The conversion was authenticated on the basis of the SRM detection of multiple product ions specific to the [,-13C2]-labeled enzyme reaction products (Fig. 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. Lignin is a phenolic polymer made by vascular plants to support secondary cell walls and to create a hydrophobic surface for water transport (1). Lignin also forms a physical barrier to restrict pathogens and resists microbial decomposition in wood (1). Lignin is typically polymerized from three phenylpropanoid monomers,p-coumaryl, coniferyl, and sinapyl alcohols, also known as the H, G, and S monolignols, respectively (2). The conversion of plant biomass into fermentable sugars or Candesartan cilexetil (Atacand) pulp and paper is largely determined by the structure, quantity, and monomer composition of lignin (3,4). Monolignol biosynthesis in angiosperms is mediated by 10 enzyme families through a metabolic grid (5,6). Three types of cytochrome P450 monooxygenases establish key structural characteristics of monolignols (5) (Fig. 1). Cinnamic acid 4-hydroxylase (C4H; CYP73A) catalyzes aromatic ring-4 hydroxylation of cinnamic acid intop-coumaric acid (7), the most direct precursor for H monolignols.p-Coumaric acid 3-hydroxylase (also calledp-coumaroyl ester 3-hydroxylase) (C3H; CYP98A) (8) hydroxylatesp-coumaroyl ester derivatives at the ring-3 position, leading to coniferaldehyde and G monolignols. Coniferaldehyde 5-hydroxylase (CAld5H; CYP84A) (9), then hydroxylates coniferyl aldehyde at the ring-5 carbon (9,10) to yield S monolignols (Fig. 1). == Fig. 1. == Proposed partial monolignol biosynthesis pathway. The 4- and 3-Hydroxylation steps are highlighted, and the dotted lines indicate multiple intermediate steps in the biosynthesis (6). Our understanding of the regulation of the monolignol biosynthetic pathway at the transcript, protein, and metabolite levels remains incomplete. Genome sequence information now allows us to investigate this regulation in greater detail. Full transcriptome analysis suggests involvement of multiple protein members of several families in monolignol biosynthesis (6,11,12). For example, inPopulus trichocarpatwoC4Hgenes,PtrC4H1(POPTR_0013s15380) andPtrC4H2(POPTR_0019s15110), sharing 85.9% DNA sequence identity, are expressed specifically and abundantly during lignification in stem differentiating xylem (SDX) (6).PtrC4H1andPtrC4H2are paralogs (not alleles) because they Candesartan cilexetil (Atacand) are located on linkage groups 13 and 19, respectively (6). The specific biochemical or regulatory roles of each PtrC4H and of each of the members of several other protein families in monolignol biosynthesis are unknown. Knowledge of such roles is central to a comprehensive understanding of the organization and regulation of the entire lignin biosynthetic pathway. The aromatic ring-3 hydroxylation was long thought to occur atp-coumaric acid to yield caffeic acid (5) (Fig. 1). Biochemical evidence indicates that 3-hydroxylation can take place atp-coumaroyl shikimic acid (Fig. 1).Arabidopsis thalianaC3H recombinant protein produced in yeast (Saccharomyces cerevisiae) convertsp-coumaroyl shikimic acid into caffeoyl shikimic acid very efficiently (kcat= 600 min1) (8). However, the yeast recombinant protein fromP. trichocarpa C3H3(PtrC3H3), the apparent ortholog of theA.thaliana C3H, has essentially no activity withp-coumaroyl shikimic acid (Results). Genetic evidence supports 3-hydroxylation at PPP1R53 thep-coumaroyl ester level.A.thalianamutants in theC3Hgene or transgenic cosuppression of this gene inA.thaliana(13), alfalfa (Medicago sativa) (14), and hybrid poplar (Populus grandidentataalba) (15) resulted in lignin enriched with H monomers. However, no accumulation of the C3H substrate,p-coumaroyl shikimic acid, Candesartan cilexetil (Atacand) was detected in theA.thaliana C3H-knockout mutant (cyp98A3) (13), inconsistent with a 3-hydroxylation path at thep-coumaroyl ester level. Clearly, our knowledge of the 3-hydroxylation pathway is incomplete and many aspects of P450 monolignol hydroxylation need to be further explored. The components of a P450 system are anchored in microsomal membranes, where the monooxygenase interacts with its redox partner, NADPH cytochrome P450 reductase (CPR), for substrate oxygenation to yield a hydroxylated product (16). In mammals, there is only one CPR species, and P450s form multiprotein complexes to interact with CPR (1720). Similarly, CPR may be a limiting factor for plant P450 reactions (21). C4H and C3H could interact to share CPR for two consecutive hydroxylation reactions, and the interaction could be.