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ABSTRACT: The stereochemical course of the dihydroceramide delta 4-(E)-desaturase from Candida albicans, cloned and expressed in the yeast Saccharomyces cerevisiae strain sur2 delta, was determined using stereospecifically labelled (2R,3S)-[2,3,4,4-2H4]-palmitic acid as a metabolic probe. Mass spectrometric analysis of the dinitrophenyl-derivatives of the labelled long-chain bases revealed elimination of a single deuterium atom from C(4) (corresponding to the C(4)-HR) along with a hydrogen atom from C(5) (corresponding to the C(5)-HS). This finding is consistent with an overall syn-elimination of the two vicinal hydrogen atoms. Besides the desaturation product sphingosine (93%) minor amounts of a 4-hydroxylated product (phytosphinganine, 7%) were identified that classify the Candida enzyme as a bifunctional desaturase/hydroxylase. Both processes, desaturation and hydroxylation proceed with loss of C(4)-HR from the chiral precursor. This finding is in agreement with a two-step process involving activation of the substrate by removal of the C(4)-HR to give a C-centred radical or radicaloid followed by either disproportionation into an olefin, water and a reduced diiron complex, or to recombination of the primary reactive intermediate with an active site-bound oxygen to yield a secondary alcohol. This result demonstrates the close mechanistic relationship between desaturation and hydroxylation as two different reaction pathways of a single enzyme and strengthens the mechanistic relationship of desaturases from fatty acid metabolism and sphingolipids.
Organic & Biomolecular Chemistry 08/2003; 1(14):2448-54. · 3.70 Impact Factor
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ABSTRACT: In contrast to 16:3 plants like rapeseed (Brassica napus), which contain alpha-linolenic acid (18:3(Delta9,12,15)) and hexadecatrienoic acid (16:3(Delta7,10,13)) as major polyunsaturated fatty acids in leaves, the silica-less diatom Phaeodactylum tricornutum contains eicosapentaenoic acid (EPA; 20:5(Delta5,8,11,14,17)) and a different isomer of hexadecatrienoic acid (16:3(Delta6,9,12)). In this report, we describe the characterization of two cDNAs having sequence homology to Delta12-fatty acid desaturases from higher plants. These cDNAs were shown to code for a microsomal and a plastidial Delta12-desaturase (PtFAD2 and PtFAD6, respectively) by heterologous expression in yeast (Saccharomyces cerevisiae) and Synechococcus, respectively. Using these systems in the presence of exogenously supplied fatty acids, the substrate specificities of the two desaturases were determined and compared with those of the corresponding rapeseed enzymes (BnFAD2 and BnFAD6). The microsomal desaturases were similarly specific for oleic acid (18:1(Delta9)), suggesting that PtFAD2 is involved in the biosynthesis of EPA. In contrast, the plastidial desaturase from the higher plant and the diatom clearly differed. Although the rapeseed plastidial desaturase showed high activity toward the omega9-fatty acids 18:1(Delta9) and 16:1(Delta7), in line with the fatty acid composition of rapeseed leaves, the enzyme of P. tricornutum was highly specific for 16:1(Delta9). Our results indicate that in contrast to EPA, which is synthesized in the microsomes, the hexadecatrienoic acid isomer found in P. tricornutum (16:3(Delta6,9,12)) is of plastidial origin.
Plant physiology 05/2003; 131(4):1648-60. · 6.53 Impact Factor
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ABSTRACT: The moss Physcomitrella patens contains high proportions of polyunsaturated very-long-chain fatty acids with up to 20 carbon atoms. Starting from preformed C18 polyunsaturated fatty acids, their biosynthesis involves a sequence of Delta6-desaturation, Delta6-elongation and Delta5-desaturation. In this report we describe for the first time the characterisation of a cDNA (PSE1) of plant origin with homology to the ELO-genes from Saccharomyces cerevisiae, encoding a component of the Delta6-elongase. Functional expression of PSE1 in S. cerevisiae led to the elongation of exogenously supplied Delta6-polyunsaturated fatty acids. By feeding experiments with different trienoic fatty acids of natural and synthetic origin, both substrate specificity and substrate selectivity of the enzyme were investigated. The activity of Pse1, when expressed in yeast, was not sensitive to the antibiotic cerulenin, which is an effective inhibitor of fatty acid synthesis and elongation. Furthermore, the PSE1 gene was disrupted in the moss by homologous recombination. This led to a complete loss of all C20 polyunsaturated fatty acids providing additional evidence for the function of the cDNA as coding for a component of the Delta6-elongase. The elimination of the elongase was not accompanied by a visible alteration in the phenotype, indicating that C20-PUFAs are not essential for viability of the moss under phytotron conditions.
The Plant Journal 09/2002; 31(3):255-68. · 6.16 Impact Factor
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ABSTRACT: The moss Physcomitrella patens contains high proportions of polyunsaturated very-long-chain fatty acids with up to 20 carbon atoms. Starting from preformed C18 polyunsaturated fatty acids, their biosynthesis involves a sequence of Δ6-desaturation, Δ6-elongation and Δ5-desaturation. In this report we describe for the first time the characterisation of a cDNA (PSE1) of plant origin with homology to the ELO-genes from Saccharomyces cerevisiae, encoding a component of the Δ6-elongase. Functional expression of PSE1 in S. cerevisiae led to the elongation of exogenously supplied Δ6-polyunsaturated fatty acids. By feeding experiments with different trienoic fatty acids of natural and synthetic origin, both substrate specificity and substrate selectivity of the enzyme were investigated. The activity of Pse1, when expressed in yeast, was not sensitive to the antibiotic cerulenin, which is an effective inhibitor of fatty acid synthesis and elongation. Furthermore, the PSE1 gene was disrupted in the moss by homologous recombination. This led to a complete loss of all C20 polyunsaturated fatty acids providing additional evidence for the function of the cDNA as coding for a component of the Δ6-elongase. The elimination of the elongase was not accompanied by a visible alteration in the phenotype, indicating that C20-PUFAs are not essential for viability of the moss under phytotron conditions.
The Plant Journal 08/2002; 31(3):255 - 268. · 6.16 Impact Factor
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Angewandte Chemie International Edition 08/2002; 41(13):2298-300. · 13.45 Impact Factor
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Stephan Diekmann,
Jennie Weston,
Ernst Anders,
Wilhelm Boland,
Bruno Schönecker,
Thomas Hettmann,
Johannes von Langen,
Stefan Erhardt,
Michael Mauksch,
Michael Bräuer, Christoph Beckmann,
Matthias Rost,
Petra Sperling,
Ernst Heinz
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ABSTRACT: The Collaborative Research Center (CRC) 436 'Metal-Mediated Reactions Modeled after Nature' was founded for the express purpose of analyzing the catalytic principles of metallo-enzymes in order to construct efficient catalysts on a chemical basis. The structure of the active center and neighboring chemical environment in enzymes serves as a focal point for developing reactivity models for the chemical redesign of catalysts. Instead of simply copying enzyme construction, we strive to achieve new chemical intuition based on the results of long-lasting natural evolution. We hope for success, since nature uses a limited set of building blocks, whereas we can apply the full repertoire of chemistry. Key substrates in this approach are small molecules, such as CO2, O2 NO3- and N2. Nature complexes these substrates, activates them and performs chemical transformations--all within the active center of a metalloenzyme. In this article, we report on some aspects and first results of the Collaborative Research Center (CRC) 436, such as nitrate reductase, sphingolipid desaturase, carbonic anhydrase, leucine aminopeptidase and dopamine beta-monooxygenase.
Journal of Biotechnology 05/2002; 90(2):73-94. · 3.05 Impact Factor
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Plant Physiology, v.131, 1648-1660 (2003).
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Organic and Biomolecular Chemistry, v.1, 2448-2454 (2003).
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Plant Journal, v.31, 255-268 (2002).
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Angewandte Chemie - International Edition, v.41, 2298-2300 (2002).
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S. Dieckmann,
J. Weston,
E Anders,
Wilhelm Boland,
B. Schönecker,
T. Hettmann,
J. von Langen,
S. Erhardt,
M. Mauksch,
M Bräuer, Christoph Beckmann,
M. Rost,
P Sperling,
E Heinz
Reviews in Molecular Biotechnology, v.90, 73-94 (2002).
