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Biotin biosynthesis was investigated in lavender cell cultures (Lavandula vera L.). Two different biological assays and two different HPLC procedures were used to identify all the intermediates involved in biotin biosynthesis. The pathway for biotin biosynthesis could be analyzed starting with [3H]pimelic acid as precursor, leading to labelled biotin and even to labelled biotinylated enzymes. Intermediates known from the bacterial pathway (7‐oxo‐8‐amino‐pelargonic acid, 7,8‐diamino‐pelargonic acid, dethiobiotin) were present in detectable amounts. Pimelic acid activation to pimeloyl‐CoA could be observed. In contrast to bacterial cells, an unknown stable labelled intermediate, named compound A, accumulated. This compound coeluted with an authentic sample of 9‐mercaptodethiobiotin from HPLC with an anion‐exchange column and was as effective as biotin in supporting the growth of the strain bioB105 of Escherichia coli. When 3H‐labelled compound A was added to the growth medium of the lavender cells it was incorporated in an acidomycin‐sensitive manner into biotin. [3H]Dethiobiotin was incorporated into both compound A and biotin. These results strongly suggest that, in higher plant cells, the reaction catalysed by biotin synthase may proceed in two distinct steps involving mercaptodethiobiotin (9‐mercaptodethiobiotin ?) as an intermediate.

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Mitochondria from green pea (Pisum sativum) leaves were purified free of peroxisomes and chlorophyll contamination and examined for their biotin content. The bulk of the bound biotin detected in plant mitochondria was shown to be associated with the matrix space to a concentration of about 13 micromolar, and no free biotin was detected. Western blot analysis of mitochondrial polypeptides using horseradish peroxidase-labeled streptavidin revealed a unique biotin-containing polypeptide with a molecular weight of 76,000. This polypeptide was implicated as being the biotinylated subunit of 3-methylcrotonyl-coenzyme A (CoA) carboxylase. Fractionation of pea leaf protoplasts demonstrated that this enzyme activity was located largely in mitochondria. The 3-methylcrotonyl-CoA carboxylase activity was latent when assayed in isotonic media. with MCase, which catalyzes the ATP-dependent carboxylation of the 3-methyl carbon of the branched acyl-CoA substrate to give 3-methylglutaconyl-CoA. MATERIALS AND METHODS ReagentsAll biochemicals were obtained from Sigma Chimie SARL, La Verpilliere, France. NaH'4CO3 (53.1 Ci/mol) was purchased from Amersham. Other chemicals were analytical grade. Plant MaterialsPea (Pisum sativum L., var Douce Provence) plants were grown from seeds in soil for 15 d under a 12-h photoperiod ofwhite light from fluorescent tubes (10-40 ,gE/m2/s) at 18°C.The plants were watered every day with tap water. Approximately 4 to 5 kg of fully expanded leaves were used for the isolation of mitochondria.Potato tubers (Solanum tuberosum L.) were obtained from a local market.In previous publications, Journet et al. (16) We also demonstrate that all the biotin present in the matrix space of the plant mitochondrion is associated 'Abbreviations: MCase, 3-methylcrotonyl-CoA carboxylase; PFP, PPi:fructose-6-phosphate-1 -phosphotransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Preparation of MitochondriaMitochondria were isolated and purified from potato tubers as described by Douce et al. (6) by using self-generating Percoll gradients. The mitochondria were found in a broad brownred band near the bottom of the tube, whereas the "microsomal" fraction, containing the yellow envelope surrounding the amyloplasts, remained near the top of the tube. The mitochondria were subsequently concentrated by differential centrifugation. To remove all the contaminating peroxisomes the purification procedure was repeated once (i.e. a second time through Percoll). Under these conditions, we have verified that catalase activity with substrate concentrations up to 200 ,M was almost negligible.Mitochondria were isolated and purified from green pea leaves as described by Douce et al. (6), using self-generating Percoll gradients and a linear gradient of0 to 10% (w/v) PVP-25 (Kca 25, Serva) (top to bottom). The mitochondria were found in a tight white band near the bottom of the tube, whereas the thylakoids remained near the top of the tube. The mitochondria were subsequently concentrated by differential centrifugation. The ...

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