Coenzyme site-directed mutants of photosynthetic A4-GAPDH show selectively reduced NADPH-dependent catalysis, similar to regulatory AB-GAPDH inhibited by oxidized thioredoxin

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB). AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH). Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2'-phosphate group of NADP. This suggested a possible involvement of these residues in the regulatory mechanism. Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties. Affinity for NADPH was decreased significantly in all mutants, and kcat(NADPH) was lowered in mutants carrying the substitution of Ser188. NADH-dependent activity was unaffected. The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme. A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4. We conclude that in the absence of interactions between Ser188 and the 2'-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH. A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH.     

Regulation of photosynthetic GAPDH dissected by mutants

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants catalyzes an NADPH-consuming reaction, which is part of the Calvin cycle. This reaction is regulated by light via thioredoxins and metabolites, while a minor NADH-dependent activity is constant and constitutive. The major native isozyme is formed by A- and B-subunits in stoichiometric ratio (A2B2, A8B8), but tetramers of recombinant B-subunits (GapB) display similar regulatory features to A2B2-GAPDH. The C-terminal extension (CTE) of B-subunits is essential for thioredoxin-mediated regulation and NAD-induced aggregation to partially inactive oligomers (A8B8, B8). Deletion mutant B(minCTE) is redox insensitive and invariably tetrameric, and chimeric mutant A(plusCTE) acquired redox sensitivity and capacity to aggregate to very large oligomers in presence of NAD. Redox regulation principally affects the turnover number, without significantly changing the affinity for either 1,3-bisphosphoglycerate or NADPH. Mutant R77A of GapB, B(R77A), is down-regulated and mimics the behavior of oxidized GapB under any redox condition, whereas mutant B(E362Q) is constantly up-regulated, resembling reduced GapB. Despite their redox insensitivity, both B(R77A) and B(E362Q) mutants are notably prone to aggregate in presence of NAD. Based on structural data and current functional analysis, a model of GAPDH redox regulation is presented. Formation of a disulfide in the CTE induces a conformational change of the GAPDH with repositioning of the terminal amino acid Glu-362 in the proximity of Arg-77. The latter residue is thus distracted from binding the 2'-phosphate of NADP, with the final effect that the enzyme relaxes to a conformation leading to a slower NADPH-dependent catalytic activity.     

Thioredoxin-dependent regulation of photosynthetic glyceraldehyde-3-phosphate dehydrogenase: autonomous vs. CP12-dependent mechanisms

Regulation of the Calvin–Benson cycle under varying light/dark conditions is a common property of oxygenic photosynthetic organisms and photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the targets of this complex regulatory system. In cyanobacteria and most algae, photosynthetic GAPDH is a homotetramer of GapA subunits which do not contain regulatory domains. In these organisms, dark-inhibition of the Calvin–Benson cycle involves the formation of a kinetically inhibited supramolecular complex between GAPDH, the regulatory peptide CP12 and phosphoribulokinase. Conditions prevailing in the dark, i.e. oxidation of thioredoxins and low NADP(H)/NAD(H) ratio promote aggregation. Although this regulatory system has been inherited in higher plants, these phototrophs contain in addition a second type of GAPDH subunits (GapB) resulting from the fusion of GapA with the C-terminal half of CP12. Heterotetrameric A₂B₂-GAPDH constitutes the major photosynthetic GAPDH isoform of higher plants chloroplasts and coexists with CP12 and A₄-GAPDH. GapB subunits of A₂B₂-GAPDH have inherited from CP12 a regulatory domain (CTE for C-terminal extension) which makes the enzyme sensitive to thioredoxins and pyridine nucleotides, resembling the GAPDH/CP12/PRK system. The two systems are similar in other respects: oxidizing conditions and low NADP(H)/NAD(H) ratios promote aggregation of A₂B₂-GAPDH into strongly inactivated A₈B₈-GAPDH hexadecamers, and both CP12 and CTE specifically affect the NADPH-dependent activity of GAPDH. The alternative, lower activity with NADH is always unaffected. Based on the crystal structure of spinach A₄-GAPDH and the analysis of site-specific mutants, a model of the autonomous (CP12-independent) regulatory mechanism of A₂B₂-GAPDH is proposed. Both CP12 and CTE seem to regulate different photosynthetic GAPDH isoforms according to a common and ancient molecular mechanism.     

Reconstitution and properties of the recombinant glyceraldehyde-3-phosphate dehydrogenase/CP12/phosphoribulokinase supramolecular complex of Arabidopsis

Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form together with the regulatory peptide CP12 a supramolecular complex in Arabidopsis (Arabidopsis thaliana) that could be reconstituted in vitro using purified recombinant proteins. Both enzyme activities were strongly influenced by complex formation, providing an effective means for regulation of the Calvin cycle in vivo. PRK and CP12, but not GapA (A(4) isoform of GAPDH), are redox-sensitive proteins. PRK was reversibly inhibited by oxidation. CP12 has no enzymatic activity, but it changed conformation depending on redox conditions. GapA, a bispecific NAD(P)-dependent dehydrogenase, specifically formed a binary complex with oxidized CP12 when bound to NAD. PRK did not interact with either GapA or CP12 singly, but oxidized PRK could form with GapA/CP12 a stable ternary complex of about 640 kD (GapA/CP12/PRK). Exchanging NADP for NAD, reducing CP12, or reducing PRK were all conditions that prevented formation of the complex. Although GapA activity was little affected by CP12 alone, the NADPH-dependent activity of GapA embedded in the GapA/CP12/PRK complex was 80% inhibited in respect to the free enzyme. The NADH activity was unaffected. Upon binding to GapA/CP12, the activity of oxidized PRK dropped from 25% down to 2% the activity of the free reduced enzyme. The supramolecular complex was dissociated by reduced thioredoxins, NADP, 1,3-bisphosphoglycerate (BPGA), or ATP. The activity of GapA was only partially recovered after complex dissociation by thioredoxins, NADP, or ATP, and full GapA activation required BPGA. NADP, ATP, or BPGA partially activated PRK, but full recovery of PRK activity required thioredoxins. The reversible formation of the GapA/CP12/PRK supramolecular complex provides novel possibilities to finely regulate GapA ("non-regulatory" GAPDH isozyme) and PRK (thioredoxin sensitive) in a coordinated manner.     

Cloning and sequence analysis of cDNAs encoding the cytosolic precursors of subunits GapA and GapB of chloroplast glyceraldehyde-3-phosphate dehydrogenase from pea and spinach

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is composed of two different subunits, GapA and GapB. cDNA clones containing the entire coding sequences of the cytosolic precursors for GapA from pea and for GapB from pea and spinach have been identified, sequenced and the derived amino acid sequences have been compared to the corresponding sequences from tobacco, maize and mustard. These comparisons show that GapB differs from GapA in about 20% of its amino acid residues and by the presence of a flexible and negatively charged C-terminal extension, possibly responsible for the observed association of the enzyme with chloroplast envelopes in vitro. This C-terminal extension (29 or 30 residues) may be susceptible to proteolytic cleavage thereby leading to a conversion of chloroplast GAPDH isoenzyme I into isoenzyme II. Evolutionary rate comparisons at the amino acid sequence level show that chloroplast GapA and GapB evolve roughly two-fold slower than their cytosolic counterpart GapC. GapA and GapB transit peptides evolve about 10 times faster than the corresponding mature subunits. They are relatively long (68 and 83 residues for pea GapA and spinach GapB respectively) and share a similar amino acid framework with other chloroplast transit peptides.     

Molecular mechanism of NADPH-glyceraldehyde-3-phosphate dehydrogenase regulation through the C-terminus of CP12 in Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) consists of four GapA subunits. This A4 GAPDH is not autonomously regulated, as the regulatory cysteine residues present on GapB subunits are missing in GapA subunits. The regulation of A4 GAPDH is provided by another protein, CP12. To determine the molecular mechanisms of regulation of A4 GAPDH, we mutated three residues (R82, R190, and S195) of GAPDH of C. reinhardtii. Kinetic studies of GAPDH mutants showed the importance of residue R82 in the specificity of GAPDH for NADPH, as previously shown for the spinach enzyme. The cofactor NADPH was not stabilized through the 2'-phosphate by the serine 195 residue of the algal GAPDH, unlike the case in spinach. The mutation of R190 also led to a structural change that was not observed in the spinach enzyme. This mutation led to a loss of activity for NADPH and NADH, indicating the crucial role of this residue in maintaining the algal GAPDH structure. Finally, the interaction between GAPDH mutants and wild-type and mutated CP12 was analyzed by immunoblotting experiments, surface plasmon resonance, and kinetic studies. The results obtained with these approaches highlight the involvement of the last residue of CP12, Asp80, in modulating the activity of GAPDH by preventing access of the cofactor NADPH to the active site. These results help us to bridge the gap between our knowledge of structure and our understanding of functional biology in GAPDH regulation.     

Evidence in favor of the symbiotic origin of chloroplasts: primary structure and evolution of tobacco glyceraldehyde-3-phosphate dehydrogenases

We report nucleotide sequences of cDNAs for the nuclear genes encoding chloroplast (GapA and GapB) and cytosolic (GapC) glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from N. tabacum. Comparison of nucleotide sequences indicates that the GapA and GapB genes evolved following duplication of an ancestral gene about 450 million years ago. However, the divergence of GapA/B and GapC occurred much earlier in evolution than the divergence of GapC and GAPDH genes of animals and fungi, suggesting that chloroplast and cytosolic GAPDHs evolved from different lineages. Comparison of amino acid sequences shows that the chloroplast GAPDHs are related to GAPDHs found in thermophilic bacteria, while the cytosolic GAPDH is related to the GAPDH found in mesophilic prokaryotes. These results strongly support the symbiotic origin of chloroplasts.     

Functional studies of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunits A and B expressed in Escherichia coli: formation of highly active A4 and B4 homotetramers and evidence that aggregation of the B4 complex is mediated by the B subunit carboxy terminus

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (phosphorylating, E.C. 1.2.1.13) (GAPDH) of higher plants exists as an A₂B₂ heterotetramer that catalyses the reductive step of the Calvin cycle. In dark chloroplasts the enzyme exhibits a molecular mass of 600 kDa, whereas in illuminated chloroplasts the molecular mass is altered in favor of the more active 150 kDa form. We have expressed in Escherichia coli proteins corresponding to the mature A and B subunits of spinach chloroplast GAPDH (GapA and GapB, respectively) in addition to a derivative of the B subunit lacking the GapB-specific C-terminal extension (CTE). One mg of each of the three proteins so expressed was purified to electrophoretic homogeneity with conventional methods. Spinach GapA purified from E. coli is shown to be a highly active homotetramer (50–70 U/mg) which does not associate under aggregating conditions in vitro to high-molecular-mass (HMM) forms of ca. 600 kDa. Since B₄ forms of the enzyme have not been described from any source, we were surprised to find that spinach GapB purified from E. coli was active (15–35 U/mg). Spinach GapB lacking the CTE purified from E. coli is more highly active (130 U/mg) than GapB with the CTE. Under aggregating conditions, GapB lacking the CTE is a tetramer that does not associate to HMM forms whereas GapB with the CTE occurs exclusively as an aggregated HMM form. The data indicate that intertetramer association of chloroplast GAPDH in vitro occurs through GapB-mediated protein-protein interaction.Abbreviations GAPDH glyceraldehyde-3-phosphate dehydrogenase - CTE carboxy-terminal extension - HMM high molecular mass - ATP adenosine triphosphate - 3PGA 3-phosphoglycerate - 1,3bisPGA 1,3-bisphosphoglycerate - HMM high-molecular mass     

The GapA/B gene duplication marks the origin of Streptophyta (charophytes and land plants)

Independent evidence from morphological, ultrastructural, biochemical, and molecular data have shown that land plants originated from charophycean green algae. However, the branching order within charophytes is still unresolved, and contradictory phylogenies about, for example,the position of the unicellular green alga Mesostigma viride are difficult to reconcile. A comparison of nuclear-encoded Calvin cycle glyceraldehyde-3-phosphate dehydrogenases (GAPDH) indicates that a crucial duplication of the GapA gene occurred early in land plant evolution. The duplicate called GapB acquired a characteristic carboxy-terminal extension (CTE) from the general regulator of the Calvin cycle CP12. This CTE is responsible for thioredoxin-dependent light/dark regulation. In this work, we established GapA, GapB, and CP12 sequences from bryophytes, all orders of charophyte as well as chlorophyte green algae, and the glaucophyte Cyanophora paradoxa. Comprehensive phylogenetic analyses of all available plastid GAPDH sequences suggest that glaucophytes and green plants are sister lineages and support a positioning of Mesostigma basal to all charophycean algae. The exclusive presence of GapB in terrestrial plants, charophytes, and Mesostigma dates the GapA/B gene duplication to the common ancestor of Streptophyta. The conspicuously high degree of GapB sequence conservation suggests an important metabolic role of the newly gained regulatory function. Because the GapB-mediated protein aggregation most likely ensures the complete blockage of the Calvin cycle at night, we propose that this mechanism is also crucial for efficient starch mobilization. This innovation may be one prerequisite for the development of storage tissues in land plants.     

Proteomic analysis of spontaneous mutants of Lactococcus lactis: Involvement of GAPDH and arginine deiminase pathway in H2O2 resistance

Lactococcus lactis, one of the most commonly used dairy starters, is often subjected to oxidative stress in cheese manufacturing. A comparative proteomic analysis was performed to identify the molecular modifications responsible for the robustness of three spontaneous H(2)O(2)-resistant (SpOx) strains. In the parental strain, glyceraldehyde-3-phosphate deshydrogenase (GAPDH) activity is ensured by GapB and the second GAPDH GapA is not produced in standard growth conditions. We showed that GapA was overproduced in the highly resistant SpOx2 and SpOx3 mutants. Its overproduction in the MG1363 strain led to an increased H(2)O(2) resistance of exponential growing cells. Upon H(2)O(2) exposure, GapB was fully inactivated by oxidation in the parental strain. In SpOx mutants, it partly remained in the reduced form sustaining partially GAPDH activity. The analysis of gapA disruption in these SpOx strains indicated that additional unraveled mechanisms likely contribute to the resistance phenotype. In the SpOx1 mutant, the arginine deiminase pathway was found to be upregulated and disruption of arcA or arcB genes abolished H(2)O(2) resistance. We concluded that arginine consumption was directly responsible for the SpOx1 phenotype. Finally, these results suggest that sustaining energy supply is a major way of leading to oxidative stress resistance in L. lactis.     

Proteome analysis of a Lactococcus lactis strain overexpressing gapA suggests that the gene product is an auxiliary glyceraldehyde 3-phosphate dehydrogenase

The sequence of the genome from the Lactococcus lactis subspecies lactis strain IL1403 shows the presence of two reading frames, gapA and gapB, putatively encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Previous proteomic analysis of the L. lactis subspecies cremoris strain MG1363 has revealed two neighbouring protein spots, GapBI and GapBII, with amino terminal sequences identical to the product of gapA from the L. lactis subspecies cremoris strain LM0230 and that of the two IL1403 sequences. In order to assign the two protein spots to their respective genes we constructed an L. lactis strain that overexpessed the gapA gene derived from MG1363 upon nisin induction. Compared to the wild-type, the overexpressing strain had a 3.4-fold elevated level of specific GAPDH activity when grown in the presence of nisin. In both MG1363 and the gapA overexpressing strain the GAPDH activity was specific for NAD. No NADP dependent activity was detected. Proteome analysis of the gapA overexpressing strain revealed two new protein spots, GapAI and GapAII, not previously detected in proteome analysis of MG1363. Results from mass spectrometry analysis of GapA and GapB and comparison with the deduced protein sequences for the GAPDH isozymes from the genome sequence of strain IL1403 allowed us to assign GapA and GapB to their apparent IL1403 homologues encoded by gapA and gapB, respectively. Furthermore, we suggest that a homologue of a gapB product, represented by GapB, is the main source of GAPDH activity in L. lactis during normal growth.     

Characterization of two active site mutations of thioredoxin reductase from Escherichia coli

Thioredoxin reductase (TRR), a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes, undergoes two sequential thiol-disulfide interchange reactions with thioredoxin during catalysis. In order to assess the catalytic role of each nascent thiol of the active site disulfide of thioredoxin reductase, the 2 cysteines (Cys-136 and Cys-139) forming this disulfide have been individually changed to serines by site-directed mutageneses of the cloned trxB gene of Escherichia coli. Spectral analyses of TRR(Ser-136,Cys-139) as a function of pH and ionic strength have revealed two pKa values associated with the epsilon 456, one of which increases from 7.0 to 8.3 as the ionic strength is increased, and a second at 4.4 which is seen only at high ionic strength. epsilon 458 of wild type TRR(Cys-136,Cys-139) and epsilon 453 of TRR(Cys-136,Ser-139) are pH-independent. A charge transfer complex (epsilon 530 = 1300 M-1 cm-1), unique to TRR(Ser-136,Cys-139), has been observed under conditions of high ammonium cation concentration (apparent Kd = 54 microM) at pH 7.6. These results suggest the assignment of Cys-139 as the FAD-interacting thiol in the reduction of thioredoxin by NADPH via thioredoxin reductase. If, as with other members of this enzyme family, the two distinct catalytic functions are each carried out by a different nascent thiol, then Cys-136 would perform the initial thiol-disulfide interchange with thioredoxin. Steady state kinetic analyses of the proteins have revealed turnover numbers of 10 and 50% of the value of the wild type enzyme for TRR(Ser-136,Cys-139) and TRR(Cys-136,Ser-139), respectively, and no changes in the apparent Km values of TR(S2) or NADPH. The finding of activity in the mutants indicates that the remaining thiol can carry out interchange with the disulfide of thioredoxin, and the resulting mixed disulfide can be reduced by NADPH via the flavin.     

Mapping of the interaction site of CP12 with glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii. Functional consequences for glyceraldehyde-3-phosphate dehydrogenase

The 8.5 kDa chloroplast protein CP12 is essential for assembly of the phosphoribulokinase/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complex from Chlamydomonas reinhardtii. After reduction of this complex with thioredoxin, phosphoribulokinase is released but CP12 remains tightly associated with GAPDH and downregulates its NADPH-dependent activity. We show that only incubation with reduced thioredoxin and the GAPDH substrate 1,3-bisphosphoglycerate leads to dissociation of the GAPDH/CP12 complex. Consequently, a significant twofold increase in the NADPH-dependent activity of GAPDH was observed. 1,3-Bisphosphoglycerate or reduced thioredoxin alone weaken the association, causing a smaller increase in GAPDH activity. CP12 thus behaves as a negative regulator of GAPDH activity. A mutant lacking the C-terminal disulfide bridge is unable to interact with GAPDH, whereas absence of the N-terminal disulfide bridge does not prevent the association with GAPDH. Trypsin-protection experiments indicated that GAPDH may be also bound to the central alpha-helix of CP12 which includes residues at position 36 (D) and 39 (E). Mutants of CP12 (D36A, E39A and E39K) but not D36K, reconstituted the GAPDH/CP12 complex. Although the dissociation constants measured by surface plasmon resonance were 2.5-75-fold higher with these mutants than with wild-type CP12 and GAPDH, they remained low. For the D36K mutation, we calculated a 7 kcal.mol(-1) destabilizing effect, which may correspond to loss of the stabilizing effect of an ionic bond for the interaction between GAPDH and CP12. It thus suggests that electrostatic forces are responsible for the interaction between GAPDH and CP12.     

Co-existence of two regulatory NADP-glyceraldehyde 3-P dehydrogenase complexes in higher plant chloroplasts.

Light/dark modulation of the higher plant Calvin-cycle enzymes phosphoribulokinase (PRK) and NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP- GAPDH-A2B2) involves changes of their aggregation state in addition to redox changes of regulatory cysteines. Here we demonstrate that plants possess two different complexes containing the inactive forms (a) of NADP-GAPDH and PRK and (b) of only NADP-GAPDH, respectively, in darkened chloroplasts. While the 550-kDa PRK/GAPDH/CP12 complex is dissociated and activated upon reduction alone, activation and dissociation of the 600-kDa A8B8 complex of NADP-GAPDH requires incubation with dithiothreitol and the effector 1,3-bisphosphoglycerate. In the light, PRK is therefore completely in its activated state under all conditions, even in low light, while GAPDH activation in the light is characterized by a two-step mechanism with 60-70% activation under most conditions in the light, and the activation of the remaining 30-40% occurring only when 1,3-bisphosphoglycerate levels are strongly increasing. In vitro studies with the purified components and coprecipitation experiments from fresh stroma using polyclonal antisera confirm the existence of these two aggregates. Isolated oxidized PRK alone does not reaggregate after it has been purified in its reduced form; only in the presence of both CP12 and purified NADP-GAPDH, some of the PRK reaggregates. Recombinant GapA/GapB constructs form the A8B8 complex immediately upon expression in E. coli.     

The internal Cys-207 of sorghum leaf NADP-malate dehydrogenase can form mixed disulphides with thioredoxin

The role of the internal Cys-207 of sorghum NADP-malate dehydrogenase (NADP-MDH) in the activation of the enzyme has been investigated through the examination of the ability of this residue to form mixed disulphides with thioredoxin mutated at either of its two active-site cysteines. The h-type Chlamydomonas thioredoxin was used, because it has no additional cysteines in the primary sequence besides the active-site cysteines. Both thioredoxin mutants proved equally efficient in forming mixed disulphides with an NADP-MDH devoid of its N-terminal bridge either by truncation, or by mutation of its N-terminal cysteines. They were poorly efficient with the more compact WT oxidised NADP-MDH. Upon mutation of Cys-207, no mixed disulphide could be formed, showing that this cysteine is the only one, among the four internal cysteines, which can form mixed disulphides with thioredoxin. These experiments confirm that the opening of the N-terminal disulphide loosens the interaction between subunits, making Cys-207, located at the dimer contact area, more accessible.     

Thioredoxin,the Processivity Factor,Sequesters an Exposed Cysteine in the Thumb Domain of Bacteriophage T7 DNA Polymerase

Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase. It achieves processivity by binding to Escherichia coli thioredoxin (trx). gp5/trx complex binds tightly to a primer-DNA template enabling the polymerization of hundreds of nucleotides per binding event. gp5 contains 10 cysteines. Under non-reducing condition, exposed cysteines form intermolecular disulfide linkages resulting in the loss of polymerase activity. No disulfide linkage is detected when Cys-275 and Cys-313 are replaced with serines. Cys-275 and Cys-313 are located on loop A and loop B of the thioredoxin binding domain, respectively. Replacement of either cysteine with serine (gp5-C275S, gp5-C313S) drastically decreases polymerase activity of gp5 on dA₃₅₀/dT₂₅. On this primer-template gp5/trx in which Cys-313 or Cys-275 is replaced with serine have 50 and 90%, respectively, of the polymerase activity observed with wild-type gp5/trx. With single-stranded M13 DNA as a template gp5-C275S/trx retains 60% of the polymerase activity of wild-type gp5/trx. In contrast, gp5-C313S/trx has only one-tenth of the polymerase activity of wild-type gp5/trx on M13 DNA. Both wild-type gp5/trx and gp5-C275S/trx catalyze the synthesis of the entire complementary strand of M13 DNA, whereas gp5-C313S/trx has difficulty in synthesizing DNA through sites of secondary structure. gp5-C313S fails to form a functional complex with trx as measured by the apparent binding affinity as well as by the lack of a physical interaction with thioredoxin during hydroxyapatite-phosphate chromatography. Small angle x-ray scattering reveals an elongated conformation of gp5-C313S in comparison to a compact and spherical conformation of wild-type gp5.     

Unique Regulation of the Calvin Cycle in the Ultrasmall Green Alga <Emphasis Type="Italic">Ostreococcus</Emphasis>

Glyceraldehyde-3-phosphate dehydrogenase (GapAB) and CP12 are two major players in controlling the inactivation of the Calvin cycle in land plants at night. GapB originated from a GapA gene duplication and differs from GapA by the presence of a specific C-terminal extension that was recruited from CP12. While GapA and CP12 are assumed to be generally present in the Plantae (glaucophytes, red and green algae, and plants), up to now GapB was exclusively found in Streptophyta, including the enigmatic green alga Mesostigma viride. However, here we show that two closely related prasinophycean green algae, Ostreococcus tauri and Ostreococcus lucimarinus, also possess a GapB gene, while CP12 is missing. This remarkable finding either antedates the GapA/B gene duplication or indicates a lateral recruitment. Moreover, Ostreococcus is the first case where the crucial CP12 function may be completely replaced by GapB-mediated GapA/B aggregation.     

Si RNA inhibition of GRP58 associated with decrease in mitomycin C-induced DNA cross-linking and cytotoxicity

The anti-cancer drug mitomycin C is metabolically activated to bind and cross-link DNA. The cross-linking contributes significantly to the cytotoxicity. The complex chemical structure of mitomycin C allows its metabolism by several known (cytosolic NAD(P)H:quinone oxidoreductase and microsomal NADPH:cytochrome P450 reductase) and unknown enzymes. The identification of new enzymes/proteins that metabolize mitomycin C and like drugs is an area of significant research interest since these studies have direct implications in drug development and clinical usage. In the present studies, we have investigated a role of cytosolic glucose regulatory protein GRP58 in mitomycin C-induced DNA cross-linking and cytotoxicity. The control and GRP58 siRNA were transfected in human colon carcinoma HCT116 cells in culture. The transfection of GRP58 siRNA but not control siRNA significantly inhibited GRP58 in human colon carcinoma HCT116 cells. The inhibition of GRP58 led to decrease in mitomycin C-induced DNA cross-linking and cytotoxicity. These results establish a role of GRP58 in mitomycin C-induced DNA cross-linking and cytotoxicity. Site-directed mutagenesis of cysteines to serines in thioredoxin domains of GRP58 and cross-linking assays revealed that both N- and C-terminal thioredoxin domains are required for GRP58-mediated mitomycin C-induced DNA cross-linking. These results suggest that GRP58 might be an important target enzyme for further studies on mitomycin C and similar drug therapy.