Dehydrogenase GapA Research Articles

Improvement of NADPH bioavailability in Escherichia coli by replacing NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase GapA with NADP+-dependent GapB from Bacillus subtilis and addition of NAD kinase

Enzymatic synthesis of some industrially important compounds depends heavily on cofactor NADPH as the reducing agent. This is especially true in the synthesis of chiral compounds that are often used as pharmaceutical intermediates to generate the correct stereochemistry in bioactive products. The high cost and technical difficulty of cofactor regeneration often pose a challenge for such biocatalytic reactions. In this study, to increase NADPH bioavailability, the native NAD(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gapA gene in Escherichia coli was replaced with a NADP(+)-dependent gapB from Bacillus subtilis. To overcome the limitation of NADP(+) availability, E. coli NAD kinase, nadK was also coexpressed with gapB. The recombinant strains were then tested in three reporting systems: biosynthesis of lycopene, oxidation of cyclohexanone with cyclohexanone monooxygenase (CHMO), and an anaerobic system utilizing 2-haloacrylate reductase (CAA43). In all the reporting systems, replacing NAD(+)-dependent GapA activity with NADP(+)-dependent GapB activity increased the synthesis of NADPH-dependent compounds. The increase was more pronounced when NAD kinase was also overexpressed in the case of the one-step reaction catalyzed by CAA43 which approximately doubled the product yield. These results validate this novel approach to improve NADPH bioavailability in E. coli and suggest that the strategy can be applied in E. coli or other bacterium-based production of NADPH-dependent compounds.

Rational design of organelle compartments in cells

Rational design of organelle compartments in cells

Interaction of GapA with HPr and Its Homologue, Crh: Novel Levels of Regulation of a Key Step of Glycolysis inBacillus subtilis?

In Bacillus subtilis cells, we identified a new partner of HPr, an enzyme of the glycolysis pathway, the glyceraldehyde-3-phosphate dehydrogenase GapA. We showed that, in vitro, phosphorylated and unphosphorylated forms of HPr and its homologue, Crh, could interact with GapA, but only their seryl-phosphorylated forms were able to inhibit its activity.

Growth-phase dependent expression of the mevalonate pathway in a terpenoid antibiotic-producing Streptomyces strain.

Streptomyces griseolosporeus MF730-N6, a terpenoid antibiotic-terpentecin (Tp) producer, has both the nonmevalonate and mevalonate pathways for the formation of IPP. The Tp biosynthetic gene (ter) and the mevalonate pathway gene cluster (mev) including an HMG-CoA reductase gene (hmgr) had previously been cloned from strain MF730-N6. In this study, two distinct dxs genes (dxs 1 and dxs 2) and a dxr gene, which encode DXP synthases and DXP reductoisomerase, and participate in the initial and the second step of the nonmevalonate pathway, respectively, were cloned. These gene products were expressed in E. coli and confirmed to have the expected activities. The dxs 1, dxs 2, dxr, mev, and ter genes were used for Northern blot and primer extension analyses to examine temporal expression of these genes together with a gap gene coding for GAP dehydrogenase, which was also cloned in this study and used as an internal control. Transcripts of the dxs 1, dxs 2, dxr, and gap genes were detected throughout the cultivation. On the other hand, messages of the mev and ter genes were not detected at early growth phase but appeared when Tp production started. These results suggested that the nonmevalonate pathway and the mevalonate pathway were mainly used for the primary metabolism and the secondary metabolism, respectively, and that both of the two dxs genes were actually transcribed in this strain.

Cloning, partial sequencing, and expression of glyceraldehyde-3-phosphate dehydrogenase gene in chick embryonic heart muscle cells.

Two recombinant plasmids containing structural gene sequences of chick embryonic heart glyceraldehyde-3-phosphate dehydrogenase (GAP dehydrogenase) were constructed and characterized. The plasmids pGAP 30 and pGAP 36 have inserts of 1200 and 950 base pairs, respectively. The identity of the clones was established by hybrid-arrested and hybrid-selection translation assays, and by immunoprecipitation of hybrid-selected translation product with GaP dehydrogenase antiserum. Hybridization of labeled pGAP 30 DNA to size-fractionated chick heart poly(A) RNA occurred at the region on the gel corresponding to the mobility of GAP dehydrogenase mRNA. Base sequence analysis of plasmid pGAP 30 and the comparison of the amino acid sequence derived from it with that of pig muscle GAP dehydrogenase revealed that the amino acid sequence of GAP dehydrogenase is strictly conserved between the chick and pig muscle tissues. Expression of GAP dehydrogenase mRNA in developing chick heart cells in cultures was monitored by in situ hybridization. The GAP dehydrogenase mRNA was present in 5-h-old dividing myoblasts, in contrast to mRNAs specific for contractile proteins, which appear late in myoblast development paralleling morphogenetic differentiation of myoblasts into myocytes (Jakowlew, S. B., Khandekar, P., Datta, K., Narula, S. K., Arnold, H. H., and Siddiqui, M. A. Q. (1982) J. Mol. Biol. 156, 673-682).

CO2 fixation and its regulation in Anacystis nidulans (Synechococcus)

Anacystis nidulans (Synechococcus) had a minimal doubling time of 5 hrs at 30 degrees C at saturating light intensity and carbon dioxide concentration. Half maximal growth rates in saturating CO2 occured at a light intensity of 0.54 mW per cm2, and there was an apparent threshold intensity of 0.13 mW per cm2 below which no growth occurred. Growth rate in saturating light was dependent on the concentration of CO2+H2CO3 in the medium, rather than on total dissolved CO2; half maximal rates were estimated at 0.1 mM CO2+H2CO3. Under saturating conditions of light and CO2, 14CO2 was fixed primarily into 3-PGA, and subsequently moved into sugar phosphates and amino acids. Incorporation into aspartate was relatively slow. CO2 fixation was strictly light-dependent. The changes in adenylate and pyridine nucleotide pools were followed in light/dark and dark/light transitions. Whereas adenylates relaxed slowly over 15-20 min to the concentrations characteristic of illuminated cells following the abrupt changes induced by darkening, the sharp drop in intracellular NADPH showed little dark recovery although rapid restoration occurred on reillumination. Other pyridine nucleotides showed no changes during these transitions. The nucleotide specificity and Km of partially purfied GAP dehydrogenase suggest a role for this enzyme in the regulation of CO2 fixation.

Dehydrogenase in carbohydrate metabolism in larvae of the silkworm, Bombyx mori L.

The dehydrogenases involved in both glycolytic and pentose phosphate pathways were studied with larval tissues of the silkworm, Bombyx mori L. The highest activity of G-6-P dehydrogenase and 6-PG dehydrogenase was found in the fat body. The purification of these two enzymes was partially carried out by precipitating with ammonium sulphate and successively by elution chromatography on a DEAE-cellulose column. The optimal pH for G-6-P dehydrogenase and 6-PG dehydrogenase was 7·7 and 8·3, respectively. The K m value of G-6-P dehydrogenase was 8·0 × 10 −5M and 3·1 × 10 −5M for G-6-P and NADP, respectively, and the K m value of 6-PG dehydrogenase was 7·0 × 10 −5M and 1·5 × 10 −5M for 6-PG and NADP, respectively. The midgut possessed high lactic dehydrogenase activity and the muscle moderate activity, whereas almost no activity was found in other tissues tested. The optimal pH for lactic dehydrogenase was 7·6, and the K m value of the enzyme for pyruvate was 1·0 × 10 −4M. The activity of lactic dehydrogenase was completely inhibited by 10 −4M of p-CMB. The activity of α-GP dehydrogenase was determined in larval tissues, and the ratio of α-GP dehydrogenase activity to lactic dehydrognase activity was obtained in several tissues. The results indicated that ratio varied specifically among different tissues. The occurrences of aldolase, phosphoglycerate kinase, and GAP dehydrogenase were also recognized in larval tissues.

Studies on the metabolism of borate. IV. Effect of borate on glyceraldehydephosphate dehydrogenase.

1. It was found that fructose 1, 6-diphosphate and fructose 6-monophosphate were accumulated by borate in anaerobic glycolysis system of liver homogenates of guinea pigs. 2. Glyceraldehydephosphate dehydrogenase*2 (GAP dehydrogenase) from yeast and in liver homogenates of guinea pigs and rats were found to be inhibited by borate. The inhibition was observed in both cases in which free D-glyceraldehyde or D-glyceraldehyde 3-phosphate was used as substrate. 3.GAP dehydrogenase was inhibited competitively by borate, and the inhibition was reversed by dialysis, but not reversed by excess sugars or polyhydroxy compounds in the experimental condition. 4. Borate has no effect on lactate dehydrogenase*6 from rabbit muscle and also in liver homogenates of guinea pigs. 5. Toxicity of borate was discussed from biochemical point of view.

Intermediary carbohydrate metabolism of Triatoma infestans (insecta; hemiptera)—I. Glycolytic and pentose phosphate pathway enzymes and the effect of DDT

1. (1) Cell-free preparations obtained from thoraces and legs of T. infestans glycolyse glucose, as well as HDP, with the production of pyruvate and α-glycerophosphate in equimolar amounts in the proportion of 1 mole of pyruvate and 1 mole of α-glycerophosphate per mole of glucose utilized. Added HDP is necessary for optimal activity, but the effect of its omission can be partially reversed by increasing the concentration of added ATP. Fluoride is not required for glucose phosphorylation. 2. (2) Cell-free extracts contain enzymes for glycolysis. GAP dehydrogenase is markedly sensitive to iodoacetate and shows appreciable activity in the absence of added l-cysteine. Lactic dehydrogenase activity is negligible, which explains the non-accumulation of lactate as an end-product of glycolysis. 3. (3) The presence of the pentose phosphate pathway in T. infestans is indicated by the presence of several reactions of this pathway in cell-free extracts; 6-PG dehydrogenase and probably G-6-P dehydrogenase appear to be rate-limiting enzymes for carbohydrate degradation through the pentose pathway. 4. (4) The enzyme content of cell-free extracts is related to the developmental stage of the insect and also to the sex. 5. (5) In vivo topically applied DDT (300 μg per specimen) inhibits the glycolytic pyruvate production by cell-free preparations of male and nymph specimens after 22 hr of exposure. Pyruvate production is restored to normal after 48 hr of exposure, but probably some degree of inhibition of α-glycerophosphate production persist in male specimens. This effect is shared by DDE, a non-toxic DDT-analog. 6. (6) Hexokinase, G-6-P dehydrogenase, 6-PG dehydrogenase, aldolase, GAP dehydrogenase, Gly-P dehydrogenase, phosphopyruvic kinase and ribolytic activity are inhibited by DDT in male specimens after 48 hr of exposure to DDT. Phosphopentose isomerase apparently is not inhibited. On the other hand, only G-6-P dehydrogenase and aldolase are inhibited in nymph specimens by DDT.