Reconstitution of the Entry Point of Plant Phenylpropanoid Metabolism in Yeast (Saccharomyces cerevisiae)

Phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and the C4H redox partner cytochrome P450 reductase (CPR) are important in allocating significant amounts of carbon from phenylalanine into phenylpropanoid biosynthesis in plants. It has been proposed that multienzyme complexes (MECs) containing PAL and C4H are functionally important at this entry point into phenylpropanoid metabolism. To evaluate the MEC model, two poplar PAL isoforms presumed to be involved in either flavonoid (PAL2) or in lignin biosynthesis (PAL4) were independently expressed together with C4H and CPR in Saccharomyces cerevisiae, creating two yeast strains expressing either PAL2, C4H and CPR or PAL4, C4H and CPR. When [3H]Phe was fed, the majority of metabolized [3H]Phe was incorporated into p-[3H]coumarate, and Phe metabolism was highly reduced by inhibiting C4H activity. PAL alone expressers metabolized very little phenylalanine into cinnamic acid. To test for intermediate channeling between PAL and C4H, we fed [3H]Phe and [14C]cinnamate simultaneously to the triple expressers, but found no evidence for channeling of the endogenously synthesized [3H]cinnamate into p-coumarate. Therefore, efficient carbon flux from Phe to p-coumarate via reactions catalyzed by PAL and C4H does not appear to require channeling through a MEC in yeast, and instead biochemical coupling of PAL and C4H is sufficient to drive carbon flux into the phenylpropanoid pathway. This may be the primary mechanism by which carbon allocation into phenylpropanoid metabolism is controlled in plants. Phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and the C4H redox partner cytochrome P450 reductase (CPR) are important in allocating significant amounts of carbon from phenylalanine into phenylpropanoid biosynthesis in plants. It has been proposed that multienzyme complexes (MECs) containing PAL and C4H are functionally important at this entry point into phenylpropanoid metabolism. To evaluate the MEC model, two poplar PAL isoforms presumed to be involved in either flavonoid (PAL2) or in lignin biosynthesis (PAL4) were independently expressed together with C4H and CPR in Saccharomyces cerevisiae, creating two yeast strains expressing either PAL2, C4H and CPR or PAL4, C4H and CPR. When [3H]Phe was fed, the majority of metabolized [3H]Phe was incorporated into p-[3H]coumarate, and Phe metabolism was highly reduced by inhibiting C4H activity. PAL alone expressers metabolized very little phenylalanine into cinnamic acid. To test for intermediate channeling between PAL and C4H, we fed [3H]Phe and [14C]cinnamate simultaneously to the triple expressers, but found no evidence for channeling of the endogenously synthesized [3H]cinnamate into p-coumarate. Therefore, efficient carbon flux from Phe to p-coumarate via reactions catalyzed by PAL and C4H does not appear to require channeling through a MEC in yeast, and instead biochemical coupling of PAL and C4H is sufficient to drive carbon flux into the phenylpropanoid pathway. This may be the primary mechanism by which carbon allocation into phenylpropanoid metabolism is controlled in plants. Carbon flux into the phenylpropanoid pathway in plants is mediated by a key gateway enzyme, phenylalanine ammonia lyase (PAL) 1The abbreviations used are: PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; CPR, NADPH:cytochrome P450 reductase; MEC, multienzyme complex; ORF, open reading frame; HPLC, high performance liquid chromatography; PA, piperonylic acid.1The abbreviations used are: PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; CPR, NADPH:cytochrome P450 reductase; MEC, multienzyme complex; ORF, open reading frame; HPLC, high performance liquid chromatography; PA, piperonylic acid. that catalyzes the non-oxidative deamination of phenylalanine (Phe) to produce trans-cinnamic acid. Trans-cinnamic acid is hydroxylated at the para-position by a cytochrome P450 monooxygenase enzyme, cinnamate-4-hydroxylase (C4H), in conjunction with NADPH:cytochrome P450 reductase (CPR). The coordinated reactions catalyzed by these enzymes account for a large fraction of the carbon flow in some specialized plant tissues. For example, in the secondary xylem of woody plants, 15–36% of assimilated carbon is channeled to lignin, a polymer of hydroxycinnamic acids derived from phenylpropanoid metabolism (1.Sarkanen K.V. Hergert H.L. Ludwig C.H. Lignin: Occurrence, Formation, Structure and Reactions. Wiley Interscience, New York1971: 43-94Google Scholar). Similarly, in vegetative tissues, soluble flavonoid or phenylpropanoid derivatives such as sinapoyl malate in Arabidopsis, or rutin and chlorogenic acid in tobacco, are known to accumulate in abundance (2.Landry L.G. Chapple C.C. Last R.L. Plant Physiol. 1995; 109: 1159-1166Crossref PubMed Scopus (502) Google Scholar, 3.Lorenzen M. Racicot V. Strack D. Chapple C. Plant Physiol. 1996; 112: 1625-1630Crossref PubMed Scopus (53) Google Scholar, 4.Mayer M.J. Narbad A. Parr A.J. Parker M.L. Walton N.J. Mellon F.A. Michael A.J. Plant Cell. 2001; 13: 1669-1682Crossref PubMed Google Scholar). Thus, PAL and subsequent enzymes in the phenylpropanoid pathway can direct significant amount of carbon into many different phenylpropanoid metabolic endproducts, as dictated by developmental programs. Since PAL resides at a metabolically important position, linking the phenylpropanoid secondary pathway to primary metabolism, the regulation of overall flux into phenylpropanoid metabolism has been suggested to be modulated by PAL as a rate-limiting enzyme (5.Hahlbrock K. Knobloch K.H. Kreuzaler F. Potts J.R. Wellmann E. Eur. J. Biochem. 1976; 61: 199-206Crossref PubMed Scopus (140) Google Scholar). How this regulation is accomplished, however, is not completely clear. Feedback inhibitory regulation of PAL activity by its own product, trans-cinnamate, has been demonstrated in vitro (6.Appert C. Logemann E. Hahlbrock K. Schmid J. Amrhein N. Eur. J. Biochem. 1994; 225: 491-499Crossref PubMed Scopus (109) Google Scholar, 7.Havir E.A. Hanson K.R. Biochemistry. 1968; 7: 1904-1914Crossref PubMed Scopus (108) Google Scholar, 8.Jorrin J. Dixon R.A. Plant Physiol. 1990; 92: 447-455Crossref PubMed Scopus (76) Google Scholar), and trans-cinnamic acid was proposed to modify transcription of PAL genes in vivo (9.Bolwell G.P. Mavandad M. Millar D. Edwards K.J. Schuch W. Dixon R.A. Phytochemistry. 1988; 27: 2109-2117Crossref Scopus (62) Google Scholar, 10.Mavandad M. Edwards R. Liang X. Lamb C.J. Dixon R.A. Plant Physiol. 1990; 94: 671-680Crossref PubMed Scopus (74) Google Scholar). In tobacco with suppressed C4H expression, reduced C4H activity was counter-intuitively correlated with a decrease in intracellular cinnamate levels, suggesting feedback inhibition (i.e. autoregulation) of PAL at a certain threshold level of endogenous cinnamate (11.Blount J.W. Korth K.L. Masoud S.A. Rasmussen S. Lamb C. Dixon R.A. Plant Physiol. 2000; 122: 107-116Crossref PubMed Scopus (166) Google Scholar). It has also been postulated that PAL can form multienzyme complexes (MECs) that include downstream enzymes such as C4H, facilitating efficient channeling of carbon into phenylpropanoid metabolism. It has been shown that a specific PAL isoform (PAL1) is physically associated with both isolated microsomes and the cytoplasmic fraction in fractionated tobacco cell extracts, supporting a model in which PAL associates with MECs in an isoform-specific manner to modulate a carbon flux into specific downstream pathways (12.Rasmussen S. Dixon R.A. Plant Cell. 1999; 11: 1537-1552Crossref PubMed Scopus (152) Google Scholar). Other data from experiments employing classical approaches such as double radioactive labeling assays (13.Czichi U. Kindl H. Planta. 1975; 125: 115-125PubMed Google Scholar, 14.Czichi U. Kindl H. Phanta. 1977; 134: 133-143Google Scholar, 15.Hrazdina G. Wagner G.J. Arch. Biochem. Biophys. 1985; 237: 88-100Crossref PubMed Scopus (153) Google Scholar), enzyme co-purification (15.Hrazdina G. Wagner G.J. Arch. Biochem. Biophys. 1985; 237: 88-100Crossref PubMed Scopus (153) Google Scholar, 16.Wagner G.J. Hrazdina G. Plant Physiol. 1984; 74: 901-906Crossref PubMed Google Scholar), and immunohistochemical localization (17.Hrazdina G. Zobel A.M. Hoch H.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8966-8970Crossref PubMed Scopus (77) Google Scholar) support the presence of MECs among enzymes in the entry point or downstream pathways. Investigation of possible MECs in phenylpropanoid metabolism has been further pursued in recent years using various direct among enzymes in the flavonoid pathway in Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar) and the of an in C.J. Dixon R.A. Plant Cell. 2001; 13: PubMed Scopus Google Scholar). is no direct evidence for to form an In poplar the entry point enzymes C4H, CPR, and of the phenylpropanoid pathway been and A. C.J. Plant Physiol. PubMed Scopus Google Scholar, D. J. C.J. 2001; Google Scholar, M. D. C.J. Plant 1999; PubMed Scopus Google Scholar, N. C.J. Plant Physiol. 2001; PubMed Scopus Google Scholar, J. C.J. Plant Physiol. PubMed Scopus Google Scholar, R. S. C.J. Plant Physiol. PubMed Scopus Google Scholar). PAL is a enzyme, the redox enzymes C4H and CPR been shown in to be N. C.J. Plant Physiol. 2001; PubMed Scopus Google Scholar, J. C.J. Plant Physiol. PubMed Scopus Google Scholar), in a MEC that In from two PAL and were shown to be highly expressed in of and but expressed in secondary xylem of poplar M. D. C.J. Plant 1999; PubMed Scopus Google Scholar, R. S. C.J. Plant Physiol. PubMed Scopus Google Scholar). The isoforms by are associated with biosynthesis of and phenylpropanoid and of the PAL with secondary xylem and are involved in lignin and a PAL expressed in secondary xylem was in for the presumed of the and PAL genes associated with lignin biosynthesis not been in In this we the of a for a PAL that the of entry point phenylpropanoid genes from the of the entry point PAL, C4H, and CPR, into the yeast Saccharomyces in different in an to the entry point of phenylpropanoid metabolism in a and we used this to evaluate the proposed MEC model and to the entry point enzymes of phenylpropanoid metabolism carbon flow from primary to secondary metabolism. and and for yeast and were from and the of were to R.A. 1995; 11: PubMed Scopus Google Scholar). The and yeast strains were and in as by the In of was to of by and at with for to were at for and in the by to for the yeast were at for with of and The cell was in the and phenylalanine as or of a PAL of and the and of the K. S. N. Plant 1995; PubMed Scopus Google Scholar) were and these two was to from a of xylem using the The were at for with of to and into a using a were to and and the enzyme of the poplar was and the was as to from poplar genes PAL2, and N. C.J. Plant Physiol. 2001; PubMed Scopus Google Scholar, R. S. C.J. Plant Physiol. PubMed Scopus Google Scholar). of poplar the E. W. D. Plant 2000; Scopus Google Scholar) was that was and a was of was was to J. Scholar). were using using a was at in a containing and were for at in with The was at in and for were using a and a and and for C4H, and CPR was as J. C.J. Plant Physiol. PubMed Scopus Google Scholar). For primary D. Phytochemistry. 1996; PubMed Scopus Google Scholar) at was used with at of PAL in the for and were from and For the ORF, a and a were and for the ORF, a and a were The were with and into the of the in the The of were by PAL PAL cell of PAL alone or yeast strains were and as were in a containing and of were at for with a The yeast were for at and the were by of of containing and for in with The yeast were in for at of containing of were used for the PAL enzyme for at in containing phenylalanine in The reactions were by of of and the were with of The were in the been The presence of cinnamic acid was and using and of PAL or C4H, and the and in of yeast were and cell and were by The was by of of and with by of the from the in a of of were at the of to of the The was to by and was used for To from yeast the yeast were in of or with of The cell and were by at for The were and The was to and of the were by The was with a and a was The for were from acid and to acid to for at a flow of When and were of the cinnamate and p-coumarate were from the and in of of these were found in cell either by or In for the experiments shown in were the from and were together for yeast and strains were and in The cell between and but of the yeast strains was to a of in of or and triple were for the and and the C4H piperonylic acid was to of the for at a of of phenylalanine was to the yeast strains were in for The from the and the were as The were fractionated a in a of acid with an of at a flow of were between and and the of fraction was by in a liquid In and triple expressers, two of activity were at and In yeast strains of were at and was independently by using the and yeast strains were by of for in were in of with together with from G. H. N. was by using containing and acid in to yeast cell were for at and were as and the radioactive were by the HPLC, using the in the [3H]Phe were that to the of and The and in the were by in a liquid with for and of PAL and alone and expressers were independently for and the with of of the PAL alone and were in of containing The or strains were also and in the as a but the of were in that the two of yeast of were in a and were by at for were through a and the of and p-coumarate was by of a PAL has been shown that different PAL isoforms may with C4H in vivo (12.Rasmussen S. Dixon R.A. Plant Cell. 1999; 11: 1537-1552Crossref PubMed Scopus (152) Google Scholar), was to PAL isoforms with in phenylpropanoid metabolism to the between PAL and C4H in K. S. N. Plant 1995; PubMed Scopus Google Scholar) a of xylem to and an poplar acid was to the poplar derived from M. D. C.J. Plant 1999; PubMed Scopus Google Scholar, R. S. C.J. Plant Physiol. PubMed Scopus Google Scholar). The was to the by this from PAL genes M. D. C.J. Plant 1999; PubMed Scopus Google Scholar, N. C.J. Plant Physiol. 2001; PubMed Scopus Google Scholar, R. S. C.J. Plant Physiol. PubMed Scopus Google Scholar). In of the was in secondary xylem and to a in and this was highly by To this the was with the the of by this was very to that R. S. C.J. Plant Physiol. PubMed Scopus Google Scholar), in and cell but little in xylem Therefore, the a in secondary is expressed in of PAL, C4H, and CPR in The for and were used together with for poplar C4H and CPR genes to the entry point of the phenylpropanoid pathway in yeast of the C4H and in the yeast are expressed of the and and together support high of C4H activity in yeast strains J. C.J. Plant Physiol. PubMed Scopus Google Scholar). To yeast strains expressing different of PAL, C4H, and CPR, the of the (PAL2) or (PAL4) were into the the of the was with the or or with these together with to different yeast two strains expressing or and two strains expressing or as and In and were to a of the genes in yeast was by using and to C4H and CPR, or using to and the was not in either or triple expressers using this an PAL enzyme assays were using from yeast The of yeast strains an activity that catalyzed the deamination of Phe to produce cinnamate as by this activity was in the not PAL in and strains were between and in and strains were between and and were no significant in PAL between and triple expressers not Therefore, is that the enzyme was in and yeast in the with which the yeast strains Phe to phenylpropanoid PAL, C4H, and CPR was for which the yeast were in containing Phe for of the and cell were fractionated by and at was of no a were to the and of these to be derived from Phe by the catalyzed by a in Phe biosynthesis A. S. M. PubMed Scopus Google Scholar) with was a in the amount of this in fed high Phe not of from or strains amounts of an as that yeast strains expressing PAL accumulate amounts of metabolism in yeast strains by PAL, C4H, and of the entry point into phenylpropanoid metabolism in this yeast reactions involved in Phe are in The pathway by of PAL, C4H, and CPR genes is in and endogenous pathways that cinnamate and p-coumarate are acid of is to carbon and reactions are by double but reactions or In to the of cinnamate in the and strains and large amounts of by a amount of cinnamate were in Thus, the of PAL, C4H, and CPR in yeast in efficient of expressers to Phe to cinnamate with In of the the amount of cinnamate and p-coumarate from cell was of that from Thus, the of the cinnamate and p-coumarate by the yeast strains was into the To yeast strains and produce p-coumarate from endogenous Phe yeast is for these strains were in Phe For yeast from the were also in with or The of these experiments are in in the of significant amounts of p-coumarate were synthesized by strains and cinnamate was the level of in yeast strains these Thus, PAL and C4H in the strains carbon from a of endogenous Phe to the secondary p-coumarate. expressing PAL the not the to significant amounts of carbon from endogenous Phe into the phenylpropanoid pathway. of amounts of Phe in in the of both cinnamate and p-coumarate by the triple expressers In expressers, the amounts of cinnamate from to that of p-coumarate was with data that Phe is a in vivo for cinnamate by strains expressing PAL but that triple expressers are to the of Phe to p-coumarate very at the Phe endogenously by of Phe in is known that S. can Phe as a J.R. The of the Saccharomyces Scholar) using a pathway the of Phe to subsequent to and of to Scopus Google Scholar). such as be synthesized from these S. is also to various acids J. Scholar, M. Lamb C.J. R. 1994; PubMed Scopus Google Scholar), for the of cinnamate and p-coumarate to and by a acid pathways and are in used assays to in carbon flux into in the yeast from the and of yeast PAL or triple were fractionated by HPLC, and the associated with fraction the was [3H]Phe was independently by of the fraction for were used to of these and The of these metabolic experiments from PAL or strains are shown in In with data in a amount of [3H]cinnamate of was found in PAL alone and and a large amount of of was found in the triple radioactive that by the were also in these yeast was in yeast strains of but not in the that cinnamate is further metabolized to this by the endogenous enzyme to the of an which be from p-coumarate by the not be in triple two radioactive specific to triple expressers were in at and with of the that may be or its metabolic The of and in expressers and triple expressers, to in triple expressers, that metabolism of cinnamate and p-coumarate to these is not a metabolic in these the of from and by of [3H]Phe the of from and by of [3H]Phe in a to the yeast strains synthesized from Phe by endogenous yeast to be a known of the Phe pathway with to and were also The intermediate was to be but the fraction in the and not be the was and this a of the of were at and with in yeast but these were not further of the from [3H]Phe to the yeast was not and was presumed to be incorporated into of the Phe was as Phe in but this to the important was that Phe was in strains and to PAL alone expressing strains or the The between the level of [3H]Phe from strains and of and that found in PAL expressing strains or in the was to the amount of that in the triple In we used piperonylic acid an and specific of C4H with a of M. F. R. D. Plant Physiol. PubMed Scopus Google Scholar), to independently the of C4H activity in this metabolic in triple When fed to C4H expressing yeast the of for the inhibition of C4H activity in vivo was found to be and at this no PAL activity not For and triple expressers in were derived from the of yeast When was to yeast strains and [3H]Phe into p-coumarate was In both triple expressers, inhibition of C4H activity in not in cinnamate and its but also in the of an metabolic pathway that Phe as an strains were also reduced in to as by in [3H]Phe found in triple expressers to the triple expressers together with in that C4H enzyme activity is a of the metabolic into phenylpropanoid metabolism in yeast by of PAL and Investigation of a in postulated in plant is possible that a MEC between PAL and C4H metabolic flux in yeast to yeast assays to between or and C4H derivatives in which the was no evidence for not we used a double labeling to [3H]cinnamate endogenously from PAL is used by C4H, to [14C]cinnamate in triple The [3H]Phe and [14C]cinnamate fed simultaneously to the strains and be incorporated into p-coumarate by of the PAL and C4H reactions and to a incorporated into via the reactions of PAL and that the endogenous yeast and plant PAL enzymes not the to in a multienzyme the of to can be used as an for the of two physically enzymes in a pathway. PAL and C4H were in a multienzyme in the yeast significant amounts of cinnamate endogenously by PAL be and to C4H, and the of to in p-coumarate be that in In two of the the of p-coumarate not of in either or but in were The of to the for that [3H]cinnamate endogenously synthesized from PAL was not used by C4H in the yeast the that a MEC between PAL and C4H in and that such a is not for efficient channeling of Phe into of of [3H]Phe and [14C]cinnamate into p-coumarate or in yeast strains and [3H]Phe and [14C]cinnamate were to strains and for of in the from two experiments are of in the from two experiments are [3H]Phe and [14C]cinnamate were to strains and for of in the from two experiments are in a this metabolic does not of PAL and C4H in yeast, we that not be to PAL and C4H in a cell to efficient of Phe to p-coumarate. To test efficient with physically PAL and we independently and either or with a expressing J. C.J. Plant Physiol. PubMed Scopus Google Scholar). the of cinnamate and p-coumarate in these two and In in of strains and was The of cinnamate and p-coumarate in and that of strains and the amount of p-coumarate that in the was of that in the triple expressers S. to key phenylpropanoid entry point PAL, C4H, and CPR, to coordinated in metabolic flux and to the proposed MEC in this efficient metabolic flux from Phe into p-coumarate was by of these a key was that biochemical coupling of PAL and C4H sufficient to and metabolic flux from Phe to p-coumarate between the two This is by data from [3H]Phe double labeling and In the double labeling is to of is that of but intermediate from to enzyme is to that from to proposed (6.Appert C. Logemann E. Hahlbrock K. Schmid J. Amrhein N. Eur. J. Biochem. 1994; 225: 491-499Crossref PubMed Scopus (109) Google Scholar, 7.Havir E.A. Hanson K.R. Biochemistry. 1968; 7: 1904-1914Crossref PubMed Scopus (108) Google Scholar, 8.Jorrin J. Dixon R.A. 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