Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized.

The cornerstone of autotrophy, the CO(2)-fixing enzyme, d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is hamstrung by slow catalysis and confusion between CO(2) and O(2) as substrates, an "abominably perplexing" puzzle, in Darwin's parlance. Here we argue that these characteristics stem from difficulty in binding the featureless CO(2) molecule, which forces specificity for the gaseous substrate to be determined largely or completely in the transition state. We hypothesize that natural selection for greater CO(2)/O(2) specificity, in response to reducing atmospheric CO(2):O(2) ratios, has resulted in a transition state for CO(2) addition in which the CO(2) moiety closely resembles a carboxylate group. This maximizes the structural difference between the transition states for carboxylation and the competing oxygenation, allowing better differentiation between them. However, increasing structural similarity between the carboxylation transition state and its carboxyketone product exposes the carboxyketone to the strong binding required to stabilize the transition state and causes the carboxyketone intermediate to bind so tightly that its cleavage to products is slowed. We assert that all Rubiscos may be nearly perfectly adapted to the differing CO(2), O(2), and thermal conditions in their subcellular environments, optimizing this compromise between CO(2)/O(2) specificity and the maximum rate of catalytic turnover. Our hypothesis explains the feeble rate enhancement displayed by Rubisco in processing the exogenously supplied carboxyketone intermediate, compared with its nonenzymatic hydrolysis, and the positive correlation between CO(2)/O(2) specificity and (12)C/(13)C fractionation. It further predicts that, because a more product-like transition state is more ordered (decreased entropy), the effectiveness of this strategy will deteriorate with increasing temperature.

Faster Rubisco is the key to superior nitrogen-use efficiency in NADP-malic enzyme relative to NAD-malic enzyme C4 grasses.

In 27 C4 grasses grown under adequate or deficient nitrogen (N) supplies, N-use efficiency at the photosynthetic (assimilation rate per unit leaf N) and whole-plant (dry mass per total leaf N) level was greater in NADP-malic enzyme (ME) than NAD-ME species. This was due to lower N content in NADP-ME than NAD-ME leaves because neither assimilation rates nor plant dry mass differed significantly between the two C4 subtypes. Relative to NAD-ME, NADP-ME leaves had greater in vivo (assimilation rate per Rubisco catalytic sites) and in vitro Rubisco turnover rates (k(cat); 3.8 versus 5.7 s(-1) at 25 degrees C). The two parameters were linearly related. In 2 NAD-ME (Panicum miliaceum and Panicum coloratum) and 2 NADP-ME (Sorghum bicolor and Cenchrus ciliaris) grasses, 30% of leaf N was allocated to thylakoids and 5% to 9% to amino acids and nitrate. Soluble protein represented a smaller fraction of leaf N in NADP-ME (41%) than in NAD-ME (53%) leaves, of which Rubisco accounted for one-seventh. Soluble protein averaged 7 and 10 g (mmol chlorophyll)(-1) in NADP-ME and NAD-ME leaves, respectively. The majority (65%) of leaf N and chlorophyll was found in the mesophyll of NADP-ME and bundle sheath of NAD-ME leaves. The mesophyll-bundle sheath distribution of functional thylakoid complexes (photosystems I and II and cytochrome f) varied among species, with a tendency to be mostly located in the mesophyll. In conclusion, superior N-use efficiency of NADP-ME relative to NAD-ME grasses was achieved with less leaf N, soluble protein, and Rubisco having a faster k(cat).

Stomatal conductance does not correlate with photosynthetic capacity in transgenic tobacco with reduced amounts of Rubisco.

High-resolution imaging of chlorophyll a fluorescence from intact tobacco leaves was used to compare the quantum yield of PSII electron transport in the chloroplasts of guard cells with that in the underlying mesophyll cells. Transgenic tobacco plants with reduced amounts of Rubisco (anti-Rubisco plants) were compared with wild-type tobacco plants. The quantum yield of PSII in both guard cells and underlying mesophyll cells was less in anti-Rubisco plants than in wild-type plants, but closely matched between the two cell types regardless of genotype. CO2 assimilation rates of anti-Rubisco plants were 4.4 micromol m(-2) s(-1) compared with 17.3 micromol m(-2) s(-1) for the wild type, when measured at a photon irradiance of 1000 micromol m(-2) s(-1) and ambient CO2 of 380 micromol mol(-1). Despite the large difference in photosynthetic capacity between the anti-Rubisco and wild-type plants, there was no discernible difference in the rate of stomatal opening, steady-state stomatal conductance or response of stomatal conductance to ambient CO2 concentration. These data demonstrate clearly that the commonly observed correlation between photosynthetic capacity and stomatal conductance can be disrupted in the long term by manipulation of photosynthetic capacity via antisense RNA technology. It was concluded that stomatal conductance is not directly determined by the photosynthetic capacity of guard cells or the leaf mesophyll.

Nitrogen-regulated hypermutator strain of Synechococcus sp. for use in in vivo artificial evolution.

Artificially evolved variants of proteins with roles in photosynthesis may be selected most conveniently by using a photosynthetic organism, such as a cyanobacterium, whose growth depends on the function of the target protein. However, the limited transformation efficiency of even the most transformable cyanobacteria wastes much of the diversity of mutant libraries of genes produced in vitro, impairing the coverage of sequence space. This highlights the advantages of an in vivo approach for generating diversity in the selection organism itself. We constructed two different hypermutator strains of Synechococcus sp. strain PCC 7942 by insertionally inactivating or nutritionally repressing the DNA mismatch repair gene, mutS. Inactivation of mutS greatly increases the mutation rate of the cyanobacterium's genes, leading to an up-to-300-fold increase in the frequency of resistance to the antibiotics rifampin and spectinomycin. In order to control the rate of mutation and to limit cellular damage resulting from prolonged hypermutation, we placed the uninterrupted mutS gene in the cyanobacterial chromosome under the transcriptional control of the cyanobacterial nirA promoter, which is repressed in the presence of NH(4)(+) as an N source and derepressed in its absence. By removing or adding this substrate, hypermutation was activated or repressed as required. As expected, hypermutation caused by repression in PnirA-mutS transformants led to an accumulation of spectinomycin resistance mutations during growth.

Photosynthesis and growth of tobacco with a substituted bacterial Rubisco mirror the properties of the introduced enzyme.

Complete replacement, by biolistic plastid transformation, of the hexadecameric ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of tobacco (Nicotiana tabacum) with the dimeric version from the bacterium, Rhodospirillum rubrum, resulted in fully autotrophic and reproductive tobacco plants that required high CO(2) concentrations to grow (Whitney SM, Andrews TJ [2001] Proc Natl Acad Sci USA 98: 14738-14743). Growth and photosynthesis of these plants was compared with that of nontransformed tobacco and other controls where the rbcL gene for the large subunit of tobacco Rubisco was linked to the aadA selectable-marker gene, simulating the gene arrangement of the transformants with R. rubrum Rubisco. An arrangement of the rbcL and aadA genes that gave rise to an abundant monocistronic rbcL transcript and a one-fifth as abundant bicistronic rbcL-aadA transcript had Rubisco levels and photosynthetic properties similar to those of nontransformed tobacco. Direct linkage of the rbcL and aadA genes, resulting in exclusive production of a bicistronic mRNA transcript analogous to that of the transformants with R. rubrum Rubisco, reduced transcript abundance and tobacco Rubisco content. The analogous transcript with the R. rubrum rbcM gene substituted for rbcL was not only reduced in abundance, but was also translated less efficiently. The photosynthetic rates of the transformants and controls were measured at high CO(2) concentrations, using a mass spectrometric method. The rates and their responses to atmospheric CO(2) concentration mirrored the amounts and the kinetic properties of the Rubiscos present. The contents of total nitrogen, carbohydrates, and photosynthetic metabolites of the leaves were also consistent with the content and type of Rubisco.

The relationship between side reactions and slow inhibition of ribulose-bisphosphate carboxylase revealed by a loop 6 mutant of the tobacco enzyme.

The first directed mutant of a higher plant ribulose-bisphosphate carboxylase/oxygenase (Rubisco), constructed by chloroplast transformation, is catalytically impaired but still able to support the plant's photosynthesis and growth (Whitney, S. M., von Caemmerer, S., Hudson, G. S., and Andrews, T. J. (1999) Plant Physiol. 121, 579-588). This mutant enzyme has a Leu to Val substitution at residue 335 in the flexible loop 6 of the large subunit, which closes over the substrate during catalysis. Its active site was intact, as judged by its barely impaired competency in the initial enolization step of the reaction sequence, and its ability to bind tightly the intermediate analog, 2'-carboxy-D-arabinitol-1,5-bisphosphate. Prompted by observations that the mutant enzyme displayed much less slow inhibition during catalysis in vitro than the wild type, its tendency to catalyze side reactions and its response to the slow inhibitor D-xylulose-1,5-bisphosphate were studied. The lessening in slow inhibition was not caused by reduced production of inhibitory side products. Except for pyruvate production, these reactions were strongly enhanced by the mutation, as was the ability to catalyze the carboxylation of D-xylulose-1,5-bisphosphate. Rather, reduced inhibition was the result of lessened sensitivity to these inhibitors. The slow isomerization phase that characterizes inhibition of the wild-type enzyme by D-xylulose-1,5-bisphosphate was completely eliminated by the mutation, and the mutant was more adept than the wild type in catalyzing the benzylic acid-type rearrangement of D-glycero-2,3-pentodiulose-1,5-bisphosphate (produced by oxidation of the substrate, D-ribulose-1,5-bisphosphate). These observations are consistent with increased flexibility of loop 6 induced by the mutation, and they reveal the underlying mechanisms by which the side reactions cause slow inhibition.

Complete spectra of the far-red chemiluminescence of the oxygenase reaction of Mn2+-activated ribulose-bisphosphate carboxylase/oxygenase establish excited Mn2+ as the source.

Chemiluminescence emitted by Mn(2+)-activated ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) while catalyzing oxygenation was analyzed to clarify the source of the emission. Using dual detectors capturing radiation over a wide range of visible and infrared wavelengths, we tested for radiation from singlet O(2) decay and found it to be essentially absent (less than 0.1% of the total luminescence intensity). Spectra were determined between 647 and 885 nm with a very sensitive, charge-coupled detector-based spectrograph to detect differences in the emission spectra between rubiscos from bacterial and higher plant sources. All Mn(2+)-activated rubiscos emitted a broad, smooth spectrum of chemiluminescence, unchanging as the reaction progressed. The spectra from higher plant rubiscos (spinach and both the wild type and an L335V mutant from tobacco), all exhibited maxima at about 800 nm. However, Mn(2+)-activated rubisco from the bacterium, Rhodospirillum rubrum, emitted at shorter wavelengths (760 nm peak), demonstrating host ligand-field influences arising from aminoacyl residue differences and/or conformational changes caused by the absence of small subunits. The findings provide strong evidence that the chemiluminescence arises from an excited state of the active-site Mn(2+) that is produced during oxygenation. We propose that the Mn(2+) becomes excited by a one-electron exchange mechanism of oxygenation that is not available to Mg(2+)-activated rubisco.

Variability of the pyrenoid-based CO2 concentrating mechanism in hornworts (Anthocerotophyta).

Hornworts (Anthocerotophyta) are the only group of land plants with pyrenoid-containing chloroplasts. CO2 exchange and carbon isotope discrimination values (Δ13C) values have previously demonstrated the presence of a CO2 concentrating mechanism (CCM) in some pyrenoid-containing species. We have examined hornwort CCM function by using a combined fluorometer/mass spectrometer based technique to compare pyrenoid-containing (PhaeocerosProsk. and Notothylas Sull.) and pyrenoid-lacking (Megaceros Campbell) hornworts, with the liverwort Marchantia polymorphaL. that has standard C3 photosynthesis and a thalloid growth form similar to hornworts. We found that Notothylas has more CCM activity than Phaeoceros, and that Megaceros has the least CCM activity. Notothylas and Phaeoceros had compensation points from 11-13 parts per million (ppm) CO2, lower K0.5(CO2) than Marchantia, negligible photorespiration, and they accumulate a pool of dissolved inorganic carbon (DIC) between 19-108 nmol mg-1 chlorophyll. Megaceroshad an intermediate compensation point of 31 ppm CO2 (compared with 64 ppm CO2 in Marchantia), a lower K0.5(CO2) than Marchantia, and some photorespiration, but no DIC pool. We also determined the catalytic rate of carboxylation per active site of Rubisco for all four species (Marchantia, 2.6 s-1; Megaceros, 3.3 s-1; Phaeoceros, 4.2 s-1; Notothylas 4.3 s-1), and found that Rubisco content was 3% of soluble protein for pyrenoid-containing species, 4% for Megaceros and 8% for Marchantia.

The relationship between CO2-assimilation rate, Rubisco carbamylation and Rubisco activase content in activase-deficient transgenic tobacco suggests a simple model of activase action.

Transgenic tobacco (Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) activase were used to examine the relationship between CO2-assimilation rate, Rubisco carbamylation and activase content. Plants used were those members of the r1 progeny of a primary transformant with two independent T-DNA inserts that could be grown without CO2 supplementation. These plants had from < 1% to 20% of the activase content of control plants. Severe suppression of activase to amounts below 5% of those present in the controls was required before reductions in CO2-assimilation rate and Rubisco carbamylation were observed, indicating that one activase tetramer is able to service as many as 200 Rubisco hexadecamers and maintain wild-type carbamylation levels in vivo. The reduction in CO2-assimilation rate was correlated with the reduction in Rubisco carbamylation. The anti-activase plants had similar ribulose-1,5-bisphosphate pool sizes but reduced 3-phosphoglycerate pool sizes compared to those of control plants. Stomatal conductance was not affected by reduced activase content or CO2-assimilation rate. A mathematical model of activase action is used to explain the observed hyperbolic dependence of Rubisco carbamylation on activase content.