I. Werner et al., RETROBIOSYNTHETIC NMR-STUDIES WITH C-13-LABELED GLUCOSE - FORMATION OF GALLIC ACID IN PLANTS AND FUNGI, The Journal of biological chemistry, 272(41), 1997, pp. 25474-25482
The biosynthesis of gallic acid was studied in cultures of the fungus
Phycomyces blakesleeanus and in leaves of the tree Rhus typhina. Funga
l cultures were grown with [1-C-13]gluose or with a mixture of unlabel
ed glucose and [U-C-13(6)]glucose. Young leaves of R. typhina were kep
t in an incubation chamber and were supplied with a solution containin
g a mixture of unlabeled glucose and [U-C-13(6)]glucose via the leaf s
tem. Isotope distributions in isolated gallic acid and aromatic amino
acids were analyzed by one-dimensional H-1 and C-13 NMR spectroscopy.
A quantitative analysis of the complex isotopomer composition of metab
olites was obtained by deconvolution of the (CC)-C-13-C-13 coupling mu
ltiplets using numerical simulation methods. This approach required th
e accurate analysis of heavy isotope chemical shift effects in a varie
ty of different isotopomers and the analysis of long range (CC)-C-13-C
-13 coupling constants. The resulting isotopomer patterns were interpr
eted using a retrobiosynthetic approach based on a comparison between
the isotopomer patterns of gallic acid and tyrosine. The data show tha
t 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 gr
oup of an early intermediate of the shikimate pathway and not from the
side chain of phenylalanine or tyrosine. It follows that the committe
d 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, caf
feic 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 incorpor
ation of uniformly C-13-labeled glucose followed by quantitative NMR a
nalysis of isotopomer patterns is suggested as a general method for bi
osynthetic studies. As shown by the plant experiment, this approach is
also applicable to systems with low incorporation rates.