Abstract
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 [(3)H]Phe was fed, the majority of metabolized [(3)H]Phe was incorporated into p-[(3)H]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 [(3)H]Phe and [(14)C]cinnamate simultaneously to the triple expressers, but found no evidence for channeling of the endogenously synthesized [(3)H]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.
Highlights
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
Cloning of a Xylem Expressed Poplar PAL Gene—Since it has been shown that different PAL isoforms may differentially associate with C4H in vivo (12), it was desirable to employ cDNAs encoding divergent PAL isoforms with potentially distinct roles in phenylpropanoid metabolism to study the interactions between PAL and C4H in yeast
The low ratio of p-[3H]coumarate/p-[14C]coumarate relative to the 3H/14C ratio for styrene indicates that [3H]cinnamate endogenously synthesized from PAL was not preferentially used by C4H in the yeast strains. These results strongly argue against the concept that a multienzyme complexes (MECs) readily forms between PAL and C4H when they co-occur in one cell, and suggest that such a complex is not required for efficient channeling of Phe into p-coumarate
Summary
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. 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). Using simple and novel analytical tools, we have used this system to evaluate the proposed MEC model and to explore how the entry point enzymes of phenylpropanoid metabolism redirect carbon flow from primary to secondary metabolism
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