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J. Biol. Chem., Vol. 279, Issue 4, 2600-2607, January 23, 2004

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

IMPLICATIONS FOR CONTROL OF METABOLIC FLUX INTO THE PHENYLPROPANOID PATHWAY^*

Dae-Kyun Ro and Carl J. Douglas

From the Department of Botany, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada

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.

Received for publication, September 8, 2003 , and in revised form, October 30, 2003.

^* This work was supported by a Discovery Grant (to C. J. D.) from the Natural Sciences and Engineering Research Council of Canada and by a University Graduate Fellowship from the University of British Columbia (to D.-K. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This article is dedicated to Dr. Towers on the occasion of his 80th birthday.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY372451.

To whom correspondence should be addressed. Tel.: 604-822-3554; Fax: 604-822-6089; E-mail: cdouglas{at}interchange.ubc.ca.

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