Bijay K. Singh

Assay of acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, has received attention recently because of the finding that it is the site of action of several new herbicides. The most commonly used assay for detecting the enzyme is spectrophotometric involving an indirect detection of the product acetolactate. The assay involves the conversion of the end product acetolactate to acetoin and the detection of acetoin via the formation of a creatine and naphthol complex. There is considerable variability in the literature as to the details of this assay. We have investigated a number of factors involved in detecting AHAS in crude ammonium sulfate precipitates using this spectrophotometric method. Substrate and cofactor saturation levels, pH optimum, and temperature optimum have been determined. We have also optimized a number of factors involved in the generation and the detection of acetoin from acetolactate. The results of these experiments can serve as a reference for new investigators in the study of AHAS.

Biosynthesis of Branched Chain Amino Acids: From Test Tube to Field

The branched chain amino acids-valine, leucine, and isoleucine-are among the 10 essential amino acids that are not synthesized in mammals. Therefore, biosynthesis of branched chain amino acids in plants is of interest due to their importance in human and animal diets. In addition, in the early 198Os,

Mutations in corn (Zea mays L.) conferring resistance to imidazolinone herbicides

SummaryThree corn (Zea mays L.) lines resistant to imidazolinone herbicides were developed by in vitro selection and plant regeneration. For all three lines, resistance is inherited as a single semidominant allele. The resistance alleles from resistant lines XA17, XI12, and QJ22 have been crossed into the inbred line B73, and in each case homozygotes are tolerant of commercial use rates of imidazolinone herbicides. All resistant selections have herbicide-resistant forms of acetohydroxyacid synthase (AHAS), the known site of action of imidazolinone herbicides. The herbicide-resistant phenotypes displayed at the whole plant level correlate directly with herbicide insensitivity of the AHAS activities of the selections. The AHAS activities from all three selections have normal feedback regulation by valine and leucine, and plants containing the mutations display a normal phenotype.

Flavin adenine dinucleotide causes oligomerization of acetohydroxyacid synthase from black Mexican sweet corn cells

Acetohydroxyacid synthase activity is stabilized and stimulated by flavin adenine dinucleotide. Flavin adenine dinucleotide was found to cause aggregation of acetohydroxyacid synthase from the dimeric to a tetrameric form. The different aggregation states of the enzyme have differential sensitivities to inhibition by branched chain amino acids as well as by imazapyr, an imidazolinone herbicide. These observations indicate that flavin adenine dinucleotide is of structural as well as of functional importance for the plant acetohydroxyacid synthase enzyme.

Biosynthesis of lysine in plants: The putative role of meso-diaminopimelate dehydrogenase

Extracts from Chlamydomonas, corn, soybean and tobacco were tested for enzymes of the lysine biosynthetic pathway. Dihydrodipicolinic acid (DHD) synthase, DHD reductase, diaminopimelate (DAP) epimerase and DAP decarboxylase were present in all. However, in contrast to the report of Wenko et al., meso-DAP dehydrogenase could not be detected in extracts prepared from soybean. Moreover, it was not found in Chlamydomonas, corn and tobacco as well. In order to set an upper limit to the amount of meso-DAP dehydrogenase that might be present, reconstruction experiments were performed with soybean and corn extracts in which the conversion of dihydrodipicolinate to lysine was made dependent on the addition of limited amounts of the meso-DAP dehydrogenase purified from Bacillus sphaericus. The presence of DAP epimerase and the absence of meso-DAP dehydrogenase indicates that the meso-DAP dehydrogenase abbreviated pathway for lysine synthesis is not operative in plants.

Determination of 2-Keto Acids and Amino Acids in Plant Extracts

Abstract 2-keto acids and amino acids were extracted using liquid nitrogen and 0.25 N HCl. The keto acids and amino acids were seperated by cation exchange chromatography on AG50W-X8 resin. The cation exchange chromatography is vital for the determination of keto acids. The keto acids were derivatized with 1,2-diamino-4,5-methylenedioxybenzene (DMB), a specific derivatizing agent for 2-keto acids. The derivatized keto acids were quantified by reversed phase high performance liquid chromatography (HPLC). This assay is highly sensitive and can measure as low as 10 fmole of the keto acids per 10 μl injection. The amino acids were analyzed by an automatic amino acid analyzer. These methods were used to show that 2-ketobutyrate (2-KB) and 2-aminobutyrate (2-AB) accumulate in plants treated with an acetohydroxyacid synthase inhibiting herbicide.

Differential metal binding interactions of the imidazolinones revealed by NMR and UV spectroscopy

NMR and UV spectroscopy and molecular modeling methods were applied to probe the interaction of the two imidazolinones, imazethapyr (5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid) and its structural isomer CL 303,135 (5-ethyl-3-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)picolinic acid), with metal ions. Both the imidazolinones inhibit the enzyme acetohydroxyacid synthase (AHAS) in vitro. However, while imazethapyr is a herbicide that is used widely in agriculture, CL303,135 does not exhibit herbicidal activity. Imazethapyr and CL303,135 exhibited considerable differences in their interactions with metals. In the metal complex of imazethapyr, the carboxyl moiety binds strongly and the pyridine nitrogen binds weakly with metals. In the case of CL303,135, both the pyridine nitrogen and the carboxyl group that are positioned ortho to each other participated strongly in the binding and were found to act together as a strong bidentate ligand to a metal ion. Both of the imidazolinones form predominantly 2:1 complexes with multivalent metal ions. However, imazethapyr binds two orders-of-magnitude more weakly (1.0 x 10 9 M -2 ) with metal ions compared to CL303,135 (1.7 x 10 11 M -2 ). The interactions of the model compounds, nicotinic acid and picolinic acid, with metals were examined similarly. It was concluded that the strong affinity of CL303,135 for metals compared to imazethapyr may affect its absorption from soil into plants, or its translocation in plants, thereby explaining the differences in herbicidal activity of imazethapyr and CL303,135.