Contribution of CoA ligases to benzenoid biosynthesis in petunia flowers.
Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the β-oxidative or nonoxidative pathways. The first step in the β-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the β-oxidative pathway.
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907, USA.
Antje
A
Klempien
Yasuhisa
Y
Kaminaga
Anthony
A
Qualley
Dinesh A
DA
Nagegowda
Joshua R
JR
Widhalm
Irina
I
Orlova
Ajit Kumar
AK
Shasany
Goro
G
Taguchi
Christine M
CM
Kish
Bruce R
BR
Cooper
John C
JC
D'Auria
David
D
Rhodes
Eran
E
Pichersky
Natalia
N
Dudareva
D001555:Benzene Derivatives / Q000737:chemistry / Q000378:metabolism*; D003066:Coenzyme A Ligases / Q000378:metabolism*; D035264:Flowers / Q000201:enzymology* / Q000378:metabolism*; D032306:Petunia / Q000201:enzymology* / Q000378:metabolism*; D013379:Substrate Specificity
D001555:Benzene Derivatives; D003066:Coenzyme A Ligases