Adenylosuccinate synthetase from maize. Purification, properties, and mechanism of inhibition by 5'-phosphohydantocidin.

Adenylosuccinate synthetase (AdSS) is the site of action hydantocidin, a potent microbial phytotoxin. A kinetic analysis of the mode of inhibition of a plant adenylosuccinate synthetase by the active metabolite 5'-phosphohydantocidin (5'-PH) was the objective of the present study. AdSS was purified 5800-fold from maize (Zea mays), to our knowledge the first purification of the enzyme from a plant source. N-terminal sequencing established the cleavage site of the previously published deduced sequence of the initial transcript. The subunit molecular mass was determined to be 48 kD and the isoelectric point was at pH 6.1. Values of the Michaelis constant for the three substrates IMP, GTP, and aspartate were 21, 16, and 335 microM, respectively. Inhibition of AdSS by 5'-PH was measurably time-dependent. The trace of the inactivation curve could not be altered by preincubating the enzyme and inhibitor in the absence of substrates but could be linearized by preincubating the enzyme with inhibitor, aspartate, GTP (or GDP), and inorganic phosphate. Inhibition of AdSS by 5'-PH was competitive with IMP, with an apparent Ki of 22 nM. Apparently, 5'-PH inhibits the enzyme by binding to the IMP site and forming a tight, dead-end complex.

Refined crystal structure of adenylosuccinate synthetase from Escherichia coli complexed with hydantocidin 5'-phosphate, GDP, HPO4(2-), Mg2+, and hadacidin.

A crystal structure of adenylosuccinate synthetase from Escherichia coli, complexed with 5'-phosphate, GDP, HPO4(2-), Mg2+, and hadacidin at 100 K, has been refined to an Rfactor of 0.195 against data to 2.6 A resolution. Bond lengths and angles deviate from expected values by 0.012 A and 1.86 degrees, respectively. Lys 16 and backbone amides 15-17 and 42 interact with the phosphates of GDP, while Ser 414, Asp 333, and backbone amides 331 and 416 interact with the base. Mg2+ is octahedrally coordinated. Oxygen atoms from GDP, phosphate, and hadacidin define the equatorial plane of coordination of the Mg2+, while backbone carbonyl 40 and the side chain of Asp 13 are the apical ligands. HPO4(2-) hydrogen bonds with Lys 16, His 41, backbone amides 13, 40, and 224, and the base moiety of the hydantocidin inhibitor. The carboxylate of hadacidin interacts with Arg 303 and Thr 301; its N-formyl group coordinates to Mg2+, and its hydroxyl group hydrogen bonds with Asp 13. The 5'-phosphate of the hydantocidin inhibitor interacts with Asn 38, Thr 129, and Thr 239 but is approximately 3.5 A from Arg 143 (related by molecular 2-fold symmetry). The base moiety of hydantocidin 5'-phosphate hydrogen bonds to Gln 224 and participates in a hydrogen-bonded network that includes the phosphate molecule, several water molecules, and Asp 13. Hydantocidin 5'-phosphate, GDP, HPO4(2-), and Mg2+ may represent a set of synergistic inhibitors even more effective than the combination of IMP, GDP, NO3-, and Mg2+.

Adenylosuccinate synthetase: site of action of hydantocidin, a microbial phytotoxin.

The site of action of hydantocidin was probed using Arabidopsis thaliana plants growing on agar plates. Herbicidal effects were reversed when the agar medium was supplemented with AMP, but not IMP or GMP, suggesting that hydantocidin blocked the two-step conversion of IMP to AMP in the de novo purine biosynthesis pathway. Hydantocidin itself did not inhibit adenylosuccinate synthetase or adenylosuccinate lyase isolated from Zea mays. However, a phosphorylated derivative of hydantocidin, N-acetyl-5'-phosphohydantocidin, was a potent inhibitor of the synthetase but not of the lyase. These results identify the site of action of hydantocidin and establish adenylosuccinate synthetase as an herbicide target of commercial potential.

A naturally occurring point mutation confers broad range tolerance to herbicides that target acetolactate synthase.

Acetolactate synthase (ALS) inhibitors are among the most commonly used herbicides. They fall into four distinct families of compounds: sulfonylureas, imidazolinones, triazolopyrimidine sulfonanilides, and pyrimidinyl oxybenzoates. We have investigated the molecular basis of imidazolinone tolerance of two field isolates of cocklebur (Xanthium sp.) from Mississippi and Missouri. In both cases, tolerance was conferred by a form of ALS that was less sensitive to inhibitors than the wild type. The insensitivity pattern of the Mississippi isolate was similar to that of a commercial mutant of corn generated in the laboratory: ICI 8532 IT. Sequencing revealed that the same residue (Ala57-->Thr) was mutated in both Mississippi cocklebur and ICI 8532 IT corn. ALS from the Missouri isolate was highly insensitive to all the ALS herbicide families, similar in this respect to another commercial corn mutant: Pioneer 3180 IR corn. Sequencing of ALS from both plants revealed a common mutation that changed Trp552 to Leu. The sensitive cocklebur ALS cDNA, fused with a glutathione S-transferase, was functionally expressed in Escherichia coli. The recombinant protein had enzymatic properties similar to those of the plant enzyme. All the possible point mutations affecting Trp552 were investigated by site-directed mutagenesis. Only the Trp-->Leu mutation yielded an active enzyme. This mutation conferred a dramatically reduced sensitivity toward representatives of all four chemical families, demonstrating its role in herbicide tolerance. This study indicates that mutations conferring herbicide tolerance, obtained in an artificial environment, also occur in nature, where the selection pressure is much lower. Thus, this study validates the use of laboratory models to predict mutations that may develop in natural populations.

Functional expression of Arabidopsis thaliana anthranilate synthase subunit I in Escherichia coli.

Anthranilate synthase is involved in tryptophan (Trp) biosynthesis. Functional expression of subunit I from Arabidopsis (ASA1) was achieved in bacteria as a protein fused with glutathione S-transferase (GST). The active product was purified in a single step on a glutathione-Sepharose column. The Vmax (45 nmol min-1mg-1), the apparent K(M) for chorismate (180 microM), and the feedback inhibition by Trp (complete inhibition by 10 microM Trp) of the purified fusion product (GST-ASA1) were comparable to anthranilate synthase purified from plants. Polyclonal antibodies raised against the fusion project and purified by affinity chromatography on a GST-ASA1-Sepharose column cross-reacted with a 61.5-kD protein in a partially purified anthranilate synthase preparation from corn seedlings. GST-ASA1 cleavage by thrombin, as well as site-directed mutagenesis modifications of the Trp allosteric site, inactivated the recombinant protein.

Catecholamines, glucagon, energy metabolism and protein degradation in rat heart.

Isoproterenol, epinephrine, phenylephrine and glucagon inhibited proteolysis in isolated perfused rat hearts. All of these agents had a positive inotropic effect, while isoproterenol and glucagon were shown to increase cyclic AMP content. The catecholamines, but not glucagon, partially depleted the adenine nucleotide pool, but the creatine-phosphate/creatine ratio was unchanged or increased. Isoproterenol markedly increased lactate production and caused release of lactate dehydrogenase. The effects of isoproterenol on these parameters, including proteolysis, were blocked by propranolol and verapamil. Isoproterenol also inhibited proteolysis when perfusate calcium was reduced from 2.5 to 0.5 mM; but, in this circumstance, isoproterenol did not deplete ATP. In hearts arrested with tetrodotoxin, neither isoproterenol nor glucagon inhibited proteolysis and neither of them depleted ATP. Both hormones still increased cyclic AMP content. These findings suggest that cyclic AMP may not be involved in the control of proteolysis, and that the effects of isoproterenol and glucagon are mediated via effects on contractility. The studies stress the importance of preventing adenine nucleotide depletion and controlling contractility in experiments on the mechanisms of inotropic agents on cardiac protein turnover.

Kinetic and regulatory properties of arogenate dehydratase in seedlings of Sorghum bicolor (L.) Moench.

Arogenate dehydratase was purified sixfold from an extract of etiolated seedlings of Sorghum bicolor. Prephenate dehydratase was not detected. The arogenate dehydratase activity displayed hyperbolic substrate kinetics with a KM for arogenate of 0.32 mM. Activity was inhibited competitively by phenylalanine and was stimulated by tyrosine. The low KI for phenylalanine (24 microM) and KA for tyrosine (2.5 microM) indicated a high affinity of the enzyme for these effectors. These results establish the routing of metabolites in phenylalanine biosynthesis in sorghum as proceeding via arogenate rather than phenylpyruvate.

Tissue Distribution and Subcellular Localization of Prephenate Aminotransferase in Leaves of Sorghum bicolor.

The tissue and subcellular distribution of prephenate aminotransferase, an enzyme of the shikimate pathway, was investigated in protoplasts from leaves of Sorghum bicolor. Activity was detected in purified epidermal and mesophyll protoplasts, and in bundle sheath strands. After fractionation of mesophyll and epidermal protoplasts by differential centrifugation, 92% of the total prephenate aminotransferase activity was detected in the plastid fraction.

Tyrosine biosynthesis in Sorghum bicolor: characteristics of prephenate aminotransferase.

A stable activity which transfers the amino group from glutamate to prephenate was extracted from 4-day old etiolated shoots of sorghum. The activity was retained on DEAE cellulose and eluted as a single peak. Prephenate aminotransferase co-eluted with a very abundant alpha-ketoglutarate: aspartate aminotransferase, but heating at 70 degrees C resulted in loss of alpha-ketoglutarate: aspartate activity with nearly full retention of prephenate: glutamate aminotransferase activity. The heated enzyme displayed high affinity and specificity for prephenate. Among 7 donors tested, only glutamate, and aspartate at less than 20% the rate with glutamate, supported prephenate aminotransferase activity. In the reverse direction, a reaction rate comparable to that in the forward direction was unchanged as the concentration of alpha-ketoglutarate was reduced from 1.0 to 0.09 mM. The apparent Km for arogenate was 0.8 mM. The forward reaction was unaffected by the inclusion of tyrosine, phenylalanine or tryptophan. Together with the discovery of arogenate dehydrogenase in sorghum [3], these data indicate that, in the sorghum plant, tyrosine derives from prephenate by transamination and aromatization, rather than the reverse sequence.

Effects of cardiac work and leucine on protein turnover.

The purpose of these experiments was to assess effects of cardiac work and leucine in hearts supplied only glucose or substrate and hormone mixtures that simulated plasma. Rates of protein degradation greatly exceeded protein synthesis in Langendorff preparations supplied glucose. This severely negative nitrogen balance was brought closer to zero by provision of more complete substrate mixtures. Cardiac work further improved the nitrogen balance by stimulating protein synthesis in hearts supplied glucose (mixture 1), glucose-insulin-glucagon-lactate-beta-hydroxybutyrate (mixture 2), or palmitate-beta-hydroxybutyrate-glucose (mixture 3) and inhibiting protein degradation in hearts supplied glucose. Cardiac work did not affect the rates of either protein synthesis or degradation in hearts provided insulin-lactate-glucose (mixture 4). The increase in protein synthesis was associated with increased rates of peptide chain initiation. Addition of 1 mM leucine had an additional effect to restore nitrogen balance to zero or to achieve positive balance in working hearts supplied substrate and hormone mixture 2.

Utilization of leucine by working rat heart.

Leucine provided substrate to partially support mechanical activity of working rat hearts; the beneficial effect on peak systolic pressure development was more marked as leucine concentration was increased to 10 mM. In Langendorff preparations that had been exposed to 5 mM [U-14C]leucine, the imposition of cardiac work was accompanied by a threefold increase in the rate of 14CO2 production within the first 30 s; however, the rate decreased 40% in the next 10 min. This transient acceleration of 14CO2 production was not observed when [1-14C]leucine was provided and appeared to be due to oxidation of a tissue pool of radioactive intermediates that was present after 10 min of Langendorff perfusion. Over a period of 1 h, oxidation of either [U-14C]- or [1-14C]leucine increased 25-40% in working compared to Langendorff preparations that were supplied 5 mM leucine and 11 mM glucose. In working hearts that were supplied a substrate and hormone mixture that simulated normal plasma and 1 mM leucine, a concentration found in the plasma of diabetic but not normal rats, leucine oxidation was accelerated 73% by work but amounted to only 3.3% of oxygen consumption.

A role for leucine in regulation of protein turnover in working rat hearts.

Effect of leucine on protein turnover was examined in perfused hearts provided with 1 (0.2 mM) or 5 times (1 mM) plasma levels of leucine and normal plasma levels of other amino acids. When hearts were perfused as Langendorff or working preparations with buffer that contained 15 mM glucose, protein degradation was 2-3 times faster than protein synthesis. As a result, the heart was in marked negative nitrogen balance. Addition of 1 mM leucine significantly improved the nitrogen balance (24-33%) by stimulating protein synthesis in Langendorff preparations (25%) and inhibiting protein degradation in both preparations (14-29%). The stimulatory effect of leucine on protein synthesis was associated with a reduction in levels of ribosomal subunits. In hearts supplied physiological levels of glucose, lactate, beta-hydroxybutyrate, insulin, and glucagon, protein synthesis was more nearly equal to protein degradation. Provision of 1 mM leucine stimulated protein synthesis only in Langendorff preparations (32%) but did not have a significant effect on protein degradation in either preparation. Although leucine did not have a significant effect on either protein synthesis or degradation in working hearts, nitrogen balance became positive with addition of 1 mM leucine. These results suggest that leucine may exert an effect on myocardial nitrogen balance in vivo under conditions that elevate plasma leucine concentrations.

Effect of leucine and metabolites of branched chain amino acids on protein turnover in heart.

Leucine, but not isoleucine or valine, inhibited protein degradation and accelerated protein synthesis in hearts perfused with buffer that contained glucose (15 mM) and normal plasma levels of other amino acids, except for the branched chain compounds. Products of leucine, isoleucine, and valine metabolism also inhibited protein degradation and stimulated protein synthesis. These compounds included the transamination and decarboxylation products, as well as acetate, acetoacetate, and propionate. In some, but not all instances, inhibition of degradation and acceleration of synthesis were accompanied by an increase in intracellular leucine. When insulin was added to the perfusate, the rate of degradation was reduced by 40%, but addition of leucine was ineffective in the presence of the hormone. Insulin, leucine (2 mM) and a mixture of branched chain amino acids at normal plasma levels increased latency of cathepsin D in hearts that were perfused with buffer containing glucose. A combination of leucine and insulin increased latency more than either substance alone. These studies indicate that leucine as well as a variety of substrates that are oxidized in the citric acid cycle are involved in regulation of protein turnover in heart muscle.