| Summary: | Cinnamate-4-hydroxylase (C4H) and its redox partner NADPHcytochrome P450 reductase
(CPR), together with phenylalanine ammonia-lyase (PAL), play central roles at the gateway
into plant phenylpropanoid metabolism. C4H catalyzes conversion of cinnamate to pcoumarate
and has been proposed to anchor a multienzyme complex (MEC) on the
endoplasmic reticulum (ER), recruiting PAL and potentially other soluble enzymes. The
formation of p-coumarate is a key step in commitment of large amounts of carbon to lignin
and soluble phenylpropanoid biosynthesis, especially in woody plants. In this thesis,
catalytic and structural roles of poplar (Populus trichocarpa x P. deltoides) C4H and CPR
were investigated.
Clones of three isoforms of CPR were isolated from poplar xylem and young leaf
cDNA libraries. Two of these cDNA clones and a previously cloned C4H cDNA were
expressed in yeast. Efficient conversion of cinnamate to p-coumarate by yeast microsomes
containing C4H confirmed the authenticity of the C4H cDNA, while co-expression of C4H
and CPR substantiated the bona fide CPR activity of the two divergent CPRs. To examine
whether the C4H and CPR are localized to the ER, C-terminal green fluorescent protein
tagged versions of C4H and the two CPRs were expressed in Arabidopsis. Confocal
microscopy analysis of Arabidopsis seedlings demonstrated predominant localization of the
chimeric proteins on ER.
To test the MEC model, an engineered yeast strain expressing PAL, C4H, and CPR
was generated, in which phenylpropanoid product formation and metabolite channeling
between enzymes could be investigated. Quantitative measurements showed that the triplegene
expresser synthesized a striking amount of p-coumarate, while PAL-alone and C4Hinhibited
triple expressers could not efficiently convert phenylalanine (Phe) to cinnamate.
When 3H-Phe and 14C-cinnamate were simultaneously fed to the triple expresser,
endogenously synthesized 3H-cinnamate was not preferred by C4H over 14C-cinnamate.
Therefore, the observed efficient carbon flow from Phe to p-coumarate via the reaction
catalyzed by PAL and C4H does not appear to require channeling through a MEC in yeast.
Analysis of the biochemical properties of the entry point reactions and enzymes suggested
instead that kinetic and thermodynamic coupling of PAL and C4H is sufficient to drive
carbon-flux from primary metabolism to the phenylpropanoid pathway.
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