Claude Anding

Design of high energy intermediate analogues to study sterol biosynthesis in higher plants

Several enzymes of plant sterol biosynthesis involve during their catalysis postulated or demonstrated carbocationic high energy intermediates (HEI). The aim of this study was to interfere with plant sterol biosynthesis by means of rationally designed species able to mimic these carbocationic HEI. It has been demonstrated previously that the design of transition state (TS) or HEI analogues could lead to powerful and specific inhibitors of enzymes. We applied this approach to the following target enzymes: 2,3-epoxy-2,3-dihydroqualene cyclase, AdoMet-cycloartenol-C-24-methyltransferase (AdoMet CMT), cycloeucalenol-obtusifoliol isomerase (COI) and Δ8-Δ7-sterol isomerase. Very potent inhibitors have been obtained in the four cases. As an example, analogues of cycloartenol substituted at C-25 by a charged heteroatom (N, As, S) have been synthesized and shown to be able to mimic the C-25 carbocationic HEI involved in the reaction catalyzed by the AdoMet CMT. These compounds were shown to be very potent and specific inhibitors of this enzyme both in vitro (Ki=2.10−8 M, Ki/Km=10−3) and in vivo. The potent inhibitors described are powerful tools to control in vivo the sterol profile of plant cells and therefore to study the structural and functional roles of sterols in cell membranes. Moreover, these compounds constitute leader molecules of a new class of rationally designed inhibitors which could be of value in plant protection.

Inhibition of δ8 → δ7-sterol isomerase and of cycloeucalenol—obtusifoliol isomerase by N-benzyl-8-aza-4α, 10-dimethyl-trans-decal-3β-ol, an analogue of a carbocationic high energy intermediate

Abstract An enzymatic assay for the δ 8 → δ 7 -sterol isomerase, an enzyme involved in sterol biosynthesis, has been developed in higher plants. This assay has been used in the study of various inhibitors. N -Benzyl-8-aza-4α, 10-dimethyl- trans -decal-3β-ol was designed to mimic the C-8 and the C-9 carbocationic high energy intermediates occurring during the reactions catalysed by the δ 8 → δ 7 -sterol isomerase and the cycloeucalenol obtusifoliol isomerase, respectively. In accordance with the ‘transition state analogues’ theory, this analogue of a high energy intermediate was found to be a very potent and specific inhibitor of the two enzymatic reactions both in vitro and in vivo .

Penetration and effects of glyphosate in isolated potato mitochondria

Abstract The penetration and effects of the sodium salt of glyphosate, N -(phosphonomethyl) glycine, in isolated potato tuber mitochondria were investigated. The penetration studyrequired the measurement of the volume of mitochondrial water space and this measurement was repeatedlycarried out using [ 14 C]dextran giving a value of 3.53 μl mg −1 protein. After a 15 minute incubation periodwith medium containing 1 μM or 1 mM glyphosate, without respiratory substrate, almost no product was found in themembrane pellet, after disruption of the organelles in distilled water. In contrast, glyphosate was found in themitochondrial water in a range of concentrations close to the external medium concentration. These results show that, at leastthe diffusion equilibrium between the medium, the intermembrane space and the matrix area was readily reached forglyphosate. The greater part of the product penetrated during the first five minutes of the incubation period.The glyphosate content, in mitochondria operating at 25°, was not changed by adding either substrate. (state IV), orsubstrate + ADP (state III). The probability of an active glyphosate transport by the mitochondrial inner membrane wastherefore very unlikely. Glyphosate, at a concentration as high as 50 mM, was unable to change therespiratory activities of isolated potato tuber mitochondria(oxygen consumption rate with different substrates, ADP/O, respiratorycontrol values, etc.).

Uptake of furconazole-cis by etiolated wheat seedlings and its effect on exogenous palmitic acid incorporation into lipids

Abstract The uptake of a14C radiolabelled fungicide and its effects on endomembranous lipid biosynthesis was studied in five-day-old etiolated wheat seedlings. An extraction procedure was performed to quantitate the amount of furconazole-cis in the underlying cells. A very fast penetration of the active ingredient was observed (20 to 35% after 30 min) and the rate of uptake was higher when the concentration of fungicide applied was low. The main effect of the fungicide was inhibition of the incorporation of [3H]palmitate into phosphatidyl choline, when the concentration of furcanazole-cis was high (87 ppm).