Retrobiosynthetic NMR Studies with 13C-Labeled Glucose

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 the13C13C 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 range13C13C 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. 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 the13C13C 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 range13C13C 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. The genetic manipulation of phenylpropanoid-derived metabolites has been proposed as a prospective target for crop improvement (1Dixon R.A. Lamb C.J. Masoud S. Sewalt V.J.H. Paiva N.L. Gene (Amst.). 1996; 179: 61-71Crossref PubMed Scopus (164) Google Scholar). A fundamental understanding of the key enzymes of the various phenylpropanoid branching pathways is therefore of increasing interest. Gallic acid (3,4,5-trihydroxybenzoic acid, compound 7, Fig.1) serves as a fundamental precursor for gallotannins and ellagitannins, abundant classes of plant secondary metabolites. Gallic acid is also formed in substantial amounts by some fungi. A considerable number of studies on the biosynthesis of tannins has been reported (for review, see Gross (2Gross G.C. Basic Life Sci. 1992; 59: 43-60Google Scholar) and Haslam (3Haslam E. Basic Life Sci. 1992; 59: 169-194Google Scholar)). Surprisingly, the biosynthesis of gallic acid, the phenolic unit of this important class of natural products, is still incompletely understood. Based mainly on radiolabeling studies, it has been proposed that gallic acid could be formed from phenylalanine (compound5) via caffeic acid (compound 8), 3,4,5-trihydroxycinnamic acid (compound 9), or protocatechuic acid (compound 6, Fig. 1) (4Kato N. Shiroya M. Yoshida S. Hasegawa M. Shokubutsugaku Zasshi. 1968; 81: 506-507Crossref Google Scholar, 5Zenk M.H. Z. Naturforsch. 1964; 19b: 83-84Crossref Scopus (15) Google Scholar, 6Neish A.C. Towers G.H.N. Chen D. El-Basyouni S.Z. Ibrahim R.K. Phytochemistry. 1964; 3: 485-492Crossref Scopus (109) Google Scholar, 7Brucker W. Hashem M. Ber. Dtsch. Bot. Ges. 1962; 75: 3-13Google Scholar, 8Brucker W. Drehmann U. Arch. Mikrobiol. 1958; 30: 396-408Crossref PubMed Scopus (2) Google Scholar, 9Brucker W. Planta. 1957; 48: 627-630Crossref Scopus (7) Google Scholar). This would imply that the carboxylic group of gallic acid is derived from the β carbon atom of phenylalanine (compound 5), and ultimately from the enoylpyruvoyl group of chorismate (compound 3, Fig.1). On the other hand, it has been argued that the carboxylic group of gallic acid is biosynthetically equivalent to the carboxylic group of shikimate (10Dewick P.M. Haslam E. Chem. Commun. 1968; : 673-675Google Scholar, 11Dewick P.M. Haslam E. Biochem. J. 1969; 113: 537-542Crossref PubMed Scopus (56) Google Scholar, 12Marigo G. Alibert G. Boudet A. C. R. Acad. Sci. Ser. D. 1969; 269: 1852-1854Google Scholar, 13Cornthwaite D.C. Haslam E. J. Chem. Soc. 1965; : 3008-3011Crossref Google Scholar, 14Conn E.E. Swain T. Chem. Ind. 1961; : 592-593Google Scholar, 15Haslam E. Haworth R.D. Knowles P.F. J. Chem. Soc. 1961; : 1854-1859Crossref Scopus (21) Google Scholar). 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) (16Tateoka T.N. Bot. Mag. 1968; 81: 103-104Crossref Google Scholar). It was also proposed that the controversial results could indicate the existence of alternative biosynthetic routes in the same organism (17Ishikura N. Hayashida S. Tazaki K. Bot. Mag. 1984; 97: 355-367Crossref Scopus (23) Google Scholar, 18Saijo R. Agric. Biol. Chem. 1983; 47: 455-460Crossref Scopus (1) Google Scholar, 19Ishikura N. Experientia. 1975; 31: 1407-1408Crossref Scopus (6) Google Scholar, 20Zaprometov M.N. Bukhlaeva V.Y. Biokhimiya. 1968; 33: 383-386PubMed Google Scholar). 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 exogenous13C-labeled glucose. Gallic acid and amino acids were obtained by hydrolysis of cell material, and the 13C distribution was analyzed by NMR spectroscopy. A central aspect of this approach was a retrobiosynthetic comparison of labeling patterns of gallic acid with those of amino acids. The results show unequivocally that gallic acid is biosynthesized in both organisms from an early intermediate of the shikimate pathway, but not via phenylalanine. [U-13C6]Glucose (99%13C) was purchased from Isotec (Miamisburg, FL), and [1-13C]glucose was purchased from Omicron (South Bend, IN). Spores of P. blakesleeanus(NRLL1555(−)) were obtained from P. Galland, Marburg, Germany. Young leaves of R. typhina were cut from a local tree between May and August. Spores of P. blakesleeanus were activated by heat treatment (48 °C) for 2 h. The culture medium contained, per liter, 2.0 g of asparagine in H2O, 5.0 g of KH2PO4, 0.5 g of MgSO4·7H2O, 0.2 ml of 14% (w/w) CaCl2 solution, 2 mg of thiamine hydrochloride, 2 mg of citric acid in H2O, 1.5 mg of Fe(NO3)3·9H2O, 1 mg of ZnSO4·7H2O, 0.3 mg of MnSO4·H2O, 50 μg of CuSO4·5H2O, 50 μg of NaMoO4·2H2O, pH 4.7. A sterile solution of 6 g of [1-13C]glucose in 5 ml of water was added to the autoclaved medium (300 ml). Alternatively, a mixture of [U-13C6]glucose (320 mg) and unlabeled glucose (7.7 g) in 5 ml of water was added to the autoclaved medium (400 ml). Erlenmeyer flasks (1 liter) containing 400 ml of culture medium were seeded with one loopful of spores and were incubated with shaking at 25 °C for 6 days. The mycelium was harvested by filtration and was washed with a small amount of water. The stems of young leaves of R. typhina (with a length of 3–4 cm) were immersed into a sterile solution of 1% (w/w) of glucose consisting of 96% of unlabeled glucose and 4% of [U-13C6]glucose. They were incubated in a dark compartment (Heraeus Vötsch climate chamber) at 18 °C and 60% humidity for 33 days. Small sections of the stem were removed with a sharp razor at intervals of 1 or 2 days. The culture medium from experiments with P. blakesleeanus was adjusted to pH 1, treated at 96 °C for 6 h, and extracted with 150 ml of diethyl ether for 12 h using a liquid/liquid perforator. Fungal biomass was treated with 1 m sulfuric acid (100 ml) at 96 °C for 6 h. The solution was filtered and was then continuously extracted with 150 ml of diethyl ether for 12 h using a liquid/liquid perforator. The organic phases obtained by extraction of culture medium and of cell mass hydrolysate of P. blakesleeanus were combined. The aqueous solution obtained by acidic treatment of biomass and subsequent extraction with ether was set aside for isolation of tyrosine and phenylalanine. The organic phase was extracted twice with 75 ml of saturated sodium bicarbonate. The aqueous solution was adjusted to pH 1 by the addition of sulfuric acid and was to extraction with 150 ml of diethyl The organic phase was to a small ml) The solution was to a of 1.5 The was with a mixture of diethyl acid Gallic acid was with the were and were to of were to a which was with a mixture of diethyl acid Gallic acid at a of and was by treatment of biomass and isolation of gallic acid was as for cell phase was with a of which was with aqueous The was were with a The of gallic acid was was isolated from cell mass hydrolysis as W. A. J. Biol. Chem. PubMed Google Scholar). and phenylalanine were isolated from the aqueous phase obtained extraction of the biomass hydrolysate with diethyl ether The aqueous phase was adjusted to 6 m and for h. The of the amino acids from the hydrolysate was as W. A. J. Biol. Chem. PubMed Google Scholar). 1H and 13C NMR were at and with a Gallic acid was in and in and phenylalanine in m The were for 1H NMR data prior to 13C NMR were as acid, data to 1H prior to for all carbon atoms of biosynthetic gallic acid, phenylalanine, and were obtained by comparison with natural This different of carbon atoms and of 13C to in 13C of carbon atoms were obtained from the 13C in the 1H The were to carbon atoms G. W. A. G. J. Biochem. 1992; PubMed Scopus Google Scholar). were based on and experiments not patterns were with the The was to unequivocally the carboxylic group of gallic acid from the carboxylic group of an early shikimate precursor or from the β carbon atom of the phenylalanine side This can be by a comparison between the labeling patterns of biosynthetic gallic acid and phenylalanine or which are obtained by hydrolysis of cell This retrobiosynthetic approach a labeling to different labeling of the carboxylic group of early shikimate and of the β carbon atom of phenylalanine or tyrosine. the carboxylic group of early shikimate and the β carbon of phenylalanine are derived from different atoms the group and the of this can be by a variety of and results were obtained with [1-13C]glucose in P. leaves of R. typhina, the incorporation of [1-13C]glucose was low to this could be by incorporation experiments using a mixture of [U-13C6]glucose and unlabeled glucose. It be shown that the of this is as with the precursor A shake flask culture of P. blakesleeanus (300 ml) was supplied with 6 g of [1-13C]glucose The culture was incubated for 6 days. Gallic acid mg) was isolated from the culture and from the cell hydrolysate as tyrosine and mg) were obtained by hydrolysis of cell mass The 13C of all carbon atoms of gallic acid (compound phenylalanine (compound 5), tyrosine (compound and Fig. was as (Fig. to the of the aromatic of phenylalanine, tyrosine and gallic acid, the carbon atoms and an 13C different biosynthetic of of and of the labeling patterns of the shikimate and the labeling of (compound can be by a retrobiosynthetic approach on of the biosynthetic pathway (Fig. The labeling of (compound 1) can be unequivocally from the labeling of the side of phenylalanine and tyrosine (Fig. The labeling patterns are on of from [1-13C]glucose is to of metabolites by the pathway and This results in the of the β of phenylalanine and tyrosine of 13C Fig. of [1-13C]glucose by the of the pathway and unlabeled (compound Fig. from the unlabeled via the pathway be the 13C labeling in the of 13C can be by via the pathways via the pathway of glucose from could 13C to the of glucose. the of glucose to (compound could also to the of glucose. The resulting could then be to via the pathway with or of The labeling in of that at of glucose is to the by The labeling of the of gallic acid with the labeling of phenylalanine (compound and tyrosine Fig. The carboxylic group of gallic acid has the β carbon atoms of tyrosine or phenylalanine are It follows that the carboxylic group is not derived from the β carbon of phenylalanine or tyrosine. As shown in Fig. 1, the carboxylic atom of early shikimate compound 4) stems from of (compound 1) which is not from [1-13C]glucose (Fig. a of 5-dehydroshikimate to gallic acid would be in with the to the same approach for the of gallic acid biosynthesis in leaves of the R. typhina, were The amount of to gallic acid and phenylalanine was low 13C in the most carbon that it was to This is to the that glucose supplied to the leaves via the leaf stem is and the NMR are by the of which were formed prior to glucose A was therefore As shown the can be by the of a mixture of [U-13C6]glucose and unlabeled glucose. This approach be with the culture and then with plant A shake flask culture of P. blakesleeanus was supplied with a mixture of [U-13C6]glucose and unlabeled glucose at a of The NMR of metabolites isolated from this culture are complex multiplets as a coupling and it was important to a of at 0.5 The central by with a 13C which were formed from the natural in the glucose The complex indicate the of various isotopomers that biosynthetic of the glucose. A analysis of the that the biosynthetic are complex of different 13C NMR of gallic acid from P. blakesleeanus incorporation of [U-13C6]glucose with unlabeled glucose for see to Fig. The patterns can be by numerical simulation of the13C13C coupling patterns for isotopomer in the As shown in Fig. the chemical shift as as to coupling show between the and It be that the of of the coupling with to the of the isotopomers is to coupling patterns and to chemical shift by heavy isotope shift The heavy isotope of in isotopomers as from comparison of with are in The various isotopomers in the mixture are by carbon A 13C atom results in a shift in the of 2 13C atoms are to a shift of the carbon in the of and coupling are also in Fig. It be that the chemical shift of the carbon atoms and 5 is by the heavy isotope shift in isotopomer (Fig. 6) has a heavy isotope shift of and has a heavy isotope shift of This results in coupling between and which are the coupling patterns the are shown in Fig. it be that the to coupling are by the The of isotopomer in the mixture can be obtained by of the isotopomers can be from different the of isotopomer can be from the of and The quantitative data obtained from different of the are in The isotopomer composition of the biosynthetic gallic acid is in Fig. The quantitative of isotopomer is by the of the 13C atoms and also by which indicate of that of the is formed via the or (Fig. that of the is via of the This is in with the labeling from [1-13C]glucose The complex multiplets of biosynthetic tyrosine obtained from the biomass grown on the of [U-13C6]glucose and unlabeled glucose was analyzed by the same The isotopomer composition is in Fig. It is that the isotopomer of the are in gallic acid and tyrosine. This is a for the of gallic acid from the shikimate isotopomers with 13C atoms in atom and the β carbon of the side chain are in the amino The of this isotopomer the of the amino acid from the atoms of shikimate and the side chain by a On the other hand, the isotopomer composition of gallic acid is by the (Fig. gallic acid were derived from phenylalanine or tyrosine by subsequent of the side the of this isotopomer could not be It follows that gallic acid is not formed via phenylalanine or tyrosine in the experiments shown that the incorporation of [1-13C]glucose in leaves of R. is The of also studies in the and is probably the major for the on gallic acid The experiments with cultures that the could be by the of glucose to the formation of The of the isotopomers are out in the by coupling and are from the of the of gallic acid with natural 13C A number of experiments were required to from glucose into the plant in the the of the plant as by the of the leaves and the of the of the length of the and the 13C in the glucose as experiments be in some be in the of other incorporation experiments with It was important to young leaves the of glucose with increasing and of the of and humidity to be to the of the plant as as 18 °C and 60% humidity most for R. typhina in the dark in 13C incorporation as with or the of glucose in R. typhina, and the biosynthetic of the plant from the plant was It was therefore to the as as to the leaves could be kept for 1 The most is the labeling of the precursor 13C-labeled glucose the 13C in studied was in On the of natural 13C small by the 13C precursor could not be with the with glucose to be experiments were with and with the of metabolites was low the incorporation of the could be by the coupling in the of gallic acid and amino acids. in the with coupling was the of the low of the metabolites this a it is that the cut leaves biosynthetic of the incubation The of the isolated metabolites been prior to the On the other hand, the been with the precursor, and this in the of precursor for formation of phenylalanine. 1% of the gallic acid been from the precursor the incubation but the small amount of formed gallic acid was from glucose. On of an with 150 young leaves of R. typhina g) which were supplied with a solution (300 ml) containing mg of [U-13C6]glucose and 6 g of natural glucose. Gallic acid mg) and amino acids were isolated by the and NMR of gallic acid and tyrosine were The complex 13C NMR of the carboxylic carbon and atom of gallic acid are shown in Fig. to the experiments with the cultures and 5), the of the coupling patterns is low as with the of isotopomers a 13C atom by which the of natural gallic The of natural gallic acid which been at the of glucose a amount was biosynthetically from unlabeled glucose in the Fig. also the and 13C NMR of natural gallic The the of the and The were to for the of the biosynthetic and the natural The 13C distribution in the natural is to a equivalent of of the central for isotopomers with 13C atoms as and are in the natural The of isotopomers with and carbon atoms in natural is The of are therefore the of in the natural gallic On the other hand, to isotopomers are in the biosynthetic and can be unequivocally on of the isotopomer deconvolution which has been (Fig. A quantitative analysis of the biosynthetic gallic acid the isotopomer composition shown in The of isotopomers with 2 and 13C isotopomers was to the for the various in the natural The natural was then from the the composition of the which was biosynthesized from the [U-13C6]glucose. The isotopomer composition of biosynthetic tyrosine was by the same approach and is also shown in Fig. The isotopomer patterns of the aromatic of biosynthetic gallic acid and tyrosine are and that the aromatic of both from the same the Gallic acid a amount of the in with the Phycomyces This isotopomer has equivalent in tyrosine the of the is low in with the based on the shikimate with the Phycomyces experiment, it can be that phenylalanine and tyrosine not as major in the gallic acid biosynthetic with 13C-labeled fundamental as or in with unlabeled is to biosynthetic formation of complex isotopomer studies, NMR to 13C-labeled isotopomers W. M.H. A. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, W. E. W. A. J. Biol. Chem. PubMed Scopus Google Scholar). the this approach would been for of the incorporation of glucose by leaves of R. typhina was shown that an analysis of isotopomer mixture is by an in analysis of coupling multiplets in one-dimensional 13C this it is important to NMR at to the chemical shift it is important to at the 0.5 to and to of of of the complex can then be the NMR of an carbon atom by the 13C coupling multiplets of different isotopomers as shown in and The deconvolution of the complex is in the most patterns a analysis by numerical simulation of the for As of the deconvolution it is to the heavy isotope shift effects and the coupling for isotopomer from the complex It be that to of can be by heavy isotope shift and coupling patterns of can this is it has the to quantitative data for isotopomer in the This isotopomer can then be for biosynthetic via comparison of isotopomer distribution patterns in different metabolites. the comparison of isotopomer patterns of the of gallic acid with those of phenylalanine and tyrosine that gallic acid is a of the shikimate The of biosynthetic studies with 13C is by the of NMR by the of natural in the the formation of metabolites from the precursor is The of and the quantitative NMR of biosynthetic isotopomers with a in the are from the natural via in the by As the of isotopomers in natural is low for and for As a of and isotopomers can be unequivocally as the of biosynthesis from the the biosynthesis of tyrosine and phenylalanine, of are for the biosynthesis of of the and via shikimate and of the side chain via the side chain of It is therefore that the isotope of [U-13C6]glucose to the side chain of tyrosine in R. typhina the of in the the by a of studies shown that plant can substantial amounts of shikimate pathway as shikimate and T. The of Scholar). from the labeling data that early shikimate were to chorismate and to tyrosine the This would in the formation of the aromatic amino acid from a at natural 13C by the addition of a side chain derived from the glucose The data in this report show that gallic acid is formed or from an early intermediate prior to or of the shikimate proposed that different via an early shikimate intermediate and via one of the aromatic amino acid could be in the same organism M.H. Z. Naturforsch. 1964; 19b: 83-84Crossref Scopus (15) Google Scholar, N. Hayashida S. Tazaki K. Bot. Mag. 1984; 97: 355-367Crossref Scopus (23) Google Scholar, 18Saijo R. Agric. Biol. Chem. 1983; 47: 455-460Crossref Scopus (1) Google Scholar, 19Ishikura N. Experientia. 1975; 31: 1407-1408Crossref Scopus (6) Google Scholar, 20Zaprometov M.N. Bukhlaeva V.Y. Biokhimiya. 1968; 33: 383-386PubMed Google Scholar). it has been that gallic acid is formed via dehydrogenation of shikimate in young leaves of Rhus but via phenylalanine in leaves of R. (17Ishikura N. Hayashida S. Tazaki K. Bot. Mag. 1984; 97: 355-367Crossref Scopus (23) Google Scholar). data show that a biosynthetic pathway via phenylalanine could of biosynthesized gallic acid in the experiments with P. blakesleeanus and in the experiments with R. typhina The biosynthesis of gallic acid (compound 7, by dehydrogenation of acid (compound 4) in both the fungus and the plant to be the major the data a pathway via protocatechuic acid (compound 6) by of acid followed by a (Fig. (4Kato N. Shiroya M. Yoshida S. Hasegawa M. Shokubutsugaku Zasshi. 1968; 81: 506-507Crossref Google Scholar). this a of acid to protocatechuic acid has been with from (16Tateoka T.N. Bot. Mag. 1968; 81: 103-104Crossref Google Scholar). to between the routes are in the of A. and with the of the M. for and and P. Galland, Marburg, for a culture of P.