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+.

The N-1-Naphthylphthalamic Acid-Binding Protein Is an Integral Membrane Protein.

N-1-Naphthylphthalmic acid (NPA)-binding protein is a plasmalemma (PM) protein involved in the control of cellular auxin efflux. We re-evaluated the spatial relationship of this protein with the PM of zucchini (Cucurbita pepo L.) hypocotyls. First, Triton X-114 partitioning indicated that the NPA-binding protein was more hydrophobic than most PM proteins. Second, the NPA-binding activity was found to be resistant to proteolytic digestion in membranes. Maximum concentrations of binding sites for NPA were virtually identical in untreated and proteinase K-treated PMs: 19.2 and 20.6 pmol [3H]NPA bound/mg protein, respectively. The insensitivity of the NPA-binding protein was not due to its presence inside tightly sealed vesicles or due to lack of protease activity in the conditions tested. This protein could be made sensitive to proteolytic degradation upon solubilization by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in the presence of sodium molybdate. Proteinase K treatment decreased the concentration of binding sites to 0.84 pmol [3H]NPA bound/mg protein from 9.2 for untreated, solubilized PM. Third, this activity could not be solubilized by chaotropic agents or sodium carbonate treatment of intact PM. This study indicates that the NPA-binding protein may be an integral membrane protein and contradicts previously reported findings that suggested that this protein was peripheral to the PM.

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.

Acetolactate synthase inhibiting herbicides bind to the regulatory site.

Acetolactate synthase from spontaneous mutants of tobacco (Nicotiana tabacum; KS-43 and SK-53) and cotton (Gossypium hirsutum; PS-3, PSH-91, and DO-2) selected in tissue culture for resistance to a triazolopyrimidine sulfonanilide showed varying degrees of insensitivity to feedback inhibitor(s) valine and/or leucine. A similar feature was evident in the enzyme isolated from chlorsulfuron-resistant weed biotypes, Kochia scoparia and Stellaria media. Dual inhibition analyses of triazolopyrimidine sulfonanilide, thifensulfuron, and imazethapyr versus feedback inhibitor leucine revealed that the three herbicides were competitive with the amino acid for binding to acetolactate synthase from wild-type cotton cultures. Acetolactate synthase inhibiting herbicides may bind to the regulatory site on the enzyme.

Properties of mutant acetolactate synthases resistant to triazolopyrimidine sulfonanilide.

Triazolopyrimidine sulfanilides are a class of highly active herbicides whose primary target is acetolactate synthase. Spontaneous mutants of tobacco (Nicotiana tabacum) (KS-43) and cotton (Gossypium hirsutum) (PS-3 and DO-2) resistant to triazolopyrimidine sulfonanilide were selected in tissue culture. Acetolactate synthase partially purified from the three mutants were 80- to 1000-fold less sensitive to inhibition by the compound compared with the corresponding wild-type enzyme. The mutants also varied in the cross-resistance pattern to other acetolactate synthase inhibiting herbicides in the sulfonylurea, imidazolinone, and pyrimidyl-oxy-benzoate chemical families. Thus, acetolactate synthase from KS-43, PS-3, and DO-2 cultures have different mutations. The affinities for pyruvate, thiamine pyrophosphate, as well as the activity of the mutant enzymes were found to be comparable to the corresponding wild-type enzymes. However, the enzyme from PS-3 was highly resistant to feedback inhibition by valine and leucine. In contrast, acetolactate synthase from KS-43 and DO-2 were inhibited by valine and leucine to nearly the same extent as the wild-type enzymes. Also, PS-3 cultures accumulated much higher levels of the branched chain amino acids compared to the wild-type cotton culture. The mutation in the PS-3 enzyme has therefore rendered it insensitive to feedback regulation by valine and leucine.