Purification and characterization of 3-dehydroquinate hydrolase and shikmate oxidoreductase. Evidence for a bifunctional enzyme

The basis for the physical association of 3-dehydroquinate dehydratase (3-dehydroquinate hydrolyase, EC 4.2.1.10) and shikimate dehydrogenase (shikimate: NADP+ 3-oxidoreductase, EC 1.1.1.25) in higher plants was investigated. The enzymes were extracted from the moss Physcomitrella patens and were purified to homogeneity. Determinations of subunit sizes were made by sodium dodecyl sulfate gel electrophoresis and gel exclusion chromatography in 6 M guanidinium chloride. Results from these studies demonstrate that both enzyme activities are carried out by a single polypeptide.     

Aggregation and separability of the shikimate pathway enzymes in yeasts

Gel filtration was employed to estimate the molecular weights and to determine possible physical aggregation of enzymes [5-dehydroquinate synthase (DHQ synthase), 5-dehydroquinase (DHQase, EC 4.2.1.10), shikimate: NADP oxidoreductase (EC 1.1.1.25), shikimate kinase (EC 2.7.1.71), 3-enolpyruvylshikimate 5-phosphate synthase (EPSP synthase)] in the shikimate pathway in eleven species of yeasts. The five enzymes were not aggregated in extracts of Hansenula henricii, H. fabianii, H. anomala, Candida utilis, Pichia guilliermondii, and Lodderomyces elongisporus. Two enzymes (DHQase and shikimate:NADP oxidoreductase) were not separable by this method and by ion exchange chromatography, and we conclude that they exist as an aggregate in these yeasts. Evidence is presented for an enzyme aggregate containing five activities, with a molecular weight of approximately 280,000 in Rhodosporidium spaerocarpum, Rh. toruloides, Rhodotorula rubra, Saccharomycopsis lipolytica, and Saccharomyces cerevisiae. Similarities between the enzymes in the shikimate pathway of plants, bacteria, and other fungi and those of investigated yeasts are discussed.     

Subcellular localization of the common shikimate-pathway enzymes in Pisum sativum L.

5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19), 3-dehydroquinate dehydratase (EC 4.2.1.10) and shikimate: NADP⁺ oxidoreductase (EC 1.1.1.25) were present in intact chloroplasts and root plastids isolated from pea seedling extracts by sucrose and modified-silica density gradient centrifugation. In young (approx. 10-d-old) seedling shoots the enzymes were predominantly chloroplastic; high-performance anion-exchange chromatography resolved minor isoenzymic activities not observed in density-gradientpurified chloroplasts. The initial enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was also associated with intact density-gradient-purified chloroplasts. 3-Dehydroquinate synthase (EC 4.6.1.3) and shikimate kinase (EC 2.7.1.71) were detected together with the other pathway enzymes in stromal preparations from washed chloroplasts. Plastidic EPSP synthase was inhibited by micromolar concentrations of the herbicide glyphosate.Abbreviations DAHP 3-deoxy-d-arabino-heptulosonate 7-phosphate - DEAE diethylaminoethyl - DHQase 3-dehydroquinate dehydratase - DTT dithiothreitol - EPSP 5-enolpyruvylshikimate 3-phosphate - SORase shikimate:NADP⁺ oxidoreductase     

Alicyclic acid metabolism in plants 8. Association, of two enzymes of the shikimate pathway in shoots of Phaseolus mungo seedlings

The association of two enzymes involved in the shikimate pathway,3-dehydroquinate hydro-lyase (EC 4.2.1.10 [EC] ) and shikimate: NADPoxidoreductase (EC 1.1.1.25 [EC] ), was studied with shoots of etiolated4-day-old Phaseolus mungo seedlings. The enzymes were not separableby ammonium sulfate fractionation, sucrose density gradientcentrifugation, polyacrylamide gel electrophoresis and chromatographyon Sephadex G-100 and DEAE-Sephadex A-50. The results are discussedin relation to the channelling function of metabolites in thealicyclic acid metabolism in higher plants. (Received October 28, 1975; )     

2. Alicyclic acid metabolism in plants Enzymes related to the shikimate pathway in developing mung bean seedlings

A study was made of chages in the activities of enzymes relatedto the biosynthesis of aromatic compounds in etiolated mungbean seedlings during their growth. Shikimate: NADP oxidoreductaseactivity in the root-shoot axes increased rapidly to attainits highest activity the 4th day after sowing, and remainedat that level over the experimental period of 7 days. 5-Dehydroquinatehydro-lyase activity continuously increased for at least 7 days.In the cotyledons, a gradual decrease in the activities of theseenzymes occurred. Phenylalanine ammonia-lyase activity in root-shootaxes gradually increased showing a maximum on the 6th day. Thehighest specific activity, on a protein basis, of this enzymewas seen in the initial stage of growth. In the cotyledons,a rise in total activity appeared on the 2nd day. Tyrosine ammonia-lyaseactivity was very low as compared with phenylalanine ammonia-lyase.The enzyme activities of light-germinated seedlings were comparedto those of dark-germinated seedlings on the 7th day. Lighthad practically no significant effect on the appearance of shikimate:NADP oxidoreductase and 5-dehydroquinate hydro-lyase activities.On the other hand, a marked effect from the light on the riseof phenylalanine ammonia-lyase and tyrosine ammonia-lyase activitieswas found, especially in the epicotyl-plumules. The results are discussed with respect to the metabolism ofalicyclic acids such as shikimic acid in the developing mungbean seedlings. ¹This work was partly supported by a grant-in-aid from the Ministryof Education.     

Association of the quinate : NAD+ oxidoreductase with one dehydroquinate hydro-lyase isoenzyme in corn seedlings

A quinate : NAD⁺ oxidoreductase has been purified from cornseedlings. This enzyme co-migrates with a dehydroquinate hydro-lyaseisoenzyme whatever separation technic is used. This is strongevidence that the two activities are associated in an enzymecomplex or in a bifunctional enzyme, in addition to the previouslycharacterized association of the shikimate : NADP⁺ oxidoreductaseand another dehydroquinate hydro-lyase isoenzyme. The purifiedquinate : NAD⁺ oxidoreductase has a poor affinity for quinicacid and is only active in the presence of NAD⁺. The associateddehydroquinate hydro-lyase isoenzyme is strongly activated invitro by shikimic acid in a pH-dependent process. The possible role of this new association is discussed in thelight of previous results from alicyclic metabolism studiesin plants and microorganisms. (Received July 18, 1980; )     

Association of the quinate : NAD+ oxidoreductase with one dehydroquinate hydro-lyase isoenzyme in corn seedlings

A quinate : NAD⁺ oxidoreductase has been purified from cornseedlings. This enzyme co-migrates with a dehydroquinate hydro-lyaseisoenzyme whatever separation technic is used. This is strongevidence that the two activities are associated in an enzymecomplex or in a bifunctional enzyme, in addition to the previouslycharacterized association of the shikimate : NADP⁺ oxidoreductaseand another dehydroquinate hydro-lyase isoenzyme. The purifiedquinate : NAD⁺ oxidoreductase has a poor affinity for quinicacid and is only active in the presence of NAD⁺. The associateddehydroquinate hydro-lyase isoenzyme is strongly activated invitro by shikimic acid in a pH-dependent process. The possible role of this new association is discussed in thelight of previous results from alicyclic metabolism studiesin plants and microorganisms. (Received July 18, 1980; )     

Gene-enzyme relationships of the arom aggregate of Schizosaccharomyces pombe

The gene-enzyme relationships of the arom multienzyme complex of Schizosaccharomyces pombe that catalyzes steps two through six in the prechorismate polyaromatic amino acid biosynthetic pathway have been studied. The various mutants were subjected to biochemical analysis by direct enzymic assays. These studies have established that aro-3A, aro-3B, aro-3C, aro-3D, and aro-3E mutants lack, respectively, the enzymic activities 5-dehydroquinate synthase, 5-dehydroquinase, shekimate kinase, 3-enolpyruvylshikimate 5-phosphate synthase, and shikimate: NADP oxidoreductase. In S. pombe lack enzymic activities for the inducible quinate catabolic pathway. The functional significance of the arom aggregate is discussed.     

Heteromerous interactions among glycolytic enzymes and of glycolytic enzymes with F-actin: effects of poly(ethylene glycol)

Interactions of glucose-6-phosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9), aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12), triose-phosphate isomerase (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1), phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1), phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.3), enolase (2-phospho-D-glycerate hydro-lyase, EC 4.2.1.11), pyruvate kinase (ATP:Pyruvate O2-phosphotransferase, EC 2.7.1.40) and lactate dehydrogenase [S)-lactate:NAD+ oxidoreductase, EC 1.1.1.27) with F-actin, among the glycolytic enzymes listed above, and with phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) were studied in the presence of poly(ethylene glycol). Both purified rabbit muscle enzymes and rabbit muscle myogen, a high-speed supernatant fraction containing the glycolytic enzymes, were used to study enzyme-F-actin interactions. Following ultracentrifugation, F-actin and poly(ethylene glycol) tended to increase and KCl to decrease the pelleting of enzymes. In general, the greater part of the pelleting occurred in the presence of both F-actin and poly(ethylene glycol) and the absence of KCl. Enzymes that pelleted more in myogen preparations than as individual purified enzymes in the presence of poly(ethylene glycol) and the absence of F-actin were tested for specific enzyme-enzyme associations, several of which were observed. Such interactions support the view that the internal cell structure is composed of proteins that interact with one another to form the microtrabecular lattice.     

Enzymes of ammonia assimilation in Rhizobium leguminosarum bacteroids.

The activities of the following enzymes were studied in connection with dinitrogen fixation in pea bacteroids: glutamine synthetase(L-glutamate: ammonia ligase (ADP-forming)(EC 6.3.1.2)(GS); glutamate dehydrogenase (NADP+)(L-glutamate: NADP+ oxidoreductase (deaminating)(EC 1.4.1.4)(GDH); glutamate synthase (L-glutamine: 2-exeglutarate aminotransferase (NADPH-oxidizing))(EC 2.6.1.53)(GOGAT). GS activity was high throughout the growth of the plant and GOGAT activity was always low. It is unlikely that GDH or the GS-GOGAT pathway can account for the incorporation of ammonia from dinitrogen fixation in the pea bacteroid,     

Purification and properties of two multiple forms of dihydrodiol dehydrogenase from guinea-pig testis

NADP+-dependent dihydrodiol dehydrogenase (trans-1,2-dihydrobenzene-1,2-diol: NADP+ oxidoreductase, EC 1.3.1.20) activity in the cytosol of guinea-pig testis was separated into two major and two minor peaks by Q-Sepharose chromatography; one minor form was immunologically cross-reacted with hepatic aldehyde reductase. The two major enzyme forms were purified to homogeneity. One form, which had the highest amount in the tissue, was a monomeric protein with a molecular weight of 32,000 and isoelectric point of 4.2, showed strict specificity for benzene dihydrodiol and NADP+, and reduced pyridine aldehydes, glyceraldehyde and diacetyl at low rates. Another form, with a molecular weight of 36,000 and isoelectric point of 5.0, oxidized n-butanol, glycerol and sorbitol as well as benzene dihydrodiol in the presence of NADP+ or NAD+, and exhibited much higher reductase activity towards various aldehydes, aldoses and diacetyl. The pI 5.0 form was more sensitive to inhibition by sorbinil and p-chloromercuriphenyl sulfonate than the pI 4.2 form and was activated by sulfate ion. The two enzymes did not catalyze the oxidation of hydroxysteroids and xenobiotic alicyclic alcohols and were immunologically different from hepatic 17 beta-hydroxysteroid-dihydrodiol dehydrogenase. The results indicate that guinea-pig testis contains at least two dihydrodiol dehydrogenases distinct from the hepatic enzymes, one of which, the pI 5.0 enzyme form, may be identical to aldose reductase.     

The levels of nicotinamide nucleotides in liver microsomes and their possible significance to the function of hexose phosphate dehydrogenase.

The concentrations of NAD and NADP have been determined in detergent extracts of washed rat liver microsomes. Precautions were taken during the preparation of the microsomes to remove nicotinamide nucleotides from their external surface both by hydrolysis by nucleotide pyrophosphatase (EC 3.6.1.9) and by washing them three times in 0.15 M-Tris/HCl, pH 8.0, to remove soluble proteins which bind these nucleotides. The mannose phosphatase was essentially completely latent, indicating that the microsomes were intact. Assuming these nucleotides are in the cisternae of the microsomes, the concentrations in the cisternae are 240 +/- 25 microM-NAD and 55 +/- 12 microM-NADP. These levels of nucleotides are compatible with both the glucose:NAD+ and the glucose 6-phosphate:NADP+ oxidoreductase activities of hexose phosphate dehydrogenase (EC 1.1.1.47). Since the organ and subcellular distributions of this dehydrogenase and glucose-6-phosphatase are similar, and Pi stimulates the glucose:NAD+ oxidoreductase activity, it is proposed that the combined action of these two enzymes leads to the reduction of both coenzymes in the lumen of the endoplasmic reticulum. A modification of the colorimetric method of Nisselbaum & Green [(1969) Anal. Biochem. 27, 212-217] for the determination of NADP+ is described. Colour formation is linear with the concentration of NADP+ and is sensitive to less than 0.3 nmol of NADP+.     

Methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase and formyltetrahydrofolate synthetase from porcine liver. Isolation of a dehydrogenase-cyclohydrolase fragment from the multifunctional enzyme

Tryptic digestion of a multifunctional enzyme from porcine liver containing methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.5), methenyltetrahydrofolate cyclohydrolase (5,10-methenyltetrahydrofolate 5-hydrolase, EC 3.5.4.9) and formyltetrahydrofolate synthetase (formate:tetrahydrofolate ligase, EC 6.3.4.3) activities destroys the synthetase. A fragment containing both dehydrogenase and cyclohydrolase activities has been isolated by affinity chromatography on an NADP+-Sepharose affinity column. The purified fragment is homogeneous on dodecyl sulfate-polyacrylamide gel electrophoresis where its molecular weight was determined as 33 000 +/- 1200 compared with 100 000 for the undigested protein. The cyclohydrolase activity retains sensitivity to inhibition by NADP+, MgATP and ATP.     

Aromatic amino acid metabolism during organogenesis in rice callus cultures

The activity during root and shoot initiation of key enzymes involved in aromatic amino acid metabolism was examined in rice ( Oryza sativa L. cv. Bala) callus cultures. Increased activities of the enzymes quinate:NAD⁺ oxidoreductase (EC 1.1.1.24), shikimate kinase (EC 2.7.1.71), chorismate mutase (EC 5.4.99.5), anthranilate synthase (EC 4.1.3.27) and tryptophan synthetase (EC 4.2.1.20) were noticed in organ-forming callus compared to proliferating callus of rice, especially prior to the visible manifestation of form. These results suggest a correlation between organogenesis and the aromatic amino acid pathway.     

Purification and properties of NADP-dependent shikimate dehydrogenase from Gluconobacter oxydans IFO 3244 and its application to enzymatic shikimate production

NADP-Dependent shikimate dehydrogenae (SKDH, EC 1.1.1.25) was purified from Gluconobacter oxydans IFO 3244. SKDH showed a single protein band on native-PAGE accompanying enzyme activity. It required NADP exclusively and catalyzed only the shuttle reaction between shikimate and 3-dehydroshikimate. The optimum pH for shikimate oxidation and 3-dehydroshikimate reduction was found at pH 10 and 7 respectively. SKDH proved to be a useful catalyst for shikimate production from 3-dehydroshikimate.     

Isolation and characterization of pig kidney mitochondrial ferredoxin:NADP+ oxidoreductase

A two-step affinity chromatography procedure, using 2',5'-ADP-agarose and adrenodoxin-Sepharose 4B affinity supports, was used to purify mitochondrial ferredoxin:NADP+ oxidoreductase (EC 1.18.1.2, formerly EC 1.6.7.1) from pig kidney. The 450:270 nm absorbance ratio of the enzyme was 0.128, and it had a specific activity of 16,305 nmol/min/mg for the reduction of cytochrome c. The mitochondrial enzyme was a monomer which contained one molecule of FAD and had calculated molecular masses of 51,500 and 48,000 daltons when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography gel exclusion chromatography, respectively. The porcine enzyme had a Km for NADPH of 0.94 microM and it expressed maximal activity when coupled with its homologous ferredoxin, although it was also active with the heterologous ferredoxin from bovine adrenal. The purified ferredoxin:NADP+ oxidoreductase supported the in vitro reduction of membrane-bound adrenal mitochondrial P-450, and it was demonstrated from immunologic studies that the enzyme shares some common epitopes with bovine adrenodoxin:NADP+ oxidoreductase.     

Modification of arginyl residues in ferredoxin-NADP+ reductase from spinach leaves.

Reaction of spinach leaves ferredoxin-NADP+ reductase (NADPH:ferredoxin oxidoreductase, EC 1.6.7.1) with alpha-dicarbonyl compounds results in a biphasic loss of activity. The rapid phase yields modified enzyme with about 30% of the original activity, but no change in the Km for NADPH. Only partial protection against inactivation is provided by NADP+, NADPH and their analogs, whereas ferredoxin affords complete protection. The reductase inactivated to 30% of original activity shows a loss of about two arginyl residues, whereas only one residue is lost in the NADP+-protected enzymes. The data suggest that the integrity of at least two arginyl residues are requested for maximal activity of ferredoxin-NADP+ reductase: one residue being located near the NADP+-binding site, the other presumably situated in the ferredoxin-binding domain.     

A thermostable shikimate 5-dehydrogenase from the archaeon Archaeoglobus fulgidus

Shikimate 5-dehydrogenase (SKDH; EC 1.1.1.25) catalyzes the reversible reduction of 3-dehydroshikimate to shikimate and is a key enzyme in the aromatic amino acid biosynthesis pathway. The shikimate 5-dehydrogenase gene, aroE, from Archaeoglobus fulgidus was cloned and overexpressed in Escherichia coli. The recombinant enzyme purified as a homodimer and yielded a maximum specific activity of 732 U/mg at 87 degrees C (with NADP+ as coenzyme). Apparent Km values for shikimate, NADP+, and NAD+ were estimated at 0.17+/-0.03 mM, 0.19+/-0.01 mM, and 11.4+/-0.4 mM, respectively. The half-life of the A. fulgidus SKDH is 2 h at the assay temperature (87 degrees C) and 17 days at 60 degrees C. Addition of 1 M NaCl or KCl stabilized the enzyme's half-life to approximately 70 h at 87 degrees C and approximately 50 days at 60 degrees C. This work presents the first kinetic analysis of an archaeal SKDH.     

Purification and Properties of NADP-Dependent Shikimate Dehydrogenase from Gluconobacter oxydans IFO 3244 and Its Application to Enzymatic Shikimate Production

NADP-Dependent shikimate dehydrogenae (SKDH, EC 1.1.1.25) was purified from Gluconobacter oxydans IFO 3244. SKDH showed a single protein band on native-PAGE accompanying enzyme activity. It required NADP exclusively and catalyzed only the shuttle reaction between shikimate and 3-dehydroshikimate. The optimum pH for shikimate oxidation and 3-dehydroshikimate reduction was found at pH 10 and 7 respectively. SKDH proved to be a useful catalyst for shikimate production from 3-dehydroshikimate.     

Inactivation of carbonyl reductase from human brain by phenylglyoxal and 2,3-butanedione: a comparison with aldehyde reductase and aldose reductase

Aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2), aldose reductase (alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21) and carbonyl reductase (secondary-alcohol:NADP+ oxidoreductase, EC 1.1.1.184) constitute the enzyme family of the aldo-keto reductases, a classification based on similar physicochemical properties and substrate specificities. The present study was undertaken in order to obtain information about the structural relationships between the three enzymes. Treatment of human aldehyde and carbonyl reductase with phenylglyoxal and 2,3-butanedione caused a complete and irreversible loss of enzyme activity, the rate of loss being proportional to the concentration of the dicarbonyl reagents. The inactivation of aldehyde reductase followed pseudo-first-order kinetics, whereas carbonyl reductase showed a more complex behavior, consistent with protein modification cooperativity. NADP+ partially prevented the loss of activity of both enzymes, and an even better protection of aldehyde reductase was afforded by the combination of coenzyme and substrate. Aldose reductase was partially inactivated by phenylglyoxal, but insensitive to 2,3-butanedione. The degree of inactivation with respect to the phenylglyoxal concentration showed saturation behavior. NADP+ partially protected the enzyme at low phenylglyoxal concentrations (0.5 mM), but showed no effect at high concentrations (5 mM). These findings suggest the presence of an essential arginine residue in the substrate-binding domain of aldehyde reductase and the coenzyme-binding site of carbonyl reductase. The effect of phenylglyoxal on aldose reductase may be explained by the modification of a reactive thiol or lysine rather than an arginine residue.