P0883 Cytochrome P450 Monooxygenases Form Protein Complexes to Regulate Key Hydroxylation Steps in Monolignol Biosynthesis

Hsi-Chuan Chen , North Carolina State University, Raleigh, NC
Quanzi Li , North Carolina State University, Raleigh, NC
Christopher M. Shuford , North Carolina State University, Raleigh, NC
Jie Liu , North Carolina State University
Ying-Chung Lin , North Carolina State University, Raleigh, NC
David Muddiman , North Carolina State University, Raleigh, NC
Ronald Sederoff , North Carolina State University, Raleigh, NC
Vincent Chiang , North Carolina State University, Raleigh, NC
Lignin is a phenolic polymer made by vascular plants to support secondary cell walls and to create a hydrophobic surface for water transport. Lignin is typically polymerized from three phenylpropanoid monomers: H, G and S monolingols. The conversion of plant biomass into fermentable sugars or pulp and paper is largely determined by the structure, quantity and monomer composition of lignin. Three types of cytochrome P450 monooxygenases establish key structural characteristics of monolignols. Cinnamic acid 4-hydroxylase (C4H) catalyzes aromatic ring-4 hydroxylation of cinnamic acid into p-coumaric acid for H monolignols. p-Coumaric acid 3-hydroxylase (also called p-coumaroyl ester 3-hydroxylase) (C3H) hydroxylates p-coumaroyl ester derivatives at the ring-3 position, leading to coniferaldehyde and G monolignols. Coniferaldehyde 5-hydroxylase (CAld5H), then hydroxylates coniferaldehyde at the ring-5 carbon to yield S monolignols. There are two C4H homologs (PtrC4H1 and PtrC4H2) and one C3H (PtrC3H3) in Populus trichocarpa. We describe the specific functions of these three hydroxylases and the discovery of their protein complexes. Protein complexes were previously postulated in monolignol biosynthesis, but with no direct evidence. Based on affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical crosslinking, reciprocal co-immunoprecipitation, and the P. trichocarpa xylem protoplast system, we showed that PtrC4H1, PtrC4H2, and PtrC3H3 form all three possible heterodimers and a heterotrimer, PtrC4H1/C4H2/C3H3. Enzyme kinetic studies revealed novel complex-enhanced activity and regulation of 4- and 3-hydroxylations that may help improve our understanding of regulatory mechanisms in monolignol biosynthesis. (This work is supported by the National Science Foundation, Plant Genome Research Program grant, DBI-0922391, to VLC.)