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.

Branched-chain 2-oxo acids which are formed by transamination of leucine, isoleucine, and valine are metabolized by the peroxisomes from mung bean (Vigna radiata L.) hypocotyls. Acylcoenzyme A (CoA) thio ester intermediates of the pathways were separated by reversed-phase high performance liquid chromatography. Retention time and cochromatography of individual acyl-CoA reference standards were used for identification of the acyl-CoA esters separated from the assay mixtures. Based on the results of identification and those of kinetic experiments, pathways of the peroxisomal degradation of 2-oxoisocaproate, 2-oxoisovalerate, and 2-oxo-3-methylvalerate are suggested.Peroxisomes are common organelles of higher plant cells. A basic metabolic function of these organelles seems to be fatty acid degradation (7). Peroxisomes degrade by ,8-oxidation saturated straight chain, long-, medium-and short-chain fatty acids (8). Unsaturated fatty acids appear also to be degraded by peroxisomes. The enzymes required to link the catabolism of unsaturated fatty acids to the fl-oxidation sequence have recently been demonstrated in glyoxysomes, the peroxisomes of lipid-storing nutrient tissues of seeds (2).Glyoxysomes are involved in the conversion of reserve lipid to sucrose during germination (1). The physiological role of the peroxisomal fatty acid degrading system in non-lipid storing tissues, i.e. in the majority of plant tissues, has yet to be elucidated. The turnover of membrane lipids has to be considered as a source of fatty acids in non-lipid storing tissues. Unsaturated fatty acids would be the main substrates for the peroxisomal fl-oxidation system. The data on the ability of glyoxysomes to degrade unsaturated fatty acids (2) support this concept.A second physiologically important source of substrate for a fatty acid degrading system in non-lipid storing tissues can result from the degradation of branched-chain amino acids in the course of steady-state protein turnover. In Lemna minor, 50 to 60% of the leucine and isoleucine resulting from protein turnover is metabolized (3). Intermediates of the catabolism of leucine, isoleucine, and valine are branched-chain 2-oxo acids. The catabolism of these acids in higher plants has received very little attention up to now ( 12). We have recently shown that the peroxisomes but not the mitochondria from a non-lipid storing tissue are able to activate by oxidative de-' Dedicated to Professor Achim Trebst on the occasion of his 60th birthday.

Peroxisomes from mung bean ( Vigna radiata L.) hypocotyls catalyze, in the presence of branched-chain 2-oxo fatty acid, CoASH and NAD, the release of C02, and the formation of NADH and acyl-CoA. The acylCoA contains one carbon atom less than the branched-chain 2-oxo fatty acid and serves as substrate for the peroxisomal acyl-CoA oxidase. CO2 release, NADH and acyl-CoA formation occur in 1:1:1 stoichiometry. For the first time the data demonstrate directly the oxidative decarboxylation of branched-chain 2-oxo fatty acids in higher plants and a location of this activity in the peroxisomes.

Peroxisomes from mung bean hypocotyl (Vigna radiata L.) degrade 2‐oxoisocaproate, the transamination product of leucine, via isobutyryl‐CoA and propionyl‐CoA to acetyl‐CoA. The methyl group at the C‐3 position forms a barrier to β‐oxidation. This barrier is overcome in the peroxisomes by several enzymatic steps. Senecioate (3‐methylcrotonate), 2‐hydroxyisovalerate, and 2‐oxoisovalerate were detected as free acid intermediates. Senecioate, formed from 3‐methylcrotonyl‐CoA, is transformed by enzymatic hydrolysis to 2‐hydroxyisovalerate. 2‐Hydroxyisovalerate is then oxidized to 2‐oxoisovalerate in an H2O2‐producing reaction, exhibiting 1:1 stoichiometry of the products, by a 2‐hydroxyacid oxidase which is different from the peroxisomal marker enzyme glycollate oxidase. 2‐oxoisovalerate is activated by an NAD‐dependent oxidative decarboxylation to isobutyryl‐CoA. Accumulation of 2‐oxoisovalerate in the presence of arsenite, an inhibitor of oxidative decarboxylations, is a feature of this latter pathway of degradation of isovaleryl‐CoA or senecioate. It is concluded that the barrier caused by the methyl group of 2‐oxoisocaproate is surmounted in higher plant peroxisomes in a manner different to that in mammalian mitochondria.

Peroxisomes from mung-bean hypocotyls catalyze, in the presence of fatty acids, CoASH, ATP, and MgCl2, the formation of acyl-CoA, AMP, and pyrophosphate in a 1:1:1 stoichiometry. This observation demonstrates that the peroxisomes of mung-bean hypocotyls possess an acyl-CoA synthetase (EC 6.2.1.3) for fatty-acid activation. Acyl-CoA synthetase activity is associated with the non-glyoxysomal peroxisomes from various tissues. The acyl-CoA synthetase of the peroxisomes of the mung-bean hypocotyl utilizes oleic, linoleic, and linolenic acid most effectively (3 nkat·mg(-1) peroxisomal protein). In contrast to the β-oxidation enzymes of the peroxisomes whith are largely solubilized in the presence of 0.2 mol·l(-1) KCl, the acyl-CoA synthetase remains associated with the membrane fraction of peroxisomes. On the basis of the latency of the enzyme and its resistance to protease treatment of the peroxisomes, it is concluded that the enzyme is located at the matrix face of the peroxisome membrane.