Purification and properties of Salmonella typhimurium acetolactate synthase isozyme II from Escherichia coli HB101/pDU9

Schloss, John V.; Dyk, Drew E. Van; Vasta, J.F.; Kutny, Rusty

doi:10.1021/bi00339a034

Cited by 148 publications

(134 citation statements)

References 61 publications

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“…The enzyme has been found in enteric bacteria (5,6,14), fungi (12), and in chloroplasts of higher plants (7,17 …”

Section: Introductionmentioning

confidence: 99%

“…The enzyme has been found in enteric bacteria (5,6,14), fungi (12), and in chloroplasts of higher plants (7,17). The catalyzed reaction is either the formation of acetohydroxy butyrate from pyruvate and aoxobutyrate or the synthesis of acetolactate from two molecules of pyruvate.…”

mentioning

confidence: 99%

“…In Escherichia coli and Salmonella typhimurium, three isozymes (ALS I, II, III) have been identified, each of them encoded by a particular gene (5,6,14). Bacterial ALS is a tetramer composed of two large (59-60 kD) and two small (9-17 kD) subunits.…”

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confidence: 99%

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Oligomeric Forms of Plant Acetolactate Synthase Depend on Flavin Adenine Dinucleotide

Durner¹,

Böger²

1990

Plant Physiol.

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Acetolactate synthase (ALS, EC 4.1.3.18) has been extracted and partially purified from etiolated barley shoots (Hordeum vulgare L.). Multiple forms of this enzyme were separated by gel filtration and/or anion-exchange chromatography using fast protein liquid chromatography. It could be demonstrated that these two species are in equilibrium, which strongly depends on the structural role of flavin adenine dinucleotide and pyruvate. With 50 micromolar of flavin adenine dinucleotide in the medium most of the ALS aggregates as a high molecular weight form (Mr = 440,000), while 50 millimolar pyruvate facilitates dissociation into the smaller form (Mr = 200,000). Data are presented to show that two enzymatically active forms are not isozymes but different oligomeric species or aggregates of the basic 58-kilodalton subunit of ALS. These different ALS species exhibit little difference in feedback inhibition by valine, leucine and isoleucine or in inhibition by the sulfonylurea herbicide chlorsulfuron. Both aggregation forms show a broad pH-optimum between 6.5 and 7. Furthermore, the affinity for pyruvate and the amount of directlyformed acetoin indicate similar properties of these separated ALS forms.Acetolactate synthase (ALS, EC 4.1.3.18; also known as acetohydroxy acid synthase, AHAS) is the first common enzyme in the biosynthesis ofthe branched-chain amino acids valine, leucine, and isoleucine. The enzyme has been found in enteric bacteria (5,6,14), fungi (12), and in chloroplasts of higher plants (7,17 MATERIALS AND METHODS Plant CultivationBarley seeds (Hordeum vulgare L.) were placed onto a nylon net, covered with filter tissue which had been soaked with distilled water, and allowed to germinate in a dark chamber at 23 'C. The roots were immersed into a medium containing 2 g/L KNO3, 34 mg/L CaSO4 and 0.25 mg/L Na2MoO4 (pH 7.0). Three d after germination the etiolated shoots were harvested (i.e. the lower half was used, which contained more than 75% of the total ALS activity), washed in distilled water, and either homogenized or stored at 70°K for later use.

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“…The enzyme has been found in enteric bacteria (5,6,14), fungi (12), and in chloroplasts of higher plants (7,17 …”

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Oligomeric Forms of Plant Acetolactate Synthase Depend on Flavin Adenine Dinucleotide

Durner¹,

Böger²

1990

Plant Physiol.

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“…Although we did not determine the exact emission maximum of light (which is between 620 and 660 nm) and also the generation of carbon radicals or alkyl peroxides should be considered [20] these results suggest the formation of singlet oxygen as a result of the oxygenase reaction of ALS. If this is an inherent property of ALS, the oxygen inactivation of the enzyme from different sources [4,21] may be due to a radical reaction. Additional experiments have been carried out with respect to the role of the flavin and metal.…”

Section: Chemiluminescence Measurementsmentioning

confidence: 99%

“…Both reactions are accompanied by the release of COZ from pyruvate [3]. As was established with the ALS II isozyme from Salmonella typhimurium, cofactors are thiamine pyrophosphate (TPP), Aavin adenine dinucleotide (FAD) and di-or trivalent metal ions [4]. The various bacterial ALS isozymes and the enzyme from higher plants differ considerably with respect to feedback inhibition, subunit composition and size [5].…”

Section: Introductionmentioning

confidence: 99%

The oxygenase reaction of acetolactate synthase detected by chemiluminescence

1994

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In addition to the synthesis of ketolacids the enzyme acetolactate synthase shows an oxygen-consuming side reaction. Partially purified acetolactate synthase from corn (Zea mays L.) and barley (Hordeum vulgare L.) exhibits chemiluminescence in the presence of oxygen, Mn" and low concentrations of pyruvate. Light emission is inhibited by azide, but not by catalase or superoxide dismutase. The data suggest the formation of singlet oxygen during the catalytic cycle, and provides a basis for a highly sensitive assay for the oxygenase reaction of acetolactate synthase. Both synthase activity and chemiluminescence are inhibited by sulfonylurea herbicides. The results add a new aspect to the irreversible inhibition of acetolactate synthase by these herbicides which may be enhanced by the presence of reactive oxygen species.

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Imazethapyr inhibition of acetolactate synthase inRhizobiumand its symbiosis with pea

et al. 1998

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: Acetolactate synthase (ALS) activity extracted from Rhizobium leguminosarum biovar. viciae has been characterized. The optimum pH for extraction was 7É6 and for the assay 7É0. The for pyruvate was 7É2 mM, and the enzyme K m was saturated at 40 mM. An obligatory requirement of TPP and Mg2`for full ALS activity was observed. Valine was the only branched-chain amino acid that caused ALS feedback inhibition. The speciÐc activity of Rhizobium ALS was nearly 20 times the activity found in pea (Pisum sativum) leaves. Bacteroids from pea nodules also showed high ALS activity, and the nodule plant fraction had higher ALS activity than other plant tissues. ALS sensitivity to imazethapyr was also dependent on the source : ALS activity of free-living Rhizobium and bacteroids was slightly more tolerant than that of other pea tissues, but the di †er-ences were less than those found in rates of speciÐc activity. It is proposed that the high ALS activity expressed by Rhizobium, both as free-living bacteria and as bacteroids, is related to the growth tolerance of rhizobia to imazethapyr and is also related to the relative tolerance of symbiotic pea plants.1998 SCI ( Pestic. Sci., 52, 372È380 (1998)

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Purification and properties of Salmonella typhimurium acetolactate synthase isozyme II from Escherichia coli HB101/pDU9

Cited by 148 publications

References 61 publications

Oligomeric Forms of Plant Acetolactate Synthase Depend on Flavin Adenine Dinucleotide

Oligomeric Forms of Plant Acetolactate Synthase Depend on Flavin Adenine Dinucleotide

The oxygenase reaction of acetolactate synthase detected by chemiluminescence

Imazethapyr inhibition of acetolactate synthase inRhizobiumand its symbiosis with pea

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