Biosynthesis of fumonisin and aal derivatives by Alternaria and Fusarium in laboratory culture.

Cultures of Fusarium moniliforme and Alternaria alternata f. sp. lycopersici were grown in the laboratory and analyzed for various fumonisin derivatives. Analyses were made by continuous flow fast atom bombardment and ionspray mass spectrometry interfaced to microcapillary HPLC. Besides FB1, FB2 and FB3 derivatives, two isomers of the one-armed FB1 (protonated molecular ion at m/z 564) and two isomers of the one-armed FB2 (m/z 548) were found. Two different isolates of A. alternata when grown in culture yielded FB1, FB2 and FB3. One of them also yielded the one-armed FB1 which was identical to that metabolite found in Fusarium and naturally infected corn. FB1, FB2 and FB3 have been found in 2 different isolates of Alternaria alternata, both obtained from tomato.

Triacylglycerol Bioassembly in Microspore-Derived Embryos of Brassica napus L. cv Reston.

Erucic acid (22:1) was chosen as a marker to study triacylglycerol (TAG) biosynthesis in a Brassica napus L. cv Reston microspore-derived (MD) embryo culture system. TAGs accumulating during embryo development exhibited changes in acyl composition similar to those observed in developing zygotic embryos of the same cv, particularly with respect to erucic and eicosenoic acids. However, MD embryos showed a much higher rate of incorporation of (14)C-erucoyl moieties into TAGs in vitro than zygotic embryos. Homogenates of early-late cotyledonary stage MD embryos (14-29 days in culture) were assessed for the ability to incorporate 22:1 and 18:1 (oleoyl) moieties into glycerolipids. In the presence of [1-(14)C]22:1-coenzyme A (CoA) and various acyl acceptors, including glycerol-3-phosphate (G-3-P), radiolabeled erucoyl moieties were rapidly incorporated into the TAG fraction, but virtually excluded from other Kennedy Pathway intermediates as well as complex polar lipids. This pattern of erucoyl incorporation was unchanged during time course experiments or upon incubation of homogenates with chemicals known to inhibit Kennedy Pathway enzymes. In marked contrast, parallel experiments conducted using [1-(14)C]18:1-CoA and G-3-P indicated that (14)C oleoyl moieties were incorporated into lyso-phosphatidic acids, phosphatidic acids, diacylglycerols, and TAGs of the Kennedy Pathway, as well as other complex polar lipids, such as phosphatidylcholines and phosphatidylethanolamines. When supplied with l-[2-(3)H(N)]G-3-P and [1-(14)C]22:1-CoA, the radiolabeled TAG pool contained both isotopes, indicating G-3-P to be a true acceptor of erucoyl moieties. Radio-high-performance liquid chromatography, argentation thin-layer chromatography/gas chromatography-mass spectrometry, and stereospecific analyses of radiolabeled TAGs indicated that 22:1 was selectively incorporated into the sn-3 position by a highly active diacylglycerol acyltransferase (DGAT; EC 2.3.1.20), while oleoyl moieties were inserted into the sn-1 and sn-2 positions. In the presence of sn-1,2-dierucin and [1-(14)C]22:1-CoA, homogenates and microsomal preparations were able to produce radiolabeled trierucin, a TAG not found endogenously in this species. A 105,000g pellet fraction contained 22:1-CoA:DGAT exhibiting the highest specific activity. The rate of 22:1-CoA:DGAT activity in vitro could more than account for the maximal rate of TAG biosynthesis observed in vivo during embryo development. In double label experiments, G-3-P was shown to stimulate the conversion of [(3)H]phosphatidylcholines to [(3)H]diacylglycerols, which subsequently acted as acceptors for (14)C erucoyl moieties. In vitro, 22:1 moieties did not enter the sn-1 position of TAGs by a postsynthetic modification or transacylation of preformed TAGs.

A simple enzymatic method for the preparation of radiolabeled erucoyl-CoA and other long-chain fatty acyl-CoAs and their characterization by mass spectrometry.

A simple two-step method for the biosynthesis of radiolabeled erucoyl-coenzyme A of high specific activity and other long-chain fatty acyl-coenzyme A (acyl-CoA) thioesters is reported. 1-14C-labeled erucic and oleic acids, as well as unlabeled ricinoleic and nervonic acids, were incubated at 35 degrees C with coenzyme A in the presence of ATP, MgCl2, and acyl-CoA synthetase (EC 6.2.1.3) from Pseudomonas spp. to yield the corresponding CoA thioesters. Following incubation, each thioester was purified by rapid passage through a disposable reverse-phase C18 extraction column. The overall yields were greater than 90% and the purities greater than 95%, based on the distribution of radioactivity, and chromatographic and spectral properties. Fast ion bombardment-mass spectrometry was employed to confirm the structures of the various acyl-CoAs.

Gas chromatography-mass spectrometry of the N(O)-heptafluorobutyryl isobutyl esters of the protein amino acids using electron impact ionisation.

The N(O)-heptafluorobutyryl isobutyl esters of the protein amino acids have been analysed by combined gas chromatography-mass spectrometry using electron impact ionisation. The spectral data have been used to confirm the structures of the amino acid derivatives.