and the respective loops in the Synechococcus and spinach enzymes. The side chains of several large-subunit residues are altered in regions previously shown by directed mutagenesis to influence CO(2)/O(2) specificity. Differences in the catalytic properties of divergent Rubisco enzymes may arise from differences in the small-subunit betaA-betaB loop. This loop may be a worthwhile target for genetic engineering aimed at improving photosynthetic CO(2) fixation.

Despite conservation of three-dimensional structure and active-site residues, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) enzymes from divergent species differ with respect to catalytic efficiency and CO 2 /O 2 specificity. A deeper understanding of the structural basis for these differences may provide a rationale for engineering an improved enzyme, thereby leading to an increase in photosynthetic CO 2 fixation and agricultural productivity. By comparing 500 active-site large subunit sequences from flowering plants with that of the green alga Chlamydomonas reinhardtii, a small number of residues were found to differ in regions previously shown by mutant screening to influence CO 2 /O 2 specificity. When directed mutagenesis and chloroplast transformation were used to change Chlamydomonas Met-42 and Cys-53 to land plant Val-42 and Ala-53 in the large subunit N-terminal domain, little or no change in Rubisco catalytic properties was observed. However, changing Chlamydomonas methylCys-256, Lys-258, and Ile-265 to land plant Phe-256, Arg-258, and Val-265 at the bottom of the ␣/␤-barrel active site caused a 10% decrease in CO 2 /O 2 specificity, largely due to an 85% decrease in carboxylation catalytic efficiency (V max /K m ). Because land plant Rubisco enzymes have greater CO 2 /O 2 specificity than the Chlamydomonas enzyme, this group of residues must be complemented by other residues that differ between Chlamydomonas and land plants. The Rubisco x-ray crystal structures indicate that these residues may reside in a variable loop of the nuclear-encoded small subunit, more than 20 Å away from the active site.The rbcL gene is one of the most sequenced genes in nature, and it is the most sequenced gene in the chloroplasts of eukaryotes. There are more than 2000 rbcL entries in the National Center for Biotechnology Information Entrez Proteins data base. Because rbcL encodes the catalytic large subunit of the ratelimiting photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, 1 EC 4.1.1.39) (reviewed in Refs. 1 and 2), it may be possible to exploit the vast sequence data base to gain a deeper understanding of the structure-function relationships of this enzyme. In particular, the values of catalytic efficiency and CO 2 /O 2 specificity vary among Rubisco enzymes from divergent species (3-5), but residues directly involved in catalysis at the ␣/␤-barrel active site are nearly 100% conserved. Thus, divergent residues, relatively far from the active site, must account for the differences in kinetic constants. The potential functional significance of these residues may be deduced by comparing the numerous Rubisco x-ray crystal structures that reside in the Protein Data Bank (6). Structures have been solved for Rubisco enzymes from evolutionarily distant prokaryotes, algae, and land plants (e.g. Refs. 7-13).Rubisco is a bifunctional enzyme that catalyzes either the carboxylation or oxygenation of RuBP, thereby initiating the Calvin cycle of photosynthesis or the photorespi...