Construction and physiological characterization of glyceraldehyde-3-phosphate dehydrogenase overproducing transformants of Aspergillus nidulans

Summary The construction and characterization of glyceraldehyde-3-phosphate-dehydrogenase (GPD) overproducing transformants of Aspergillus nidulans and their behaviour in acetate-limited continuous cultures and glucose-grown batch cultures are described. The A. nidulans acetamidase deletion strain MH1277 was transormed with the homologous gpdA gene on a vector with the homologous acetamidase-gene (amdS) as a selection marker. Transformant Al contains about nine integrated copies of the gpdA gene, and shows a proportional gene-dosage GPD production of about 22% of the total soluble cell protein. Compared to the wild-type MH1277, Al has higher growth yields and reaches higher specific growth rates on both acetate and glucose, which could be due to the key position of GPD in glycolysis and gluconeogenesis.Offprint requests to: P. P. F. Hanegraaf     

Modulation of glyceraldehyde-3-phosphate dehydrogenase inAnacystis nidulans by glutathione

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13; GAPDH) from the cyanobacteriumAnacystis nidulans was activated up to five-fold by reduced glutathione (GSH) in the physiological concentration range (0.1–2 mM GSH). Non-physiological reductants, like dithiothreitol (DTT) and -mercaptoethanol, also activated the enzyme. Oxidized glutathione (GSSG) had no effect on the cyanobacterial GAPDH but treatment with H₂O₂ led to a rapid, reversible deactivation of both untreated and GSH-treated enzyme preparations. GSH reversed the inhibition induced by H₂O₂. An oligomeric form of the enzyme (apparentM _r440,000) was dissociated by GSH into a lower-M _r, more active enzyme form (M _r200,000). The enzyme was shown to obey regular Michaelis-Menten kinetics. The activation of GAPDH by GSH was associated with a decrease inK _m and an increase inV _max values of the enzyme for 3-phosphoglycerate. GSH had virtually no effect on a GAPDH preparation isolated from corn chloroplasts and studied for comparison.Abbreviations GAPDH glyceraldehyde-3-phosphate dehydrogenase - GSH reduced glutathione - GSSG oxidized glutathione - DTT dithiothreitol     

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.     

Mechanism of glyceraldehyde-3-phosphate transfer from aldolase to glyceraldehyde-3-phosphate dehydrogenase

The catalytic interaction of glyceraldehyde-3-phosphate dehydrogenase with glyceraldehyde 3-phosphate has been examined by transient-state kinetic methods. The results confirm previous reports that the apparent Km for oxidative phosphorylation of glyceraldehyde 3-phosphate decreases at least 50-fold when the substrate is generated in a coupled reaction system through the action of aldolase on fructose 1,6-bisphosphate, but lend no support to the proposal that glyceraldehyde 3-phosphate is directly transferred between the two enzymes without prior release to the reaction medium. A theoretical analysis is presented which shows that the kinetic behaviour of the coupled two-enzyme system is compatible in all respects tested with a free-diffusion mechanism for the transfer of glyceraldehyde 3-phosphate from the producing enzyme to the consuming one.     

A multi-component upstream activation sequence of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter

Summary The majority of the activation potential of the Saccharomyces cerevisiae TDH3 gene promoter is contained within nucleotides –676 to –381 (relative to the translation initiation codon). An upstream activation sequence (UAS) in this region has been characterized by in vitro and in vivo assays and demonstrated to be composed of two small, adjacent DNA sequence elements. The essential determinant of this upstream UAS is a general regulatory factor 1 (GRF1) binding site at nucleotides –513 to –501. A synthetic DNA element comprising this sequence, or an analogue in which two of the degenerate nucleotides of the GRF1 site consensus sequence were altered, activated 5 deleted TDH3 and CYC1 promoters. The second DNA element of the UAS is a 7 by sequence which is conserved in the promoters of several yeast genes encoding glycolytic enzymes and occurs at positions –486 to –480 of the TDH3 promoter. This DNA sequence represents a novel promoter element: it contains no UAS activity itself, yet potentiates the activity of a GRF1 UAS. The potentiation of the GRFl UAS by this element occurs when placed upstream from the TATA box of either the TDH3 or CYC1 promoters. The characteristics of this element (termed GPE for GRF1 site potentiator element) indicate that it represents a binding site for a different yeast protein which increases the promoter activation mediated by the GRF1 protein. Site-specific deletion and promoter reconstruction experiments suggest that the entire activation potential of the –676 to –381 region of the TDH3 gene promoter may be accounted for by a combination of the GRF1 site and the GPE.     

Sturgeon glyceraldehyde-3-phosphate dehydrogenase.

The formation of binary complexes between sturgeon apoglyceralddhyde-3-phosphate dehydrogenase, coenzymes (NAD+ and NADH) and substrates (phosphate, glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate) has been studied spectrophotometrically and spectrofluorometrica-ly. Coenzyme binding to the apoenzyme can be characterized by several distinct spectroscopic properties: (a) the low intensity absorption band centered at 360 nm which is specific of NAD+ binding (Racker band); (b) the quenching of the enzyme fluorescence upon coenzyme binding; (c) the quenching of the fluorescence of the dihydronicotinamide moiety of the reduced coenzyme (NADH); (D) the hypochromicity and the red shift of the absorption band of NADH centered at 338 nm; (e) the coenzyme-induced difference spectra in the enzyme absorbance region. The analysis of these spectroscopic properties shows that up to four molecules of coenzyme are bound per molecule of enzyme tetramer. In every case, each successively bound coenzyme molecule contributes identically to the total observed change. Two classes of binding sites are apparent at lower temperatures for NAD+ Binding. Similarly, the binding of NADH seems to involve two distinct classes of binding sites. The excitation fluorescence spectra of NADH in the binary complex shows a component centered at 260 nm as in aqueous solution. This is consistent with a "folded" conformation of the reduced coenzyme in the binary complex, contradictory to crystallographic results. Possible reasons for this discrepancy are discussed. Binding of phosphorylated substrates and orthophosphate induce similar difference spectra in the enzyme absorbance region. No anticooperativity is detectable in the binding of glyceraldehyde 3-phosphate. These results are discussed in light of recent crystallographic studies on glyceraldehyde-3-phosphate dehydrogenases.     

Improvement of the transformation efficiency of Flammulina velutipes Fv-1 using the glyceraldehyde-3-phosphate dehydrogenase gene promoter

To improve the expression level of heterologous genes in Flammulina velutipes Fv-1, we constructed new vectors having glyceraldehydes-3-phosphate dehydrogenase (gpd) gene promoter to control the expression of target genes. When the hygromycin B phosphotransferase (hph) gene from Escherichia coli was controlled by the gpd promoter, transformation efficiency was 3-fold higher than the case of that controlled by the tryptophan synthetase gene (trp1) promoter.     

Ascorbate-induced oxidation of glyceraldehyde-3-phosphate dehydrogenase

Oxidation of the essential cysteins of glyceraldehyde-3-phosphate dehydrogenase into the sulfenic acid derivatives was observed in the presence of ascorbate, resulting in a decrease in the dehydrogenase activity and the appearance of the acylphosphatase activity. The oxidation was promoted by EDTA, NAD(+), and phosphate, and blocked in the presence of deferoxamine. The ascorbate-induced oxidation was suppressed in the presence of catalase, suggesting the accumulation of hydrogen peroxide in the conditions employed. The data indicate the metal-mediated mechanism of the oxidation due to the presence of metal traces in the reaction medium. Physiological importance of the mildly oxidized GAPDH is discussed in terms of its ability to uncouple glycolysis and to decrease the ATP level in the cell.