title: Membrane protein complexes catalyze both 4- and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis.
abstract: The hydroxylation of 4- and 3-ring carbons of cinnamic acid derivatives during monolignol biosynthesis are key steps that determine the structure and properties of lignin. Individual enzymes have been thought to catalyze these reactions. In stem differentiating xylem (SDX) of Populus trichocarpa, two cinnamic acid 4-hydroxylases (PtrC4H1 and PtrC4H2) and a p-coumaroyl ester 3-hydroxylase (PtrC3H3) are the enzymes involved in these reactions. Here we present evidence that these hydroxylases interact, forming heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane protein complexes. Enzyme kinetics using yeast recombinant proteins demonstrated that the enzymatic efficiency (V(max)/k(m)) for any of the complexes is 70-6,500 times greater than that of the individual proteins. The highest increase in efficiency was found for the PtrC4H1/C4H2/C3H3-mediated p-coumaroyl ester 3-hydroxylation. Affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical cross-linking, and reciprocal coimmunoprecipitation provide further evidence for these multiprotein complexes. The activities of the recombinant and SDX plant proteins demonstrate two protein-complex-mediated 3-hydroxylation paths in monolignol biosynthesis in P. trichocarpa SDX; one converts p-coumaric acid to caffeic acid and the other converts p-coumaroyl shikimic acid to caffeoyl shikimic acid. Cinnamic acid 4-hydroxylation is also mediated by the same protein complexes. These results provide direct evidence for functional involvement of membrane protein complexes in monolignol biosynthesis.
authors.affiliation: Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Hsi-Chuan
authors.initials: HC
authors.lastname: Chen
authors.forename: Quanzi
authors.initials: Q
authors.lastname: Li
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Shuford
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.forename: David C
authors.initials: DC
authors.lastname: Muddiman
authors.forename: Ronald R
authors.initials: RR
authors.lastname: Sederoff
authors.forename: Vincent L
authors.initials: VL
authors.lastname: Chiang
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D003373:Coumaric Acids; D017931:DNA Primers / Q000235:genetics; D006900:Hydroxylation; D047468:Immunoprecipitation; D007700:Kinetics; D008031:Lignin / Q000096:biosynthesis*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D015394:Molecular Structure; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D010636:Phenols; D010666:Phenylpropionates; D010957:Plasmids / Q000235:genetics; D032107:Populus / Q000378:metabolism*; D011422:Propionates; D050564:Trans-Cinnamate 4-Monooxygenase / Q000737:chemistry / Q000378:metabolism*; D052584:Xylem / Q000378:metabolism*; D015003:Yeasts
chemical_list: D003373:Coumaric Acids; D017931:DNA Primers; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D010636:Phenols; D010666:Phenylpropionates; D011422:Propionates; C496130:sinapyl alcohol; D008031:Lignin; C010559:coniferyl alcohol; D050564:Trans-Cinnamate 4-Monooxygenase; D002265:Carboxylic Ester Hydrolases; C067271:trans-4-coumaroyl esterase; C495469:p-coumaric acid