[Show abstract][Hide abstract] ABSTRACT: Soybeans (Glycine max) contain an α-galactosidase that makes up a small fraction of the total protein of the seed. The properties of this enzyme are of interest because of its potential to convert the galactooligosaccharides, stachyose and raffinose, in soybean meal to sugars digestible in the human gastro intestinal tract and thereby increase potential uses of this vegetable protein source in human and animal foods. Study of this enzyme required the isolation of milligram quantities of electrophoretically pure protein from ground soybeans and therefore, scaleup of laboratory procedures by a factor of 300 times. Large scale acid precipitation, ammonium sulfate precipitation, and centrifugal recovery of the precipitated protein allowed α-galactosidase to be isolated from 45.5 kg soybean meal containing 17.1 kg protein, to obtain an enzyme extract with a specific activity of 90 to 100. A novel combination of strong anion exchange and cation exchange chromatography followed by Concanavalin-A affinity chromatography with a methyl α-D mannoside gradient gave α-galactosidase with an average specific activity of 56,000. Ion exchange chromatography preceding Concanavalin-A affinity chromatography allowed elimination of a relatively costly melibiose affinity chromatography step (which followed the Concanavalin-A column In the laboratory procedure) thereby making scaleup practical.
[Show abstract][Hide abstract] ABSTRACT: A cDNA encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 18.104.22.168) from potato (Solanum tuberosum L.) presumably specifies a chloroplast transit sequence near its 5'-end. In order to show the function of this transit sequence, we constructed a plasmid that contains the entire coding region of the cDNA downstream from a T7 promoter. Using this plasmid as template, DAHP synthase mRNA was synthesized in vitro with T7 RNA polymerase. The resulting mRNA served as template for the in vitro synthesis of a 59-kDa polypeptide. This translation product was identified as the DAHP synthase precursor by immunoprecipitation with a monospecific polyclonal antibody raised against pure tuber DAHP synthase and by radiosequencing of the [(3)H]leucine-labeled translation product. Incubation of the 59-kDa polypeptide with isolated spinach (Spinacia oleracea L.) chloroplasts resulted in a 53-kDa polypeptide that was resistant to protease treatment. Fractionation of chloroplasts, reisolated after import, showed the mature DAHP synthase in the stroma fraction. Incubation of the 59-kDa polypeptide with a chloroplast precursor-processing enzyme cleaved the precursor between Ser49 and Ala50, generating a mature DAHP synthase of 489 residues. The uptake of the DAHP synthase precursor into isolated chloroplasts was inhibited by anti-DAHP synthase, and the precursor was not processed cotranslationally by canine microsomal membranes. We conclude that the transit sequence is able to direct DAHP synthase into chloroplasts.
[Show abstract][Hide abstract] ABSTRACT: The cDNA for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Arabidopsis encodes a polypeptide with an amino-terminal signal sequence for plastid import. A cDNA fragment encoding the processed form of the enzyme was expressed in Escherichia coli. The resulting protein was purified to electrophoretic homogeneity. The enzyme requires Mn(2+) and reduced thioredoxin (TRX) for activity. Spinach (Spinacia oleracea) TRX f has an apparent dissociation constant for the enzyme of about 0.2 microM. The corresponding constant for TRX m is orders of magnitude higher. In the absence of TRX, dithiothreitol partially activates the enzyme. Upon alkylation of the enzyme with iodoacetamide, the dependence on a reducing agent is lost. These results indicate that the first enzyme in the shikimate pathway of Arabidopsis appears to be regulated by the ferredoxin/TRX redox control of the chloroplast.
[Show abstract][Hide abstract] ABSTRACT: The shikimate pathway links metabolism of carbohydrates to biosynthesis of aromatic compounds. In a sequence of seven metabolic steps, phosphoenolpyruvate and erythrose 4-phosphate are converted to chorismate, the precursor of the aromatic amino acids and many aromatic secondary metabolites. All pathway intermediates can also be considered branch point compounds that may serve as substrates for other metabolic pathways. The shikimate pathway is found only in microorganisms and plants, never in animals. All enzymes of this pathway have been obtained in pure form from prokaryotic and eukaryotic sources and their respective DNAs have been characterized from several organisms. The cDNAs of higher plants encode proteins with amino terminal signal sequences for plastid import, suggesting that plastids are the exclusive locale for chorismate biosynthesis. In microorganisms, the shikimate pathway is regulated by feedback inhibition and by repression of the first enzyme. In higher plants, no physiological feedback inhibitor has been identified, suggesting that pathway regulation may occur exclusively at the genetic level. This difference between microorganisms and plants is reflected in the unusually large variation in the primary structures of the respective first enzymes. Several of the pathway enzymes occur in isoenzymic forms whose expression varies with changing environmental conditions and, within the plant, from organ to organ. The penultimate enzyme of the pathway is the sole target for the herbicide glyphosate. Glyphosate-tolerant transgenic plants are at the core of novel weed control systems for several crop plants.
[Show abstract][Hide abstract] ABSTRACT: The shikimate pathway, a collection of seven enzymatic reactions whose end product is chorismate, has been studied for many years in a variety of microorganisms and plants. In microbial systems, the end product of the pathway is used primarily for the synthesis of aromatic amino acids. In plants, chorismate is the precursor not only for the synthesis of aromatic amino acids, but also for many secondary metabolites with diverse physiological roles. Because of the dual involvement of the shikimate pathway in primary and secondary metabolism, there are long-standing questions regarding its subcellular location, and its potential coordinate regulation with other pathways that use chorismate or its products in response to environmental or developmental demands.
Trends in Plant Science 01/1997; · 11.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Potato (Solanum tuberosum L.) cells were transformed with an antisense DNA construct encoding part of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 22.214.171.124), the first enzyme of the shikimate pathway, to examine the role(s) of this protein in plant growth and development. Chimeric DNA constructs contained the transcript start site, the first exon, and part of the first intron of the shkA gene in antisense or sense orientations under the control of the cauliflower mosaic virus 35S promoter. Some, but not all, of the transgenic plants expressing antisense DAHP synthase RNA showed reduced levels of wound-induced DAHP synthase enzyme activity, polypeptide, and mRNA 12 and 24 h after wounding. No alteration in the wound induction of DAHP synthase gene expression was observed in transgenic potato tubers containing the chimeric sense construct. Reduced steady-state levels of DAHP synthase mRNA were observed in stem and shoot tip tissue. Some plants with the chimeric antisense construct had reduced stem length, stem diameter, and reduced stem lignification.
[Show abstract][Hide abstract] ABSTRACT: Tomato (Lycopersicon esculentum L. cv. UC82b) was found to contain two distinct 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase genes that are differentially expressed. The corresponding cDNAs were isolated and characterized. Both genes code for putative plastidic DAHP synthase isoforms. The deduced amino acid sequences are 79% identical. A comparison of the known Solanaceae DAHP synthases indicates two distinct conserved isoforms. The steady-state levels of transcripts of the two tomato genes differ in all organs analysed.
[Show abstract][Hide abstract] ABSTRACT: Expression of a potato (Solanum tuberosum L.) cDNA encoding 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway, complemented an Escherichia coli mutant devoid of the enzyme. While the potato cDNA encodes a protein with an amino terminal putative transit sequence for chloroplast import, immunocytochemistry localized the E. coli synthesized potato enzyme to the bacterial cytosol.
[Show abstract][Hide abstract] ABSTRACT: Soybeans contain the enzyme alpha-galactosidase, which hydrolyzes alpha-1, 6 linkages in stachyose and raffinose to give sucrose and galactose. We have found that galactose, a competitive product inhibitor of alpha-galactosidase, strongly promotes the heat stability of the tetrameric form of the enzyme at pH 4.0 and at temperatures of up to 70 degrees C for 60 min. Stachyose and raffinose also protect alpha-galactosidase from denaturation at pH 4.0 although to a lesser extent. Glucose and mannose have little effect. At pH 7.0 the enzyme is a monomer, and galactose has no effect on the heat stability of the enzyme. In the absence of heat protection of the enzyme by added sugars, a series deactivation mechanism was found to describe the deactivation data. In comparison, a unimolecular, non-first order deactivation model applies at pH 4.0, where heat protection effects were observed. At a temperature above 60 degrees C, simple deactivation is a suitable model. The results suggest that alpha-galactosidase conformation and heat stability are directly related.
Enzyme and Microbial Technology 09/1992; 14:609-13. · 2.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Potato (Solanum tuberosum L.) tubers contain two isoenzymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 126.96.36.199), the enzyme that catalyzes the first step of aromatic amino acid biosynthesis. One of the isoenzymes is specifically activated by Mn(2+), and the other requires Co(2+), Mg(2+), or another divalent cation for activity. Monospecific polyclonal antibodies against the Mn(2+)-activated isoenzyme do not cross-react with the other isoenzyme. Wounding of potato tubers induces the Mn(2+)-activated form but not the other. We conclude that two different genes encode two different isoenzymes that catalyze the first step in the shikimate pathway.
[Show abstract][Hide abstract] ABSTRACT: Light and fungal elicitor induce mRNA encoding 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase in suspension cultured cells of parsley (Petroselinum crispum L.). The kinetics and dose response of mRNA accumulation were similar for DAHP synthase and phenylalanine ammonia-lyase (PAL). Six micrograms of elicitor from Phytophthora megasperma f. glycinia gave a detectable induction within 1 hour. Induction of DAHP synthase and PAL mRNAs by light was transient, reaching maximal levels at 4 hours and returning to pretreatment levels after 24 hours. Our data suggest that either light or fungal elicitor transcriptionally activate DAHP synthase. A coordinate regulation for key enzymes in the synthesis of primary and secondary metabolites is indicated.
[Show abstract][Hide abstract] ABSTRACT: The levels of the tryptophan-sensitive isoenzyme of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Escherichia coli, encoded by the aroH gene, were elevated in tyrR and/or trpR mutants. The effect of tyrR and trpR lesions on aroH expression was confirmed by using a lacZ reporter system. The mutational elimination of either repressor led to a threefold increase in beta-galactosidase.
Journal of Bacteriology 07/1991; 173(12):3930-2. · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In Escherichia coli, genes aroF+, aroG+, and aroH+ encode isoenzymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that are feedback inhibited by tyrosine, phenylalanine, and tryptophan, respectively. A single base pair change in aroF causes a Pro-148-to-Leu-148 substitution and results in a tyrosine-insensitive enzyme.
Journal of Bacteriology 12/1990; 172(11):6581-4. · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Salmonella typhimurium aroF gene, encoding the tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, was localized to a chromosomal PstI fragment by Southern blotting with an Escherichia coli aroF probe. This fragment was cloned by screening a plasmid library for complementation of an E. coli aroF mutant. The nucleotide sequence of S. typhimurium aroF was determined and compared with its E. coli homolog. The nucleotide sequences are 85.1% identical, and the corresponding amino acid sequences are 96.1% identical. The E. coli genes encoding the three DAHP synthase isoenzymes are evolutionarily more distant from one another than are the homologous aroF genes of E. coli and S. typhimurium. The S. typhimurium aroF regulatory region contains three imperfect palindromes, two upstream of the promoter and one overlapping the promoter, that are nearly identical to operators aroFo1, aroFo2, and TyrR box 1 of E. coli. The aroFo1 and aroFo2 sequences of the two organisms are each separated by three turns of the DNA helix with no sequence similarity. The 5' ends of the aroF transcripts for both organisms contain untranslated regions with potential stem-loop structures. Translational fusions of the aroF regulatory regions to lacZ were constructed and then introduced in single copy into the E. coli chromosome. beta-Galactosidase assays for tyrR-mediated regulation of aroF-lacZ expression revealed that the E. coli TyrR repressor apparently recognizes the operators of both organisms with about equal efficiency.
Journal of Bacteriology 06/1990; 172(5):2259-66. · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: alpha-Galactosidase from soybean (Glycine max) was purified by a five-step procedure. The enzyme's natural substrates, raffinose and stachyose, have K(m)'s of 3. 0 mM and 4. 79 mM, respectively. The products, galactose and sucrose, were measured after separation by liquid chromatography. Galactose is a competitive product inhibitor of stachyose and raffinose hydrolysis with a K(i) of 0. 12 mM. We determined these parameters by an integral kinetic approach. Stachyose hydrolysis gives a nearly constant level of raffinose shortly after hydrolysis begins. Thus, cleavage of the first alpha-(1,6)-bond in the tetrasaccharide is the rate-limiting step. Since the stachyose hydrolysis yields raffinose, soybean alpha-galactosidase simultaneously hydrolyzes two substrates. We present a novel approach for analyzing simultaneous substrate hydrolysis with competitive product inhibition by a modified integral rate expression. The experimentally found kinetic parameters are confirmed by solving the simultaneous equations which describe the hydrolysis. This technique may be applicable to other hydrolytic enzymes with multiple substrates.
Biotechnology and Bioengineering 02/1990; 35(1):15-22. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A cDNA encoding potato (Solanum tuberosum L.) 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway, was cloned into phage lambda gt11. The clone represents the first cDNA for this enzyme from any eukaryotic source. The nucleotide sequence of the cDNA was determined, and its identity was confirmed through partial amino acid sequence analysis of the encoded enzyme. The cDNA contains a 1527-base pair open reading frame that encodes a polypeptide with a calculated molecular weight of 56,153. The amino terminus of the deduced polypeptide resembles a chloroplast transit sequence. Amino acid sequence identities between the mature potato enzyme and the homologous isoenzymes from Escherichia coli are only about 22%. The potato cDNA hybridized to various plant mRNAs that are all about 2 kilobases in size.
Journal of Biological Chemistry 02/1990; 265(3):1608-14. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A series of tobacco (Nicotiana tabacum L.) cell lines have been selected for growth in the presence of normally lethal concentrations of glyphosate, up to 20 mM. Activity of 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), which is inhibited by glyphosate, is elevated in tolerant cells. However, EPSPS isolated from tolerant cells is still inhibited by the herbicide. Increased activity of EPSPS is the result of increased levels of the enzyme in tolerant cells, as determined by immunoblotting, rather than a change in enzyme activity. RNA blots with a petunia EPSPS cDNA demonstrate a correlation between levels of tolerance and steady state level of EPSPS mRNA. Increased expression of EPSPS mRNA results from amplification of at least two genes encoding this enzyme in the tobacco genome. The degree of gene amplification also increases with level of glyphosate tolerance. Selected cells grown in the absence of glyphosate have an elevated EPSPS mRNA abundance and maintain some gene amplification, indicating that selection has resulted in a stable genetic modification.