Abstract
The biosynthesis of gallic acid was studied in cultures of the fungus Phycomyces blakesleeanus and in leaves of the tree Rhus typhina. Fungal cultures were grown with [1-13C]glucose or with a mixture of unlabeled glucose and [U-13C6]glucose. Young leaves of R. typhina were kept in an incubation chamber and were supplied with a solution containing a mixture of unlabeled glucose and [U-13C6]glucose via the leaf stem. Isotope distributions in isolated gallic acid and aromatic amino acids were analyzed by one-dimensional 1H and 13C NMR spectroscopy. A quantitative analysis of the complex isotopomer composition of metabolites was obtained by deconvolution of the 13C13C coupling multiplets using numerical simulation methods. This approach required the accurate analysis of heavy isotope chemical shift effects in a variety of different isotopomers and the analysis of long range 13C13C coupling constants. The resulting isotopomer patterns were interpreted using a retrobiosynthetic approach based on a comparison between the isotopomer patterns of gallic acid and tyrosine. The data show that both in the fungus and in the plant all carbon atoms of gallic acid are biosynthetically equivalent to carbon atoms of shikimate. Notably, the carboxylic group of gallic acid is derived from the carboxylic group of an early intermediate of the shikimate pathway and not from the side chain of phenylalanine or tyrosine. It follows that the committed precursor of gallic acid is an intermediate of the shikimate pathway prior to prephenate or arogenate, most probably 5-dehydroshikimate. A formation of gallic acid via phenylalanine, the lignin precursor, caffeic acid, or 3,4, 5-trihydroxycinnamic acid can be ruled out as major pathways in the fungus and in young leaves of R. typhina. The incorporation of uniformly 13C-labeled glucose followed by quantitative NMR analysis of isotopomer patterns is suggested as a general method for biosynthetic studies. As shown by the plant experiment, this approach is also applicable to systems with low incorporation rates.
Highlights
The biosynthesis of gallic acid was studied in cultures of the fungus Phycomyces blakesleeanus and in leaves of the tree Rhus typhina
Since the carboxylic group of early shikimate derivatives and the  carbon of phenylalanine are derived from different atoms of pyruvate, this condition can be fulfilled by a variety of precursors, and unequivocal results were obtained with [1-13C]glucose in P. blakesleeanus
FIG. 4. 13C NMR signature of C-4 of gallic acid from P. blakesleeanus cultured with [U-13C6]glucose diluted with unlabeled glucose (1:25; w/w)
Summary
The biosynthesis of gallic acid was studied in cultures of the fungus Phycomyces blakesleeanus and in leaves of the tree Rhus typhina. The carboxylic group of gallic acid is derived from the carboxylic group of an early intermediate of the shikimate pathway and not from the side chain of phenylalanine or tyrosine. It has been argued that the carboxylic group of gallic acid is biosynthetically equivalent to the carboxylic group of shikimate (10 –15) This would imply that gallic acid is formed from an early shikimate intermediate, e.g. directly from 5-dehydroshikimate (compound 4) by dehydrogenation or via protocatechuic acid (compound 6) as an intermediate (Fig. 1) [16]. This report describes studies with shake flask cultures of the fungus, Phycomyces blakesleeanus, and with young leaves of the tree, Rhus typhina, which were supplied with exogenous 13C-labeled glucose. A fundamental understanding of the key enzymes of the various phenylpropanoid branching pathways is of increasing interest
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