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Studies on the Inhibition of Biotin-Containing Carboxylases by Acetyl-Coa Carboxylase Inhibitors

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Studies on the Inhibition of Biotin-Containing Carboxylases by Acetyl-Coa Carboxylase Inhibitors Studies on the Inhibition of Biotin-Containing Carboxylases by Acetyl-CoA Carboxylase Inhibitors Anja Motel, Simone Günther, Martin Clauss, Klaus Kobek, Manfred Focke, and Hartmut K. Lichtenthaler Botanisches Institut (Pflanzenphysiologie und Pflanzenbiochemie), Universität Karlsruhe, Kaiserstraße 12, D-W-7500 Karlsruhe, Bundesrepublik Deutschland Z. Naturforsch. 48c, 294-300 (1993); received November 11, 1992 Acetyl-CoA Carboxylase, Cycloxydim, Diclofop, Graminicides, Propionyl-CoA Carboxylase In higher plants the biosynthetic machinery of de novo fatty acid biosynthesis, measured as [14C]acetate incorporation into fatty acids, is predominantly located in plastids. A key enzyme in this pathway is the biotin-containing acetyl-CoA carboxylase (ACC, EC 6.4.1.2) which catalyzes the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. The ACC from Poaceae is very efficiently blocked by two herbicide classes, the cyclohexane-l,3-diones (e.g. sethoxydim, cycloxydim) and the aryloxyphenoxy-propionic acids (e.g. diclofop, fluazifop). It is shown that within the Poaceae not only different species but also different varieties exist which exhibit an altered sensitivity and tolerance towards both herbicide classes, which points to a mutation of the target enzyme ACC. In purifying the ACC we extended our research to the possible presence of other biotin-containing plant enzymes. In protein preparations from maize, oat, barley, pea and lentil we were able to demonstrate the carboxylation of acetyl-CoA, propionyl-CoA and methylcrotonyl-CoA. The two herbicide classes not only block the ACC, but also the activity of the propionyl-CoA carboxylase (PCC), whereas the methylcrotonyl- CoA carboxylase (MCC), a distinct biotin-containing enzyme from mitochondria, is not affected. MCC may play a role in isoprenoid catabolism. Whether PCC is a separate plastid enzyme or only a side activity of ACC is under current investigation. The efficiency of the graminicides in sensitive Poaceae is then not only determined by the inhibition of ACC, malonyl-CoA and fatty acid biosynthesis, but also by the exclusion of the PCC-catalyzed metabolic pathways of the plant cell. Introduction tion of the amino acids valine, isoleucine, methio­ nine and odd-numbered fatty acids, whereas MCC The enzyme acetyl-CoA carboxylase ACC is the may play a role in the degradation of leucine and key enzyme of the de novo fatty acid biosynthesis ß-methyl-branched keto acids (isoprenoid resi­ in plant plastids and also supplies various other dues). biosynthetic pathways with malonyl-CoA (such as All biotin-dependent carboxylases consist of flavonoid and polyketide biosynthesis). It is the three domains: a) the biotin carboxylase (BC), major biotin-containing enzyme of plants. Until which catalyzes the ATP-dependent carboxylation 1990 it was assumed that ACC was the only biotin- of biotin with hydrogencarbonate; b) the carboxyl containing carboxylase known in plants [1], In the transferase (CT), which transfers the activated car­ green algae Chlorella additionally a urea carboxy­ bon dioxide from the biotin to an acceptor mole­ lase (UC) was detected [2], cule to acetyl-CoA in the case of ACC); c) the More recently the activity of other biotin-con­ (e.g. biotin carboxyl carrier protein (BCCP), which is taining carboxylases, e.g. propionyl-CoA carboxy­ covalently linked to the prosthetic biotin group by lase (PCC), methylcrotonyl-CoA carboxylase a mobile spacer, which allows the biotin to move (MCC) and pyruvate carboxylase (PC), has been between the two catalytic centers. The coupling detected in non-purified preparations of sun­ site of biotin to BCCP is the e-amino group of a flower, carrot, rape and maize [3] and the MCC ac­ lysyl side chain [6]. This modified amino acid resi­ tivity also in pea, maize and oat [4, 5]. The enzyme due is also called biocytin. The biotin-containing PCC may function as in animals in the degrada- carboxylases work in a two-step mechanism, as is Reprint requests to Prof. Dr. H. K. Lichtenthaler. shown below for the ACC. In the first step the biotin carboxylase catalyzes Verlag der Zeitschrift für Naturforschung, D-W-7400 Tübingen the carboxylation of the biotinylated prosthetic 0939-5075/93/0300-0294 $01.30/0 group at the biotin carboxyl carrier protein (reac- A. Motel et al. ■ Inhibition of Acetyl-CoA Carboxylase 295 tion center I). After translocation of this group by CoA at the second step. The mechanism of this en­ a 1.4 mm long swivel arm to reaction center II, the zyme reaction can be described as a two-site ping- carboxyl transferase catalyzes the carboxyl trans­ pong bi-bi uni-uni reaction [7], The ACC reaction fer from biotin to acetyl-CoA forming malonyl- is A T P - and M g2+-dependent: M g2+ ACC-biotin + ATP + H C 03~ ACC-biotin-COO" + ADP + P; ACC-biotin-COO- + acetyl-CoA ACC-biotin + malonyl-CoA Acetyl-CoA + ATP + HCOf -» A D P + P; + m alonyl-C oA . It had been shown before that two groups of 7 days and then kept for 8 days in the dark for the chemically different herbicides specifically inhibit assay of MCC activity. For ACC purification bar­ the plant acetyl-CoA carboxylase ACC [8-13]. ley was grown for 5 days in the dark. Maize (Zea These are derivatives of either aryloxyphenoxy- mays L. var. Mona), lentil (Lens culinaris L .) and propionic acids (e.g. diclofop, fluazifop, haloxy- pea (Pisum sativum L. var. Lisa) were grown for fop) or cyclohexane-l,3-diones (e.g. sethoxydim, about 10-12 days in the light. cycloxydim). Both herbicide groups are very effec­ For enzyme isolation and developmental studies tive in many grasses, whereas monocotyledonous the seedlings were cut above the ground and either plants outside the Poaceae and dicotyledonous analyzed directly or the different leaves were care­ plants appear to be resistant. Different species of fully separated including the leaf base and sepa­ the same genus exhibit different sensitivity to­ rately analyzed. wards both herbicide groups, whereby the one is sensitive (e.g. Poa pratensis) and the other toler­ Purification o f the ACC ant (e.g. Poa annua) [14]. Certain Poaceae are able The purification of ACC from barley leaf homo- to develop resistance against diclofop by an in­ genates [20] was performed using different HPLC creased metabolization of the herbicide in the techniques (Table I). An essential step in this chro­ cytoplasm, whereas the isolated ACC enzyme is matographic purification was an anionic ion sensitive. In oat the hydroxylation product is change chromatography of protein fractions pre­ 5-OH-diclofop, which in barley, however, is found cipitated by ammonium sulfate (0-40% ) using a only in small amounts [15]. The ring hydroxylation special chromatographic material (Fractogel, is catalyzed by a NADPH-cytochrome c (P 4 5 0 ) Merck, Germany). A second chromatographic reductase [16]. 5-O H -diclofop is much less phyto­ step was dye ligand HPLC chromatography (Frac­ toxic than diclofop itself [10], togel TSK AF-Red A, Merck, Germany) [17]. In this paper we have tried to separate the three Thereafter the ACC enzyme was further purified biotin-containing carboxylases ACC, PCC and using a self-fabricated affinity column with the im­ MCC of plants and to test whether all of them are mobilized herbicide diclofop bound to an agarose sensitive to the ACC inhibitors such as cycloxy­ matrix, which is proved to be very specific for the dim, sethoxydim and the aryloxyphenoxypropio- binding of the ACC. The elution was performed nic acid derivative diclofop. Furthermore we de­ using buffer containing 200-250 mM KC1. termined the ho -values for cycloxydim and diclo­ fop in chloroplasts of different varieties of the Assay for carboxylase activity tolerant plant Festuca rubra in order to see wheth­ Assays of the different carboxylase activities er the ACC tolerance was variable or constant within one plant species. were performed by measuring the heat and acid- stable incorporation of [l4C]hydrogen carbonate Materials and Methods into one of the offered thioester (acetyl-CoA, pro- pionyl-CoA, methylcrotonyl-CoA). Enzyme frac­ Plant growth tions from leaf material of young lentil, pea, barley Barley (Hordeum vulgare L.) was grown on a and maize seedlings were prepared by ammonium mineral-containing peat (TKS II) in the light for sulfate fractioning (0-40% precipitation) and 296 A. Motel et al. • Inhibition of Acetyl-CoA Carboxylase partly chromatographic separation on an anionic The purity of this preparation was proven with exchange column (Fractogel, Merck) as described SDS-PAGE and showed a band at 220 kDa and [10]. two smaller bands at about 40 kDa and 50 kDa. Determination of different kinetic constants and Its biotin content was made visible after a Western inhibition studies for ACC and PCC was per­ blot with an extravidin/alkaline phosphatase. Ad­ formed with a post anion exchange chromato­ dition of avidin to the enzymic assay inhibited the graphic fraction, which represents an acceptable ACC activity as expected. For the purified ACC combination of ACC purity and stability. The ap­ preparation we determined the A^-values as 55 |im plied herbicides were gifts from different firms: (acetyl-CoA), 3.3 m M (HC03 ) and 192 jim (ATP/ diclofop from Hoechst GmbH, Germany, cycloxy- Mg2+). This preparation also exhibited PCC activi­ dim from BASF AG, Germany, and sethoxydim ty, but with different A^m-values as 32 ^m (propion- from Nippon Soda AG, Japan. yl-CoA), 4.5 mM (HC03 ) and 90 |^m (ATP/M g2+). This purified ACC/PCC enzyme preparation did not show any MCC activity. Results and Discussion In attempts to purify the ACC we dried to sepa­ Studies on the enzyme activities of ACC, PCC and rate the ACC from the PCC activity which car- M CC in plants boxylates propionyl-CoA. This is essential with re­ In the course of our investigations on the start­ spect to the question whether the carboxylation of ing enzymes of de novo fatty acid biosynthesis we propionyl-CoA is catalyzed by the ACC as a side purified ACC from barley to near homogeneity.
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