Heinrich Sandermann

Plant metabolism of xenobiotics

Metabolism of foreign chemicals (xenobiotics) by plants generally proceeds in three phases: transformation, conjugation and compartmentation. The participating enzymes have numerous similarities not only to the enzymes of normal secondary plant metabolism, but also to those of xenobiotic metabolism in mammalian liver. Plants may therefore be considered as a green liver, acting as an important global sink for environmental chemicals.

Use of plant cell cultures to study the metabolism of environmental chemicals

The metabolism of the following environmental chemicals has been studied in cell suspension cultures of wheat (Triticum aestivum L.) and soybean (Glycine max L.):2, 4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), hexachlorobenzene, pentachlorophenol, diethylhexylphthalate , benzo [alpha] pyrene, and DDT. All chemicals tested, including the persistent ones, were partially metabolized. Polar conjugates predominated in all cases. A covalent incorporation into lignin could be demonstrated for 2,4-D and pentachlorophenol. A specific deposition in the cellular vacuole could be demonstrated for the beta-D-glucopyranoside conjugates derived from 2,4-D. A rapid assay procedure to evaluate the metabolism of a given 14C-labeled chemical in plant cell suspension cultures is described. This procedure requires about 1 week, and the reproducibility of the results obtained has been assessed.

Plant biochemistry of xenobiotics: Isolation and properties of soybean O- and N-glucosyl and O- and N-malonyltransferases for chlorinated phenols and anilines

O-Glucosyltransferase (O-GT), O-malonyltransferase (O-MAT), N-glucosyltransferase (N-GT), and N-malonyltransferase (N-MAT) activities have been detected in cultured soybean cells, using pentachlorophenol and 3,4-dichloroaniline as xenobiotic standard substrates. The O-GT was purified approximately 1000-fold, and the N-MAT approximately 70-fold. There was an extensive copurification of O-GT and O-MAT. The following functional molecular weight values were obtained, 47 kDA (O-GT), 48 kDA (O-MAT) 43 kDa (N-GT), and 48 kDa (N-MAT). O-GT and N-MAT appeared to be monomeric polypeptides with isoelectric points of approximately 4.8 and approximately 6.1, respectively. The O-GT, N-GT, and N-MAT activities had marked substrate specificities for chlorinated aromatic xenobiotics and thus illustrate the existence of plant isoenzymes with specificity for xenobiotics.

β-d-Glucosyl and O-malonyl-β-d-glucosyl conjugates of pentachlorophenol in soybean and wheat: Identification and enzymatic synthesis

Abstract The polar metabolite fraction formed by soybean cell suspension cultures from [ 14 C]pentachlorophenol (PCP) was fractionated by repeated high-performance liquid chromatography. The β- d -glucopyranosyl conjugate of PCP was identified by cochromatography, by chemical and enzymatic degradation, and by mass spectroscopic comparison with the authentic compound. The O -(malonyl)-β- d -glucopyranosyl conjugate of PCP was identified by chromatographic and electrophoretic comparison with the enzymatically synthesized compound, by specific chemical and enzymatic degradation, and by mass spectroscopy. Soluble protein extracts from both soybean and wheat cells catalyzed (a) the UDPG-dependent conversion of PCP to the β- d -glucopyranosyl conjugate, and (b) the malonyl-SCoA-dependent further conversion to the O -(malonyl)-β- d -glucopyranosyl conjugate. These enzymes were partially purified and had apparent molecular weights between 40,000 and 48,000. Free PCP was again released from the conjugates upon incubation with plant malonyl esterase and/or β-glucosidase preparations.

Oxygenation of benzo[a]pyrene by plant microsomal fractions.

Benzo [a] pyrene is a highly carcinogenic compound that occurs universally [ 1,2]. An estimated 894 tons are released annually in the US alone, mainly in combustion processes [ 11. BP is extensively metabolized in mammalian systems, in particular by the mixed-function oxygenase system of liver microsomes [3]. Species of cytochrome P450 are involved in this process and lead via epoxide intermediates to various phenolic and dihydrodiol metabolites. A defined diol-epoxide is though to act as the ultimate carcinogen by binding covalently to cellular DNA [3]. BP is a relatively stable compound [ 1,2] which is, for example, completely inert under waste treatment conditions [4] but it has been found to be metabolized by certain microorganisms [5], by intact plants [6,7] and by cultured plant cells [8,9]. The biochemical reactions leading to the various plant metabolite fractions of BP are as yet unknown although certain analogies with the liver microsomal system have been postulated on the basis of HPLC data [lo]. It is now reported that plant microsomal fractions catalyze the oxygenation of BP and that this process differs considerably from the oxygenation of BP by liver microsomal fractions.

Lipid solvation and kinetic cooperativity of functional membrane proteins

Abstract Lipid solvation is required for the functional activity of many membrane proteins. The kinetic cooperativity observed can arise from non-cooperative lipid/protein interactions.

Biosynthesis of D-apiose V. Nad+-dependent biosynthesis of UDP-apiose and UDP-xylose from UDP-D-glucuronic acid with an enzyme preparation from Lemna minor L.

1. n1. The nature of the products formed from UDP-glucuronic acid with a protein preparation from Lemna minor L. has been reinvestigated with an enzyme preparation which gives a much higher yield of the products than our previous preparation. n n2. n2. Besides UDP-apiose and UDP-D-xylose, a product was formed the properties of which are consistent with its formulation as cyclic apiose 1, 2-phosphate. The latter compound is very probably a nonenzymic breakdown product of UDP-apiose. n n3. n3. The formation of apiose and xylose was strongly stimulated by NAD+. NADP+ was much less effective than NAD+ as cofactor, and NADH strongly inhibited the reaction.

Plant metabolism of chlorinated anilines: Isolation and identification of N-glucosyl and N-malonyl conjugates

Abstract Cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.), as well as intact wheat plants, were incubated with 4-[ring-U-14C]chloroaniline and 3,4-[ring-U-14C]dichloroaniline, respectively. In soybean cells, a single metabolite identified as the N-malonyl conjugate was in both cases formed in over 90% yield and was largely excreted. In wheat cells and wheat plants, incorporation into the insoluble residue predominated. Several soluble metabolites were also formed, including acid labile N-glucosyl conjugates. A chemical mechanism is proposed to explain the known variation in acid sensitivity of xenobiotic N-glucosyl conjugates.

Plant metabolism of herbicides with CP bonds: Phosphinothricin

Plants have been reported not to metabolize phosphinothricin. In order to reexamine these literature findings, cell suspension cultures of soybean (Glycine max L.), wheat (Triticum aestivum L.), and maize (Zea mays L.) were incubated with l-[3,4-14C]phosphinothricin. In maize cells which took up up to 50% of the applied radioactivity four different metabolites were detected. They were identified as 4-methylphosphinico-2-oxo-butyric acid, 4-methylphosphinico-2-hydroxybutyric acid, 4-methylphosphinico-butyric acid, and 3-methylphosphinico-propionic acid, respectively. A new HPLC separation method was developed in order to identify these unusually polar metabolites. In soybean and wheat cultures, 10 and 6%, respectively, of the applied radioactivity was taken up. In soybean only one metabolite, 3-methylphosphinico-propionic acid, could be detected whereas in wheat 4-methyl-phosphinico-butyric acid was additionally present. Radioactivity in the culture media could be attributed to unchanged herbicide in all cases. 14CO2-evolution from soybean and maize cultures was very low (<0.1% of the applied radioactivity within 4 days). Incorporation of radioactivity from applied [14C]phosphinothricin into bound residues also reached only small amounts (<0.1%). A metabolism scheme for phosphinothricin is proposed.

beta-D-Galactoside transport in Escherichia coli. Reversible inhibition by Aprotic Solvents and its Reconstitution in transport-negative membrane vesicles.

At relatively low concentrations (less than 3M) the aprotic solvents: dimethylsulfoxide, N-methylpyrrolidone, tetramethylurea and hexamethylphosphoric triamide, inhibited beta-D-galactoside transport by whole cells, and the derived membrane vesicles of Escherichia coli. Inhibition was not due to gross leakiness of the membrane and could be largely reversed by a simple washing procedure...