Use of rationally designed inhibitors to study sterol and triterpenoid biosynthesis (original) (raw)
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Biochemical and Biophysical Research Communications, 1986
SUMYGKRY : N-] (l,5,9)-trimethyl-decyl) [-4~,10-dimethyl-8-aza-trans-decal-3~-oi 9 was designed to mimic the C9 or C8 high energy carbocationic intermediates postulated during the enzymic cyclization of 2,3-oxidosqualene to different triterpenes. The structurally new molecule 9 inhibits stronglv cycloartenol and lanosterol cvclases in maize seedlings and rat liver microsomes respectively, whereas it does not inhibit ~-amyrin cyclase in the plant system. For the first time 2,3-oxidosqualene cycloartenol cyclase and ~-amyrin cyclase have been differentiated in the same plant material by use of a specific inhibitor.
1987
Several enzymes of plant sterol or triterpenoid biosynthesis involve during their catalysis postulated or demonstrated carbocationic high energy intermediates. It has been demonstrated previously that the design of transition state or high energy intermediate analogues could lead to powerful and specific inhibitors of enzymes. The aim of the present study was to interfere with plant sterol or triterpenoid biosynthesis by means of rationally designed species able to mimic the carbocationic high energy intermediates. We applied this approach to the following target enzymes : 2,3-oxidosqualene cyclase, 5-adenosyl methionine , cycloeucalenol-obtusifoliol isomerase and t8 ' t7-sterol isomerase. Very potent inhibitors have been obtained in each case. As an example N-substituted-8-azadecalins were shown to inhibit strongly Lri vLt'w the cycloeucalenol-obtusifoliol isomerase and the A8-L17-sterol isomerase (Ki/Km = 103). N-I (1,5,9)trimethyl-decyl I-4cx, 1O-dimethyl-8-aza-trans-decal-3-ol was shown to inhibit also the 2(3)-oxido-squalene-cycloartenol cyclase in cell-free extracts from maize (Zecz mcty4) seedlings ; in the same plant material, the 2(3)-oxido-squalene-amyrin cyclase was not inhibited. Hence for the first time, these two cyclases have been discriminated by use of a specific inhibitor.
Design of high energy intermediate analogues to study sterol biosynthesis in higher plants
Lipids, 1986
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.
Plant molecular biology, 2001
A vast array of triterpenes are found in living organisms in addition to lanosterol and cycloartenol, which are involved in sterol biosynthesis in non-photosynthetic and photosynthetic eukaryotes respectively. The chemical structure of these triterpenes is determined by a single step catalysed by 2,3-oxidosqualene-triterpene cyclases. The present study describes cloning and functional expression in yeast of several OS-triterpene cyclases. Three Arabidopsis thaliana cDNAs encoding proteins (ATLUP1, ATLUP2, ATPEN1) 57%, 58% and 49% identical to cycloartenol synthase from the same plant were isolated. Expression of these cDNAs in yeast showed that the recombinant proteins catalyse the synthesis of various pentacyclic triterpenes. Whereas ATLUP1 is essentially involved in the synthesis of lupeol, ATLUP2 catalyses the production of lupeol, beta- and alpha-amyrin (in a 15:55:30 ratio). ATLUP2 is therefore a typical multifunctional enzyme. Under the same conditions, ATPEN1 did not lead to ...
SpringerPlus, 2013
Centella asiatica is a well-known medicinal plant, produces large amount of triterpenoid saponins, collectively known as centelloids, with a wide-spectrum of pharmacological applications. Various strategies have been developed for the production of plant secondary metabolites in cell and tissue cultures; one of these is modular metabolic engineering, in which one of the competitive metabolic pathways is selectively suppressed to channelize precursor molecules for the production of desired molecules by another route. In plants the precursor 2,3oxidosqualene is shared in between two competitive pathways involved with two isoforms of oxidosqualene cyclases. One is primary metabolic route for the synthesis of phytosterol like cycloartenol by cycloartenol synthase; another is secondary metabolic route for the synthesis of triterpenoid like β-amyrin by β-amyrin synthase. The present work is envisaged to evaluate specific negative modulators for cycloartenol synthase, to channelize the precursor molecule for the production of triterpenoids. As there are no experimentally determined structures for these enzymes reported in the literature, we have modeled the protein structures and were docked with a panel of ligands. Of the various modulators tested, ketoconazole has been evaluated as the negative modulator of primary metabolism that inhibits cycloartenol synthase specifically, while showing no interaction with β-amyrin synthase. Amino acid substitution studies confirmed that, ketoconazole is specific modulator for cycloartenol synthase, LYS728 is the key amino acid for the interaction. Our present study is a novel approach for identifying a suitable specific positive modulator for the over production of desired triterpenoid secondary metabolites in the cell cultures of plants.
European Journal of Biochemistry, 1989
The molecular features of 19 synthetic substrates and ground-state analogues of cycloeucalenol, the natural substrate of cycloeucalenolobtusifoliol isomerase, a membrane-bound enzyme specific to higher plants, and of 9 synthetic carbocationic analogues of the high-energy intermediate occurring during the reaction catalyzed by the isomerase, were related to their ability to be transformed by this enzyme (catalytical competence) and their potency as inhibitors of this enzyme. With substrates and ground-state analogues it has been possible to determine at least two critical domains: significant binding requires the presence of the 3j-hydroxyl group on the ring A with the correct stereochemistry together with absence of a 4P-methyl group. Moreover initial enzyme-substrate interaction appears to be dependent upon the accessibility of the 3P-oxygen. Substitutions on the ring B do not preclude binding whereas they are of great influence on substrate transformation. Modifications of the ring A domain affects binding of high-energy intermediate analogues to a much lesser extent. The effects of these and other modifications suggest that ground-state and high-energy intermediate analogues bind two different conformations of the isomerase active site.