IntroductionUsing glass capillary gas-liquid ,chromatography and mass spectrometry the alkaloid patterns of Lupinus polyphyllus --~ LINDL. plants and cell suspension cultures were analyzed. Sparteine, angustifoline,lupanine, 13-hydroxylupanine, 17-0x0lupanine, 13-cinnamoyllupanine (new for L. polphyllws), 13-tigloyllupanine and its cis-isomer 13-angel~~llupanine (as a new alkaloid), and a dehydrolupanine were found in L. polyphyllus leaves ' = 13-hydroxylupanine; 8/9 = tigloyl-and angeloyllupanine.

The alkaloid composition of cell suspension cultures and differentiated plants of Lupinus polyphyllus was evaluated using quartz capillary gas-liquid chromatography, GLC-MS and FD-MS. Lupanine (97% of total alkaloids), sparteine, 13-angeloyloxylupanine and 13-tigloyloxylupanine were detected in alkaloid extracts of L. polyphyllus cell suspension cultures. Lupanine, 13-cis and 13-trans-cinnamoyloxylupanine were found in the spent cell culture medium. No significant difference was found in the alkaloid composition of photomixotrophic and heterotrophic cell strains although the alkaloid content was 5 to 10 times higher in photomixotrophic cell strains. In the respective plants we could identify 18 alkaloids which include the following esters of 13-hydroxylupanine: 13-tigloyloxylupanine, 13-angeloyloxylupanine, 13-cis-cinnamoyloxylupanine, 13-trans-cinnamoyloxylupanine, 13-benzoyloxylupanine, 13-(2-methylbutyryl)-oxylupanine; and 13-vanilloyloxylupanine.

Biosynthesis of heme d1, the essential prosthetic group of the dissimilatory nitrite reductase cytochrome cd1, requires the methylation of the tetrapyrrole precursor uroporphyrinogen III at positions C‐2 and C‐7. We produced Pseudomonas aeruginosa NirE, a putative S‐adenosyl‐l‐methionine (SAM)‐dependent uroporphyrinogen III methyltransferase, as a recombinant protein in Escherichia coli and purified it to apparent homogeneity by metal chelate and gel filtration chromatography. Analytical gel filtration of purified NirE indicated that the recombinant protein is a homodimer. NirE was shown to be a SAM‐dependent uroporphyrinogen III methyltransferase that catalyzes the conversion of uroporphyrinogen III into precorrin‐2 in vivo and in vitro. A specific activity of 316.8 nmol of precorrin‐2 h−1·mg−1 of NirE was found for the conversion of uroporphyrinogen III to precorrin‐2. At high enzyme concentrations NirE catalyzed an overmethylation of uroporphyrinogen III, resulting in the formation of trimethylpyrrocorphin. Substrate inhibition was observed at uroporphyrinogen III concentrations above 17 μm. The protein did bind SAM, although not with the same avidity as reported for other SAM‐dependent uroporphyrinogen III methyltransferases involved in siroheme and cobalamin biosynthesis. A P. aeruginosa nirE transposon mutant was not complemented by native cobA encoding the SAM‐dependent uroporphyrinogen III methyltransferase involved in cobalamin formation. However, bacterial growth of the nirE mutant was observed when cobA was constitutively expressed by a complementing plasmid, underscoring the special requirement of NirE for heme d1 biosynthesis.

ABsm.icT.-Alkaloid extracts from Biifilisifi fiztstrtilis leaves, analyzed by capillary gas chromatography and gc/ms, were shown to contain sparteine ( l ) , lupanine (6), cytisine (3), A'-methylcytisine (2), anagyrine (U), 17-ososparteine (ll), 13-hydroxyanagyrine (lo), Fi,C~dehydrolupanine (4), tinctorine (7) (new for Bnplzsiti), isot inctorine ( 5 ) (new alkaloid) and 13-acetoxyanagyrine (9) (new alkaloid).The alkaloid content of cell suspension criltures was three orders lower than that (Jf the differentiated plants; lripanine figured as the only alkaloid. A biogenetic pathway w*ith lripanine biosynthesis as the central part, from which the a-pyridone alkaloids derive via 5,Gdehydrolupanine (4), is discussed.

During heme biosynthesis the oxygen-independent coproporphyrinogen III oxidase HemN catalyzes the oxidative decarboxylation of the two propionate side chains on rings A and B of coproporphyrinogen III to the corresponding vinyl groups to yield protoporphyrinogen IX. Here, the sequence of the two decarboxylation steps during HemN catalysis was investigated. A reaction intermediate of HemN activity was isolated by HPLC analysis and identified as monovinyltripropionic acid porphyrin by mass spectrometry. This monovinylic reaction intermediate exhibited identical chromatographic behavior during HPLC analysis as harderoporphyrin (3-vinyl-8,13,17-tripropionic acid-2,7,12,18-tetramethylporphyrin). Furthermore, HemN was able to utilize chemically synthesized harderoporphyrinogen as substrate and converted it to protoporphyrinogen IX. These results suggest that during HemN catalysis the propionate side chain of ring A of coproporphyrinogen III is decarboxylated prior to that of ring B.