Compositional analysis of glyphosate-tolerant soybeans treated with glyphosate.

The compositional analyses and safety assessment of glyphosate-tolerant soybeans (GTS) were previously described. These analyses were extensive and included addressing the potential effects on seed composition from the genetic modification. Detailed compositional analyses established that GTS, which had not been treated with glyphosate, were comparable to the parental soybean line and to other conventional soybeans. In this study, two GTS lines, 40-3-2 and 61-67-1, were treated with commercial levels of glyphosate, the active ingredient in Roundup herbicide. The composition of the seed from soybeans sprayed with glyphosate was compared to that of a nonsprayed parental control cultivar, A5403. The nutrients measured in the seed included protein, oil, ash, fiber, carbohydrates, and amino acids. The concentration of isoflavones (also referred to as phytoestrogens) was also measured as these compounds are derived from the same biochemical pathway that was engineered for glyphosate tolerance. The analytical results from these studies demonstrate that the GTS soybeans treated with glyphosate were comparable to the parental soybean cultivar, A5403, and other conventional soybean varieties.

Metabolic engineering of Arabidopsis and Brassica for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production.

Poly(hydroxyalkanoates) are natural polymers with thermoplastic properties. One polymer of this class with commercial applicability, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) can be produced by bacterial fermentation, but the process is not economically competitive with polymer production from petrochemicals. Poly(hydroxyalkanoate) production in green plants promises much lower costs, but producing copolymer with the appropriate monomer composition is problematic. In this study, we have engineered Arabidopsis and Brassica to produce PHBV in leaves and seeds, respectively, by redirecting the metabolic flow of intermediates from fatty acid and amino acid biosynthesis. We present a pathway for the biosynthesis of PHBV in plant plastids, and also report copolymer production, metabolic intermediate analyses, and pathway dynamics.

PHA production, from bacteria to plants.

The genes encoding the polyhydroxyalkanoate (PHA) biosynthetic pathway in Ralstonia eutropha (3-ketothiolase, phaA or bktB; acetoacetyl-CoA reductase, phaB; and PHA synthase, phaC) were engineered for plant plastid targeting and expressed using leaf (e35S) or seed-specific (7s or lesquerella hydroxylase) promoters in Arabidopsis and Brassica. PHA yields in homozygous transformants were 12-13% of the dry mass in homozygous Arabidopsis plants and approximately 7% of the seed weight in seeds from heterozygous canola plants. When a threonine deaminase was expressed in addition to bktB, phaB and phaC, a copolyester of 3-hydroxybutyrate and 3-hydroxyvalerate was produced in both Arabidopsis and Brassica.

Metabolic modeling as a tool for evaluating polyhydroxyalkanoate copolymer production in plants.

The production of polyhydroxyalkanoates in plants is an interesting commercial prospect due to lower carbon feedstock costs and capital investments. The production of poly-(3-hydroxybutyrate) has already been successfully demonstrated in plant plastids, and the production of more complex polymers is under investigation. Using a mathematical simulation model this paper outlines the theoretical prospects of producing the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-3HV)] in plant plastids. The model suggests that both the 3HV/3HB ratio and the copolymer production rate will vary considerably between dark and light conditions. Using metabolic control analysis we predict that the beta-ketothiolase predominately controls the copolymer production rate, but that the activity of all three enzymes influence the copolymer ratio. Dynamic simulations further suggest that controlled expression of the three enzymes at different levels may enable desirable changes in both the copolymer production rate and the 3HV/3HB ratio. Finally, we illustrate that natural variations in substrate and cofactor levels may have a considerable impact on both the production rate and the copolymer ratio, which must be taken into account when constructing a production system.

Multiple beta-ketothiolases mediate poly(beta-hydroxyalkanoate) copolymer synthesis in Ralstonia eutropha.

Polyhydroxyalkanoates (PHAs) are a class of carbon and energy storage polymers produced by numerous bacteria in response to environmental limitation. The type of polymer produced depends on the carbon sources available, the flexibility of the organism's intermediary metabolism, and the substrate specificity of the PHA biosynthetic enzymes. Ralstonia eutropha produces both the homopolymer poly-beta-hydroxybutyrate (PHB) and, when provided with the appropriate substrate, the copolymer poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (PHBV). A required step in production of the hydroxyvalerate moiety of PHBV is the condensation of acetyl coenzyme A (acetyl-CoA) and propionyl-CoA to form beta-ketovaleryl-CoA. This activity has generally been attributed to the beta-ketothiolase encoded by R. eutropha phbA. However, we have determined that PhbA does not significantly contribute to catalyzing this condensation reaction. Here we report the cloning and genetic analysis of bktB, which encodes a beta-ketothiolase from R. eutropha that is capable of forming beta-ketovaleryl-CoA. Genetic analyses determined that BktB is the primary condensation enzyme leading to production of beta-hydroxyvalerate derived from propionyl-CoA. We also report an additional beta-ketothiolase, designated BktC, that probably serves as a secondary route toward beta-hydroxyvalerate production.

The composition of glyphosate-tolerant soybean seeds is equivalent to that of conventional soybeans.

One important aspect of the safety assessment of genetically engineered crops destined for food and feed uses is the characterization of the consumed portion of the crop. One crop currently under development, glyphosate-tolerant soybeans (GTS), was modified by the addition of a glyphosate-tolerance gene to a commercial soybean cultivar. The composition of seeds and selected processing fractions from two GTS lines, designated 40-3-2 and 61-67-1, was compared with that of the parental soybean cultivar, A5403. Nutrients measured in the soybean seeds included macronutrients by proximate analyses (protein, fat, fiber, ash, carbohydrates), amino acids and fatty acids. Antinutrients measured in either the seed or toasted meal were trypsin inhibitor, lectins, isoflavones, stachyose, raffinose and phytate. Proximate analyses were also performed on batches of defatted toasted meal, defatted nontoasted meal, protein isolate, and protein concentrate prepared from GTS and control soybean seeds. In addition, refined, bleached, deodorized oil was made, along with crude soybean lecithin, from GTS and control soybeans. The analytical results demonstrated the GTS lines are equivalent to the parental, conventional soybean cultivar.

The expressed protein in glyphosate-tolerant soybean, 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4, is rapidly digested in vitro and is not toxic to acutely gavaged mice.

The safety of 5-enolpyruvylshikimate-3-phosphate synthase enzyme derived from Agrobacterium sp. strain CP4 (CP4 EPSPS) was assessed. CP4 EPSPS is the only protein introduced by genetic manipulation that is expressed in glyphosate-tolerant soybeans, which are being developed to provide new weed-control options for farmers. Expression of this protein in plants imparts high levels of glyphosate tolerance. The safety of CP4 EPSPS was ascertained by evaluating both physical and functional characteristics. CP4 EPSPS degrades readily in simulated gastric and intestinal fluids, suggesting that this protein will be degraded in the mammalian digestive tract upon ingestion as a component of food or feed, There were no deleterious effects due to the acute administration of CP4 EPSPS to mice by gavage at a high dosage of 572 mg/kg body wt, which exceeds 1000-fold tha anticipated consumption level of food products potentially containing CP4 EPSPS protein. CP4 EPSPS does not pose any important allergen concerns because this protein does not possess characteristics typical of allergenic proteins. These data, in combination with seed compositional analysis and animal feeding studies, support the conclusion that glyphosate-tolerant soybean are as safe and nutritious as traditional soybeans currently being marketed.

The feeding value of soybeans fed to rats, chickens, catfish and dairy cattle is not altered by genetic incorporation of glyphosate tolerance.

Animal feeding studies were conducted with rats, broiler chickens, catfish and dairy cows as part of a safety assessment program for a soybean variety genetically modified to tolerate in-season application of glyphosate. These studies were designed to compare the feeding value (wholesomeness) of two lines of glyphosate-tolerant soybeans (GTS) to the feeding value of the parental cultivar from which they were derived. Processed GTS meal was incorporated into the diets at the same concentrations as used commercially; diary cows were fed 10 g/100 g cracked soybeans in the diet, a level that is on the high end of what is normally fed commercially. In a separate study, laboratory rats were fed 5 and 10 g unprocessed soybean meal 100 g diet. The study durations were 4 wk (rats and dairy cows), 6 wk (broilers) and 10 wk (catfish). Growth, feed conversion (rats, catfish, broilers), fillet composition (catfish), and breast muscle and fat pad weights (broilers) were compared for animals fed the parental and GTS lines. Milk production, milk composition, rumen fermentation and nitrogen digestibility were also compared for dairy cows. In all studies, measured variables were similar for animals fed both GTS lines and the parental line, indicating that the feeding value of the two GTS lines is comparable to that of the parental line. These studies support detailed compositional analysis of the GTS seeds, which showed no meaningful differences between the parental and GTS lines in the concentrations of important nutrients and antinutrients. They also confirmed the results of other studies that demonstrated the safety of the introduced protein, a bacterial 5-enolpyruvyl-shikimate-3-phosphate synthase from Agrobacterium sp. strain CP4.

Site-directed mutagenesis of a conserved region of the 5-enolpyruvylshikimate-3-phosphate synthase active site.

The active site of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) has been probed using site-directed mutagenesis and inhibitor binding techniques. Replacement of a specific glycyl with an alanyl or a prolyl with a seryl residue in a highly conserved region confers glyphosate tolerance to several bacterial and plant EPSPS enzymes, suggesting a high degree of structural conservation between these enzymes. The glycine to alanine substitution corresponding to Escherichia coli EPSPS G96A increases the Ki(app) (glyphosate) of petunia EPSPS 5000-fold while increasing the Km(app)(phosphoenolpyruvate) about 40-fold. Substitution of this glycine with serine, however, abolishes EPSPS activity but results in the elicitation of a novel EPSP hydrolase activity whereby EPSP is converted to shikimate 3-phosphate and pyruvate. This highly conserved region is critical for the interaction of the phosphate moiety of phosphoenolpyruvate with EPSPS.

Structure and topological symmetry of the glyphosate target 5-enolpyruvylshikimate-3-phosphate synthase: a distinctive protein fold.

5-enol-Pyruvylshikimate-3-phosphate synthase (EPSP synthase; phosphoenolpyruvate:3-phosphoshikimate 1-carboxyvinyltransferase, EC 2.5.1.19) is an enzyme on the pathway toward the synthesis of aromatic amino acids in plants, fungi, and bacteria and is the target of the broad-spectrum herbicide glyphosate. The three-dimensional structure of the enzyme from Escherichia coli has been determined by crystallographic techniques. The polypeptide backbone chain was traced by examination of an electron density map calculated at 3-A resolution. The two-domain structure has a distinctive fold and appears to be formed by 6-fold replication of a protein folding unit comprising two parallel helices and a four-stranded sheet. Each domain is formed from three of these units, which are related by an approximate threefold symmetry axis; in each domain three of the helices are completely buried by a surface formed from the three beta-sheets and solvent-accessible faces of the other three helices. The domains are related by an approximate dyad, but in the present crystals the molecule does not display pseudo-symmetry related to the symmetry of point group 32 because its approximate threefold axes are almost normal. A possible relation between the three-dimensional structure of the protein and the linear sequence of its gene will be described. The topological threefold symmetry and orientation of each of the two observed globular domains may direct the binding of substrates and inhibitors by a helix macrodipole effect and implies that the active site is located near the interdomain crossover segments. The structure also suggests a rationale for the glyphosate tolerance conferred by sequence alterations.

Arginine chemical modification of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase.

Reaction of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) with the arginine reagents phenylglyoxal (PGO) and p-hydroxyphenylglyoxal (HPGO) leads to inactivation of the enzyme. Inactivation with HPGO leads to modification of approximately 3 mol of arginine per mole of enzyme. The modification reaction follows pseudo-first-order kinetics with a t1/2 of 1 min at 5 mM p-hydroxyphenylglyoxal in 0.1 M triethanolamine HCl, pH 7.8. By titration of HPGO-modified enzyme with 5,5'-bis(dithio-2-nitrobenzoic acid), the possibility of cysteine modification by the arginine reagent was ruled out. While shikimate 3-phosphate (S3P) afforded partial protection to the enzyme against inactivation by HPGO, complete protection could be obtained by using a mixture of S3P and glyphosate. Under the latter conditions, only 1 mol arginine was modified per mole of enzyme. This pattern of reactivity suggests that two arginines may be involved in the binding of S3P and glyphosate to EPSP synthase. A third reactive arginine appears to be nonessential for EPSPS activity. Labeling of EPSP synthase with [14C]phenylglyoxal, peptic digestion, HPLC mapping, and amino acid sequencing indicate that Arg-28 and Arg-131 are two of the reactive arginines labeled with [14C]PGO.

Mechanism of the EPSP synthase catalyzed reaction: evidence for the lack of a covalent carboxyvinyl intermediate in catalysis.

In order to detect covalent reaction intermediates in the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase reaction, we have investigated the interaction of EPSP synthase with the reaction product EPSP. An exchange of EPSP-methylene protons could be demonstrated by incubating EPSPS with EPSP in D2O. Since trace amounts of contaminating Pi would lead to reversal of EPSPS reaction and hence methylene proton exchange, we added pyruvate kinase, ADP, Mg++ and K+. Under these conditions, any contaminating Pi that is converted to PEP is trapped as ATP. No exchange of EPSP protons with those of the solvent could be detected in the presence of this trap system, suggesting that enzyme-bound EPSP is unable to form a covalent tetrahedral complex. Incorporation of [14C] from [14C]-S3P and [14C]-PEP into EPSP could be detected, but only in the absence of a PEP (or Pi) trap system. This indicates that for the exchange reaction, Pi is required, and also indicates the absence of a covalent intermediate, unless the carboxyvinyl-enzyme-bound S3P is completely restricted from exchange.

Identification of the reactive cysteines of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase and their nonessentiality for enzymatic catalysis.

Reaction of 5-enolpyruvylshikimate-3-phosphate synthase of Escherichia coli with the thiol reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) leads to a modification of only 2 of the 6 cysteines of the enzyme, with a significant loss of its enzymatic activity. Under denaturing conditions, however, all 6 cysteines of 5-enolpyruvylshikimate-3-phosphate synthase react with DTNB, indicating the absence of disulfide bridges in the native protein. In the presence of shikimate 3-phosphate and glyphosate, only 1 of the 2 cysteines reacts with the reagent, with no loss of activity, suggesting that only 1 of these cysteines is at or near the active site of the enzyme. Cyanolysis of the DTNB-inactivated enzyme with KCN leads to elimination of 5-thio-2-nitrobenzoate, with formation of the thiocyano-enzyme. The thiocyano-enzyme is fully active; it exhibits a small increase in its I50 for glyphosate (6-fold) and apparent Km for phosphoenolpyruvate (4-fold) compared to the unmodified enzyme. Its apparent Km for shikimate 3-phosphate is, however, unaltered. These results clearly establish the nonessentiality of the active site-reactive cysteine of E. coli 5-enolpyruvylshikimate-3-phosphate synthase for either catalysis or substrate binding. Perturbations in the kinetic constants for phosphoenolpyruvate and glyphosate suggest that the cysteine thiol is proximal to the binding site for these ligands. By N-[14C]ethylmaleimide labeling, tryptic mapping, and N-terminal sequencing, the 2 reactive cysteines have been identified as Cys408 and Cys288. The cysteine residue protected by glyphosate and shikimate 3-phosphate from its reaction with DTNB was found to be Cys408.

Bacterial expression and isolation of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase.

5-enol-Pyruvylshikimate-3-phosphate synthase (EPSP synthase, EPSPS), an in vivo enzyme target of the herbicide glyphosate (N-phosphonomethyl glycine), was purified from a Petunia hybrida suspension culture line, MP4-G, by a small-scale high-performance chromatographic purification procedure. The cDNA encoding the mature petunia EPSPS (lacking the chloroplast transit sequence) was cloned into a plasmid, pMON342, for expression in Escherichia coli. This clone complemented the EPSPS deficiency of an E. coli aroA- mutant, and the plant enzyme constituted approximately 1% of the total extractable protein. Large-scale purification of the enzyme from E. coli cells resulted in a highly active protein which was homogeneous as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino terminal sequencing. Antibodies raised against the purified enzyme also reacted with the E. coli EPSPS in Western analyses. The availability of large quantities of the plant enzyme will significantly facilitate mechanistic investigations as well as a comparative study with EPSPS from bacteria and fungi.

Olefin oxygenation and N-dealkylation by dopamine beta-monooxygenase: catalysis and mechanism-based inhibition.

In an initial communication [May, S. W., Mueller, P. W., Padgette, S. R., Herman, H. H., & Phillips, R. S. (1983) Biochem. Biophys. Res. Commun. 110, 161-168], we reported that 1-phenyl-1-(aminomethyl)ethene hydrochloride (PAME) is an olefinic substrate for dopamine beta-monooxygenase (DBM; EC 1.14.17.1) which inactivates the enzyme in an apparent mechanism-based manner. The present study further characterizes this reaction. The inactivation reaction yields kinact = 0.23 min-1 at pH 5.0 and 37 degrees C and is strictly dependent on reductant (ascorbate) and oxygen. The DBM/PAME substrate reaction (apparent kcat = 14 s-1), shown to be stimulated by fumarate, gives the corresponding epoxide as product, identified by derivatization with 4-(p-nitrobenzyl)pyridine. However, the lack of DBM inhibition by alpha-methylstyrene oxide, and the observation of identical PAME/DBM inactivation rates in the absence and presence of preformed enzymatic PAME epoxide, indicates that free epoxide is not the inactivating species. A structure-activity study revealed that 4-hydroxylation of PAME (to give 4-HOPAME) increases both kinact (0.81 min-1) and apparent kcat (56 s-1) values, while 3-hydroxylation (to give 3-HOPAME) greatly diminishes inactivation activity while retaining substrate activity (apparent kcat = 47 s-1). 4-Hydroxy-alpha-methylstyrene was found to be a DBM inhibitor (kinact = 0.53 min-1) with weak substrate activity (apparent kcat = 0.71 s-1), while 3-hydroxy-alpha-methylstyrene and alpha-(cyanomethyl) styrene were found not to exhibit detectable DBM substrate activity and only weak inhibitory activity. 3-Phenylpropargylamine hydrochloride showed no detectable DBM substrate activity but rapidly inactivated the enzyme. A new substrate activity for DBM was discovered, N-dealkylation of N-phenylethylenediamine and N-methyl-N-phenylethylenediamine, and the lack of O-dealkylation activity with phenyl 2-aminoethyl ether and 4-hydroxyphenyl 2-aminoethyl ether indicates that DBM N-dealkylation proceeds via initial one-electron abstraction from the benzylic nitrogen heteroatom. With this new substrate and inhibitor reactivity information in hand, along with the other known substrate reactions, a DBM oxygenation mechanism analogous to that for cytochrome P-450 is proposed.

Antihypertensive activities of phenyl aminoethyl sulfides, a class of synthetic substrates for dopamine beta-hydroxylase.

Four sulfur-containing analogues of phenylpropylamine were synthesized and evaluated as substrates for dopamine beta-hydroxylase (DBH) and monoamine oxidase (MAO). All four phenyl aminoethyl sulfides were shown to be good substrates for DBH whereas only the two analogues not possessing a methyl group alpha to the terminal amino group were substrates for MAO. All four analogues were tested for acute antihypertensive activity in an animal model for hypertension, the spontaneously hypertensive rat (SHR). Two of the analogues, both of which should partition readily across the blood-brain barrier, did not appreciably reduce systemic blood pressure in the 6-h testing period. However, the two analogues that were designed to be relatively restricted to peripheral sites of action caused a dramatic drop in blood pressure in SHR of 25% within 1-1.5-h postinjection, with the analogue designed to be both restricted to the periphery and MAO inactive, causing a more prolonged antihypertensive activity.

Effects of phenyl-2-aminoethyl sulfide, a novel dopamine-beta-hydroxylase substrate, on the cardiovascular system of the anesthetized dog.

In previous work we have established that phenyl-2-aminoethyl sulfide (PAES) is a novel substrate for dopamine-beta-hydroxylase (DBH) which is stereospecifically oxygenated by the enzyme to the corresponding sulfoxide, (S)-phenyl-2-aminoethyl sulfoxide (PAESO). We now report that PAES possesses very little, if any, direct adrenergic agonist activity, but exhibits indirect sympathomimetic activity at relatively high doses (approximately 4 mg/kg). This assertion, that PAES is a new indirect sympathomimetic, is supported by our finding that pretreatment with cocaine completely abolishes the sympathomimetic activity of PAES. Furthermore, the effects of PAES are diminished with consecutive administration. In addition to its indirect sympathomimetic activity, we have also observed that PAES infusion almost completely blocks the reflex response elicited by hydralazine, a direct vasodilator. In contrast, we have found that PAESO possesses neither direct nor indirect sympathomimetic activity at doses as high as 6 mg/kg. Since PAES should be readily converted in vivo into PAESO, the implications of these findings in terms of potential antihypertensive action of PAES are discussed.

Dopamine-B-hydroxylase: suicide inhibition by the novel olefinic substrate, 1-phenyl-1-aminomethylethene.

Dopamine-B-hydroxylase [E.C.1.14.17.1] plays a key role in the biosynthetic interconversion of neurotransmitters. It is now demonstrated for the first time that dopamine-B-hydroxylase also catalyzes the oxygenation of an olefinic substrate, 1-phenyl-1-aminomethylethene, producing 2,3-dihydroxy-2-phenylpropylamine after acid workup. This reaction gives the normal oxygenase stoichiometry of electrons to O2 to product of 2:1:1, and is kinetically comparable to other oxygenase activities of dopamine-B-hydroxylase, with a kcat value of 10 sec-1 and a KM of 8.3 mM. 1-Phenyl-1-aminomethylethene is also a time-dependent, first-order inactivator of dopamine-B-hydroxylase. The inactivation process exhibits the characteristics of mechanism-based, irreversible inactivation, giving a KI value of 13 mM and a kinac of 0.04 min-1. The central role of dopamine-B-hydroxylase in catecholamine metabolism suggests possible pharmacological uses for olefinic inhibitors of this enzyme.